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Sample records for cell line h-1

  1. Characterization of stimulus-secretion coupling in the human pancreatic EndoC-βH1 beta cell line.

    Directory of Open Access Journals (Sweden)

    Lotta E Andersson

    Full Text Available Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay, gene expression (Gene Chip array, metabolite levels (GC/MS, respiration (Seahorse XF24 Extracellular Flux Analyzer, glucose utilization (radiometric, lactate release (enzymatic colorimetric, ATP levels (enzymatic bioluminescence and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the

  2. [Adenovirus-mediated delivery of nm23-H1 gene inhibits growth of colorectal carcinoma cell line Lovo].

    Science.gov (United States)

    Wang, Qi; He, Xueling; Liu, Yan; Yin, Hailin

    2010-12-01

    This experimental study sought to find out the inhibitory effects of Ad-GFP-nm23-H1 on proliferation and metastasis of human colorectal carcinoma cell line Lovo, and, further, to gain an insight into some theoretical and methodical basis for instituting nm23-H1 gene therapy of cancers. MTT assay and Transwell chamber were used to detect the rates of proliferation and invasion as well as the adhesion of Lovo cells in vitro. The results demonstrated that the proliferation inhibition rates of Lovo cells treated with Ad-GFP-nm23-H1 of 10(10) PFU/ml, 10(9) PFU/ml and 10(8) PFU/ml were 84.9% +/- 1.51%, 48.5% +/- 7.23% and 22.5% +/- 5.47%, that the adherence inhibition rates of Lovo cells treated with Ad-GFP-nm23-H1 of 10(10) PFU/ml, 10(9) PFU/ml and 10(8) PFU/ml were 70.3% +/- 2.40%, 60.1% +/- 5.68% and 18.5% +/- 3.61%, and that the invasiveness inhibition rates of Lovo cells treated with Ad-GFP-nm23-H1 of 10(10) PFU/ml, 10(9) PFU/ml and 10(8) PFU/ml were 83.2% +/- 5.71%, 52.2% +/- 6.94% and 28.1% +/- 8.21%. These data suggested that Ad-GFP-nm23-H1 exerted significant inhibitory effects on the proliferation and metastasis of human colorectal carcinoma cell line Lovo in a dose-dependent way.

  3. Cell Lines Expressing Nuclear and/or Mitochondrial RNAse H1 | NCI Technology Transfer Center | TTC

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    The National Institute of Child Health & Human Development (NICHD), Program in Genomics of Differentiation, seeks interested parties to further co-develop small molecule inhibitors of RNase H1, especially in regards to genome instability, transcription, and translation.

  4. Establishment of a functional cell line expressing both subunits of H1a and H2c of human hepatocyte surface molecule ASGPR.

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    Hu, Bin; Yang, Yan; Liu, Jia; Ma, Zhiyong; Huang, Hongping; Liu, Shenpei; Yu, Yuan; Hao, Youhua; Wang, Baoju; Lu, Mengji; Yang, Dongliang

    2010-10-01

    To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR H1b) on ASGPR ligands' binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH1a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector pIRES2EGFP, pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRH1a and H2c in 4-1-6 were confirmed by RT-PCR, Western blotting and immunofluorescence. The endocytosis function of the artificial "ASGPR" on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H1b/pCDNA3.1 (neo) was transfected into cell line 4-1-6, H1b did not down-regulate the ligand binding ability of ASGPR. The eukaryotic expression plasmid H1b/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single H1b nor H1b and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both H1a and H2c stably was established. The new split variant H1b has no effect on ASGPR binding to ASOR. ASGPRH1b alone can't bind to ASOR, it yet can't form functional complex with ASGPRH2c.

  5. Distinct differences in the responses of the human pancreatic β-cell line EndoC-βH1 and human islets to proinflammatory cytokines.

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    Oleson, Bryndon J; McGraw, Jennifer A; Broniowska, Katarzyna A; Annamalai, Mani; Chen, Jing; Bushkofsky, Justin R; Davis, Dawn B; Corbett, John A; Mathews, Clayton E

    2015-09-01

    While insulinoma cells have been developed and proven to be extremely useful in studies focused on mechanisms controlling β-cell function and viability, translating findings to human β-cells has proven difficult because of the limited access to human islets and the absence of suitable insulinoma cell lines of human origin. Recently, a human β-cell line, EndoC-βH1, has been derived from human fetal pancreatic buds. The purpose of this study was to determine whether human EndoC-βH1 cells respond to cytokines in a fashion comparable to human islets. Unlike most rodent-derived insulinoma cell lines that respond to cytokines in a manner consistent with rodent islets, EndoC-βH1 cells fail to respond to a combination of cytokines (IL-1, IFN-γ, and TNF) in a manner consistent with human islets. Nitric oxide, produced following inducible nitric oxide synthase (iNOS) expression, is a major mediator of cytokine-induced human islet cell damage. We show that EndoC-βH1 cells fail to express iNOS or produce nitric oxide in response to this combination of cytokines. Inhibitors of iNOS prevent cytokine-induced loss of human islet cell viability; however, they do not prevent cytokine-induced EndoC-βH1 cell death. Stressed human islets or human islets expressing heat shock protein 70 (HSP70) are resistant to cytokines, and, much like stressed human islets, EndoC-βH1 cells express HSP70 under basal conditions. Elevated basal expression of HSP70 in EndoC-βH1 cells is consistent with the lack of iNOS expression in response to cytokine treatment. While expressing HSP70, EndoC-βH1 cells fail to respond to endoplasmic reticulum stress activators, such as thapsigargin. These findings indicate that EndoC-βH1 cells do not faithfully recapitulate the response of human islets to cytokines. Therefore, caution should be exercised when making conclusions regarding the actions of cytokines on human islets when using this human-derived insulinoma cell line.

  6. Effect of Maillard reaction on biochemical properties of peanut 7S globulin (Ara h 1) and its interaction with a human colon cancer cell line (Caco-2)

    NARCIS (Netherlands)

    Teodorowicz, M.; Fiedorowicz, E.; Kostyra, H.; Wichers, H.J.; Kostyra, E.

    2013-01-01

    Purpose The purpose of this study was to determine the influence of Maillard reaction (MR, glycation) on biochemical and biological properties of the major peanut allergen Ara h 1. Methods Three different time/temperature conditions of treatment were applied (37, 60, and 145 °C). The extent of MR wa

  7. VP2 capsid domain of the H-1 parvovirus determines susceptibility of human cancer cells to H-1 viral infection.

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    Cho, I-R; Kaowinn, S; Song, J; Kim, S; Koh, S S; Kang, H-Y; Ha, N-C; Lee, K H; Jun, H-S; Chung, Y-H

    2015-05-01

    Although H-1 parvovirus is used as an antitumor agent, not much is known about the relationship between its specific tropism and oncolytic activity. We hypothesize that VP2, a major capsid protein of H-1 virus, determines H-1-specific tropism. To assess this, we constructed chimeric H-1 viruses expressing Kilham rat virus (KRV) capsid proteins, in their complete or partial forms. Chimeric H-1 viruses (CH1, CH2 and CH3) containing the whole KRV VP2 domain could not induce cytolysis in HeLa, A549 and Panc-1 cells. However, the other chimeric H-1 viruses (CH4 and CH5) expressing a partial KRV VP2 domain induced cytolysis. Additionally, the significant cytopathic effect caused by CH4 and CH5 infection in HeLa cells resulted from preferential viral amplification via DNA replication, RNA transcription and protein synthesis. Modeling of VP2 capsid protein showed that two variable regions (VRs) (VR0 and VR2) of H-1 VP2 protein protrude outward, because of the insertion of extra amino-acid residues, as compared with those of KRV VP2 protein. This might explain the precedence of H-1 VP2 protein over KRV in determining oncolytic activity in human cancer cells. Taking these results together, we propose that the VP2 protein of oncolytic H-1 parvovirus determines its specific tropism in human cancer cells.

  8. Killing of p53-deficient hepatoma cells by parvovirus H-1 and chemotherapeutics requires promyelocytic leukemia protein

    Institute of Scientific and Technical Information of China (English)

    Maike Sieben; Markus Moehler; Kerstin Herzer; Maja Zeidler; Vera Heinrichs; Barbara Leuchs; Martin Schuler; Jan J Cornelis; Peter R Galle; Jean Rommelaere

    2008-01-01

    AIM: To evaluate the synergistic targeting and killing of human hepatocellular carcinoma (HCC) cells lacking p53 by the oncolytic autonomous parvovirus (PV) H-1 and chemotherapeutic agents and its dependence on functional promyelocytic leukemia protein (PML).METHODS: The role of p53 and PML in regulating cytotoxicity and gene transfer mediated by wild-type (wt)PV H-1 were explored in two pairs of isogenic human hepatoma cell lines with different p53 status.Furthermore,H-1 PV infection was combined with cytostatic drug treatment.RESULTS: While the HCC cells with different p53 status studied were all susceptible to H-1 PV-induced apoptosis,the cytotoxicity of H-1 PV was more pronounced in p53-negative than in p53-positive cells.Apoptosis rates in p53-negative cell lines treated by genotoxic drugs were further enhanced by a treatment with H-1 PV.In flow cytometric analyses,H-1 PV infection resulted in a reduction of the mitochondrial transmembrane potential.In addition,H-1 PV cells showed a significant increase in PML expression.Knocking down PML expression resulted in a striking reduction of the level of H-1 PV infected tumor cell death.CONCLUSION: H-1 PV is a suitable agent to circumvent the resistance of p53-negative HCC cells to genotoxic agents,and it enhances the apoptotic process which is dependent on functional PML.Thus,H-1 PV and its oncolytic vector derivatives may be considered as therapeutic options for HCC,particularly for p53-negative tumors.

  9. Design, spectral characterization, thermal, DFT studies and anticancer cell line activities of Co(II), Ni(II) and Cu(II) complexes of Schiff bases derived from 4-amino-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-thiol.

    Science.gov (United States)

    Tyagi, Prateek; Chandra, Sulekh; Saraswat, B S; Yadav, Deepak

    2015-06-15

    A series of two biologically active Schiff base ligands L(1), L(2) have been synthesized in equimolar reaction of 4-amino-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-thiol with thiophene-2-carbaldehyde and furan-2-carbaldehyde. The synthesized Schiff bases were used for complexation with different metal ions like Co(II), Ni(II) and Cu(II) by using a molar ratio of ligand: metal as 1:1 and 2:1. The characterization of Schiff bases and metal complexes was done by (1)H NMR, UV-Vis, TGA, IR, mass spectrometry and molar conductivity studies. The in DFT studies the geometries of Schiff bases and metal complexes were fully optimized with respect to the energy using the 6-31+g(d,p) basis set. On the basis of the spectral studies an octahedral geometry has been assigned for Co(II), Ni(II) and Cu(II) complexes. The effect of these complexes on proliferation of human breast cancer cell line (MCF-7) and human hepatocellular liver carcinoma cell line (Hep-G2) were studied and compared with those of free ligand. The anticancer cell line results reveal that all metal complexes show moderate to significant % cytotoxicity on cell line HepG2 and MCF-7.

  10. Design, spectral characterization, thermal, DFT studies and anticancer cell line activities of Co(II), Ni(II) and Cu(II) complexes of Schiff bases derived from 4-amino-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-thiol

    Science.gov (United States)

    Tyagi, Prateek; Chandra, Sulekh; Saraswat, B. S.; Yadav, Deepak

    2015-06-01

    A series of two biologically active Schiff base ligands L1, L2 have been synthesized in equimolar reaction of 4-amino-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-thiol with thiophene-2-carbaldehyde and furan-2-carbaldehyde. The synthesized Schiff bases were used for complexation with different metal ions like Co(II), Ni(II) and Cu(II) by using a molar ratio of ligand: metal as 1:1 and 2:1. The characterization of Schiff bases and metal complexes was done by 1H NMR, UV-Vis, TGA, IR, mass spectrometry and molar conductivity studies. The in DFT studies the geometries of Schiff bases and metal complexes were fully optimized with respect to the energy using the 6-31+g(d,p) basis set. On the basis of the spectral studies an octahedral geometry has been assigned for Co(II), Ni(II) and Cu(II) complexes. The effect of these complexes on proliferation of human breast cancer cell line (MCF-7) and human hepatocellular liver carcinoma cell line (Hep-G2) were studied and compared with those of free ligand. The anticancer cell line results reveal that all metal complexes show moderate to significant % cytotoxicity on cell line HepG2 and MCF-7.

  11. Caveolin-1 influences human influenza A virus (H1N1 multiplication in cell culture

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    Hemgård Gun-Viol

    2010-05-01

    Full Text Available Abstract Background The threat of recurring influenza pandemics caused by new viral strains and the occurrence of escape mutants necessitate the search for potent therapeutic targets. The dependence of viruses on cellular factors provides a weak-spot in the viral multiplication strategy and a means to interfere with viral multiplication. Results Using a motif-based search strategy for antiviral targets we identified caveolin-1 (Cav-1 as a putative cellular interaction partner of human influenza A viruses, including the pandemic influenza A virus (H1N1 strains of swine origin circulating from spring 2009 on. The influence of Cav-1 on human influenza A/PR/8/34 (H1N1 virus replication was determined in inhibition and competition experiments. RNAi-mediated Cav-1 knock-down as well as transfection of a dominant-negative Cav-1 mutant results in a decrease in virus titre in infected Madin-Darby canine kidney cells (MDCK, a cell line commonly used in basic influenza research as well as in virus vaccine production. To understand the molecular basis of the phenomenon we focussed on the putative caveolin-1 binding domain (CBD located in the lumenal, juxtamembranal portion of the M2 matrix protein which has been identified in the motif-based search. Pull-down assays and co-immunoprecipitation experiments showed that caveolin-1 binds to M2. The data suggest, that Cav-1 modulates influenza virus A replication presumably based on M2/Cav-1 interaction. Conclusion As Cav-1 is involved in the human influenza A virus life cycle, the multifunctional protein and its interaction with M2 protein of human influenza A viruses represent a promising starting point for the search for antiviral agents.

  12. H1foo Has a Pivotal Role in Qualifying Induced Pluripotent Stem Cells

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    Akira Kunitomi

    2016-06-01

    Full Text Available Embryonic stem cells (ESCs are a hallmark of ideal pluripotent stem cells. Epigenetic reprogramming of induced pluripotent stem cells (iPSCs has not been fully accomplished. iPSC generation is similar to somatic cell nuclear transfer (SCNT in oocytes, and this procedure can be used to generate ESCs (SCNT-ESCs, which suggests the contribution of oocyte-specific constituents. Here, we show that the mammalian oocyte-specific linker histone H1foo has beneficial effects on iPSC generation. Induction of H1foo with Oct4, Sox2, and Klf4 significantly enhanced the efficiency of iPSC generation. H1foo promoted in vitro differentiation characteristics with low heterogeneity in iPSCs. H1foo enhanced the generation of germline-competent chimeric mice from iPSCs in a manner similar to that for ESCs. These findings indicate that H1foo contributes to the generation of higher-quality iPSCs.

  13. File list: Oth.ALL.05.Suv39h1.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.05.Suv39h1.AllCell mm9 TFs and others Suv39h1 All cell types SRX604254,SRX6...04255 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.05.Suv39h1.AllCell.bed ...

  14. File list: Oth.ALL.10.Suv39h1.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.10.Suv39h1.AllCell mm9 TFs and others Suv39h1 All cell types SRX604254,SRX6...04255 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.10.Suv39h1.AllCell.bed ...

  15. File list: Oth.PSC.50.Suv39h1.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.50.Suv39h1.AllCell mm9 TFs and others Suv39h1 Pluripotent stem cell SRX6042...54,SRX604255 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.50.Suv39h1.AllCell.bed ...

  16. File list: Oth.ALL.20.Suv39h1.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.20.Suv39h1.AllCell mm9 TFs and others Suv39h1 All cell types SRX604254,SRX6...04255 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.20.Suv39h1.AllCell.bed ...

  17. File list: His.ALL.50.H1PERIOD2.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.ALL.50.H1PERIOD2.AllCell hg19 Histone H1.2 All cell types SRX395615,SRX395614,S...RX329005,SRX329002,SRX329003 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.ALL.50.H1PERIOD2.AllCell.bed ...

  18. File list: His.ALL.05.H1PERIOD2.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.ALL.05.H1PERIOD2.AllCell hg19 Histone H1.2 All cell types SRX395615,SRX395614,S...RX329003,SRX329002,SRX329005 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.ALL.05.H1PERIOD2.AllCell.bed ...

  19. File list: His.ALL.20.H1PERIOD2.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.ALL.20.H1PERIOD2.AllCell hg19 Histone H1.2 All cell types SRX395614,SRX395615,S...RX329005,SRX329002,SRX329003 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.ALL.20.H1PERIOD2.AllCell.bed ...

  20. Extracellular vesicles shed by melanoma cells contain a modified form of H1.0 linker histone and H1.0 mRNA-binding proteins

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    Schiera, Gabriella; Di Liegro, Carlo Maria; Puleo, Veronica; Colletta, Oriana; Fricano, Anna; Cancemi, Patrizia; Di Cara, Gianluca; Di Liegro, Italia

    2016-01-01

    Extracellular vesicles (EVs) are now recognized as a fundamental way for cell-to-cell horizontal transfer of properties, in both physiological and pathological conditions. Most of EV-mediated cross-talk among cells depend on the exchange of proteins, and nucleic acids, among which mRNAs, and non-coding RNAs such as different species of miRNAs. Cancer cells, in particular, use EVs to discard molecules which could be dangerous to them (for example differentiation-inducing proteins such as histone H1.0, or antitumor drugs), to transfer molecules which, after entering the surrounding cells, are able to transform their phenotype, and even to secrete factors, which allow escaping from immune surveillance. Herein we report that melanoma cells not only secrete EVs which contain a modified form of H1.0 histone, but also transport the corresponding mRNA. Given the already known role in tumorigenesis of some RNA binding proteins (RBPs), we also searched for proteins of this class in EVs. This study revealed the presence in A375 melanoma cells of at least three RBPs, with apparent MW of about 65, 45 and 38 kDa, which are able to bind H1.0 mRNA. Moreover, we purified one of these proteins, which by MALDI-TOF mass spectrometry was identified as the already known transcription factor MYEF2. PMID:27633859

  1. How Does Influenza A (H1N1 Infection Proceed in Allogeneic Stem Cell Transplantation Recipients?

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    Sinem Civriz Bozdağ

    2012-03-01

    Full Text Available Clinical course of H1N1 infection in Allogeneic Hematopoietic Stem Cell Transplantation (AHSCT patients is contraversial. We report three AHSCT patients who were infected with Influenza A/H1N1 infection. All of the patients were diagnosed with different hematological diagnosis and were at different stages of transplantation.All of them were treated with oseltamivir,zanamivir was switched with oseltamivir in one patient. All of the three patients were survived without any complication. Swine flu, can display with different courses and progress with bacterial or other viral infections in immunsupressed patients.

  2. Characterization of the 2009 pandemic A/Beijing/501/2009 H1N1 influenza strain in human airway epithelial cells and ferrets.

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    Penghui Yang

    Full Text Available BACKGROUND: A novel 2009 swine-origin influenza A H1N1 virus (S-OIV H1N1 has been transmitted among humans worldwide. However, the pathogenesis of this virus in human airway epithelial cells and mammals is not well understood. METHODOLOGY/PRINCIPAL FINDING: In this study, we showed that a 2009 A (H1N1 influenza virus strain, A/Beijing/501/2009, isolated from a human patient, caused typical influenza-like symptoms including weight loss, fluctuations in body temperature, and pulmonary pathological changes in ferrets. We demonstrated that the human lung adenocarcinoma epithelial cell line A549 was susceptible to infection and that the infected cells underwent apoptosis at 24 h post-infection. In contrast to the seasonal H1N1 influenza virus, the 2009 A (H1N1 influenza virus strain A/Beijing/501/2009 induced more cell death involving caspase-3-dependent apoptosis in A549 cells. Additionally, ferrets infected with the A/Beijing/501/2009 H1N1 virus strain exhibited increased body temperature, greater weight loss, and higher viral titers in the lungs. Therefore, the A/Beijing/501/2009 H1N1 isolate successfully infected the lungs of ferrets and caused more pathological lesions than the seasonal influenza virus. Our findings demonstrate that the difference in virulence of the 2009 pandemic H1N1 influenza virus and the seasonal H1N1 influenza virus in vitro and in vivo may have been mediated by different mechanisms. CONCLUSION/SIGNIFICANCE: Our understanding of the pathogenesis of the 2009 A (H1N1 influenza virus infection in both humans and animals is broadened by our findings that apoptotic cell death is involved in the cytopathic effect observed in vitro and that the pathological alterations in the lungs of S-OIV H1N1-infected ferrets are much more severe.

  3. Autophagy suppresses cell migration by degrading GEF-H1, a RhoA GEF

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    Tanaka, Masato; Shimizu, Shigeomi

    2016-01-01

    Cell migration is a process crucial for a variety of biological events, such as morphogenesis and wound healing. Several reports have described the possible regulation of cell migration by autophagy; however, this remains controversial. We here demonstrate that mouse embryonic fibroblasts (MEFs) lacking autophagy protein 5 (Atg5), an essential molecule of autophagy, moved faster than wild-type (WT) MEFs. Similar results were obtained for MEFs lacking Atg7 and unc-51-like kinase 1 (Ulk1), which are molecules required for autophagy. This phenotype was also observed in Atg7-deficient macrophages. WT MEFs moved by mesenchymal-type migration, whereas Atg5 knockout (KO) MEFs moved by amoeba-like migration. This difference was thought to be mediated by the level of RhoA activity, because Atg5 KO MEFs had higher RhoA activity, and treatment with a RhoA inhibitor altered Atg5 KO MEF migration from the amoeba type to the mesenchymal type. Autophagic regulation of RhoA activity was dependent on GEF-H1, a member of the RhoA family of guanine nucleotide exchange factors. In WT MEFs, GEF-H1 directly bound to p62 and was degraded by autophagy, resulting in low RhoA activity. In contrast, the loss of autophagy increased GEF-H1 levels and thereby activated RhoA, which caused cells to move by amoeba-like migration. This amoeba-like migration was cancelled by the silencing of GEF-H1. These results indicate that autophagy plays a role in the regulation of migration by degrading GEF-H1. PMID:27120804

  4. Autophagy suppresses cell migration by degrading GEF-H1, a RhoA GEF.

    Science.gov (United States)

    Yoshida, Tatsushi; Tsujioka, Masatsune; Honda, Shinya; Tanaka, Masato; Shimizu, Shigeomi

    2016-06-07

    Cell migration is a process crucial for a variety of biological events, such as morphogenesis and wound healing. Several reports have described the possible regulation of cell migration by autophagy; however, this remains controversial. We here demonstrate that mouse embryonic fibroblasts (MEFs) lacking autophagy protein 5 (Atg5), an essential molecule of autophagy, moved faster than wild-type (WT) MEFs. Similar results were obtained for MEFs lacking Atg7 and unc-51-like kinase 1 (Ulk1), which are molecules required for autophagy. This phenotype was also observed in Atg7-deficient macrophages. WT MEFs moved by mesenchymal-type migration, whereas Atg5 knockout (KO) MEFs moved by amoeba-like migration. This difference was thought to be mediated by the level of RhoA activity, because Atg5 KO MEFs had higher RhoA activity, and treatment with a RhoA inhibitor altered Atg5 KO MEF migration from the amoeba type to the mesenchymal type. Autophagic regulation of RhoA activity was dependent on GEF-H1, a member of the RhoA family of guanine nucleotide exchange factors. In WT MEFs, GEF-H1 directly bound to p62 and was degraded by autophagy, resulting in low RhoA activity. In contrast, the loss of autophagy increased GEF-H1 levels and thereby activated RhoA, which caused cells to move by amoeba-like migration. This amoeba-like migration was cancelled by the silencing of GEF-H1. These results indicate that autophagy plays a role in the regulation of migration by degrading GEF-H1.

  5. Storage of cell lines.

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    Parker, Katharine A

    2011-01-01

    The successful storage of cell lines depends upon many factors, including the condition of the cells to be frozen and the experience of the operator. Attempting to freeze down unhealthy, contaminated or poorly labelled cells can have huge implications for a research laboratory. This chapter outlines the importance of good record keeping, vigilant monitoring, aseptic technique, and high-quality reagents in the successful storage and downstream propagation of cell lines.

  6. Vaccination with Leishmania histone H1-pulsed dendritic cells confers protection in murine visceral leishmaniasis.

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    Agallou, Maria; Smirlis, Despina; Soteriadou, Ketty P; Karagouni, Evdokia

    2012-07-20

    Visceral leishmaniasis is the most severe form of leishmaniases affecting millions of people worldwide often resulting in death despite optimal therapy. Thus, there is an urgent need for the development of effective anti-infective vaccine(s). In the present study, we evaluated the prophylactic value of bone marrow-derived dendritic cells (BM-DCs) pulsed with the Leishmania (L.) infantum histone H1. We developed fully mature BM-DCs characterized by enhanced capacity of IL-12 production after ex vivo pulsing with GST-LeishH1. Intravenous administration of these BM-DCs in naive BALB/c mice resulted in antigen-specific spleenocyte proliferation and IgG1 isotype antibody production and conferred protection against experimental challenge with L. infantum independently of CpG oligonucleotides (ODNs) co-administration. Protection was associated with a pronounced enhancement of parasite-specific IFNγ-producing cells and reduction of cells producing IL-10, whereas IL-4 production was comparable in protected and non-protected mice. The polarization of immune responses to Th1 type was further confirmed by the elevation of parasite-specific IgG2a/IgG1 ratio in protected mice. The above data indicate the immunostimulatory capacity of Leishmania histone H1 and further support its exploitation as a candidate protein for vaccine development against leishmaniasis.

  7. Nm23H1 mediates tumor invasion in esophageal squamous cell carcinoma by regulation of CLDN1 through the AKT signaling

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    Kuo, K-T; Chen, C-L; Chou, T-Y; Yeh, C-T; Lee, W-H; Wang, L-S

    2016-01-01

    Esophageal cancer is a lethal malignancy worldwide. Previously, low expression of metastasis suppressor Nm23H1 and tight junction (TJ) protein claudin-1 (CLDN1) have been known to correlate with poor prognosis in esophageal squamous cell carcinoma (ESCC). However, the molecular interaction between them has not been clarified. In the present study, we first examined the expression of Nm23H1 and CLDN1 in 74 surgical ESCC samples by immunohistochemistry (IHC) to verify their clinicopathologic significance. The biologic effects of Nm23H1 gene silencing or overexpression in ESCC cell lines were then studied by migration and invasion studies, and its regulation on CLDN1 expression was also investigated by western blot analysis. Moreover, the expression of Nm23H1 and CLDN1 at the same invasion front of ESCC tumors was verified by immunofluorescence. The results showed a significantly positive correlation between the expression of Nm23H1 and CLDN1 (γ=0.296, P=0.011) in surgical specimens, especially for the 34 tumors with lymph-node metastasis (γ=0.455, P=0.007). In ESCC cell lines, silencing of Nm23H1 expression markedly enhanced cell invasiveness, accompanied by increased Akt phosphorylation and decreased CLDN1 expression. Conversely, Nm23H1-expressed transfectants exhibited reduced invasiveness, decreased Akt phosphorylation and correspondingly increased CLDN1 expression. Regain of CLDN1 expression in ESCC cells significantly suppressed invasiveness, but did not influence the Akt phosphorylation. Moreover, treating Nm23H1-depleted cells with the AKT inhibitor MK2206 recovered CLDN1 expression, and diminished the invasiveness of ESCC cells. Finally, decreased expressions of both CLDN1 and E-cadherin were observed at the invasive front of the Nm23H1-negative tumors. Overall, our current study documented that reduced Nm23H1 expression activates the AKT signaling pathway, results in diminished CLDN1 expression and potentiates invasiveness of ESCC cells. Enhancement of Nm

  8. Leukemia SH-1 Cells Purged by ZnPcH1-Based Photodynamic Therapy%ZnPcH1介导的光动力疗法净化白血病SHI-1细胞

    Institute of Scientific and Technical Information of China (English)

    林晓岚; 黄慧芳; 陈万紫

    2012-01-01

    本研究探讨新型酞菁类光敏剂二磺基二邻苯二甲酰亚胺甲基酞菁锌(ZnPcH1)介导的光动力疗法(PDT)对急性单核细胞白血病SHI-1细胞的杀伤作用及其机制,为PDT应用于白血病自体骨髓体外净化提供理论依据.以SHI-1细胞作为研究对象,应用MTT比色法检测细胞增殖.采用AO/EB复合染色、TUNEL、DNA二倍体分析、Annexin-V -FITC/PI双染法等分析细胞的死亡方式.SHI-1细胞与正常骨髓单个核细胞(MNC)以1∶100-1∶10000混合建立混合细胞模型,通过巢式RT-PCR检测SHI-1细胞融合基因MLL/AF6 mRNA的表达以研究ZnPcH1-PDT对骨髓MNC中所掺入的SHI-1细胞的净化效果.结果表明,ZnPcH1-PDT对SHI-1细胞有杀伤作用,且呈量效关系.ZnPcH1-PDT能诱导细胞凋亡,且呈时间依赖性.0.5 μmol/L ZnPcH1-PDT可完全杀灭模拟缓解骨髓中掺入的SHI-1细胞.结论:ZnPcH1-PDT有希望成为新的高效而简便的骨髓净化手段.%The objective of this study was to investigate the effect of a novel Zinc phthalocyanine (ZnPcH1 ) based photodynamic therapy (PDT) on acute monocytic leukemia cell lines SHI-1 and its mechanism, so as to provide theory basis for bone marrow purging in vino for patients with leukemia. The killing effect of ZnPcH, -PDT on SHI-1 cells were assessed by MTT method; the SHI-1 cell death patterns were analyzed by AO/EB fluorescence staining, TdT-mediated dUTP nick end labeling (TUNEL), DNA ploidy analysis,and Annexin V-FITC/P1 double staining. Cell mixture was established by integrating SHI-1 cells with normal bone marrow MNC ( by 1:100 - 1:10 000). Purging effect of ZnPcH1-PDT against SHI-1 mixed into normal MNC was assessed by analyzing the expression of fusion gene MLL/AF6 mRNA using nested RT-PCR. The results showed that ZnPcH, -PDT could effectively inhibit SHI-1 cell proliferation in dose-dependent manner, and ZnPcH, -PDT could induce cell apoptosis in time-dependent manner. 0.5 μmol/L ZnPcH, -PDT could completely

  9. Deficient expression of enhanced reactivation of parvovirus H-1 in ataxia telangiectasia cells irradiated with X-rays or u. v. light

    Energy Technology Data Exchange (ETDEWEB)

    Hilgers, G.; Chen, Y.Q.; Cornelis, J.J.; Rommelaere, J.

    1987-02-01

    Cells of patients with ataxia telangiectasia (AT), an inherited disease characterized by a high propensity to cancer, are hypersensitive to ionizing radiation. We investigated whether the hyper-radiosensitivity of AT cells correlated with a defect in their constitutive and/or conditional ability to rescue a damaged exogenous virus. For that purpose, parvovirus H-1, a single-stranded DNA virus whose intranuclear replication mostly relies on host cell functions, was used as a probe. The survival of u.v.- or gamma-irradiated H-1 was measured in X-, u.v.- or mock-irradiated human cells of normal (NB-E) or AT (AT5BIVA) origin. gamma-Irradiated H-1 survived to similar extents in untreated normal and AT cell lines. Both X- and u.v.-irradiation induced normal cells to achieve an enhanced reactivation (ER) of gamma- or u.v.-damaged H-1. In contrast, neither dose-effect curves nor time course revealed significant levels of ER expression after X- or u.v.-irradiation in AT5BIVA cells. Our results suggest that the impairment of ER of damaged parvoviruses may constitute a marker of the AT cell phenotype and be related to the radiosensitivity of AT cells.

  10. Expression and significance of B7-H1 in peripheral blood dendritic cells from patients with bladder cancer

    Institute of Scientific and Technical Information of China (English)

    Chuanbiao Ji; Yonghua Wang; Qinchao Yu; Jing Liu; Yanan Liu; Jie Cui

    2013-01-01

    Objective: The aim of this study was to study the expression and the clinical significance of B7-H1 on dendritic cells (DCs) in peripheral blood from patients with bladder cancer. Methods: Peripheral blood mononuclear cell were disparted from 30 bladder cancer patients and 7 healthy controls by density gradient centrifugation and then co-cultured. The expression of B7-H1 on DCs were analyzed by flow cytometry. Results: Expression of B7-H1 on DCs in bladder cancer was higher than healthy controls (P < 0.01). And the expression were strongly associated with the pathological grade and clinical stage of bladder cancer (P < 0.05). Conclusion: The up-regulation of B7-H1 on DCs was strongly associated with neoplastic progression of bladder cancer. B7-H1/programmed death (PD)-1 signal pathway may also play an important role in immune escape of bladder cancer during initial phase of T cell immune response.

  11. Pediatric and Adult High-Grade Glioma Stem Cell Culture Models Are Permissive to Lytic Infection with Parvovirus H-1.

    Science.gov (United States)

    Josupeit, Rafael; Bender, Sebastian; Kern, Sonja; Leuchs, Barbara; Hielscher, Thomas; Herold-Mende, Christel; Schlehofer, Jörg R; Dinsart, Christiane; Witt, Olaf; Rommelaere, Jean; Lacroix, Jeannine

    2016-05-19

    Combining virus-induced cytotoxic and immunotherapeutic effects, oncolytic virotherapy represents a promising therapeutic approach for high-grade glioma (HGG). A clinical trial has recently provided evidence for the clinical safety of the oncolytic parvovirus H-1 (H-1PV) in adult glioblastoma relapse patients. The present study assesses the efficacy of H-1PV in eliminating HGG initiating cells. H-1PV was able to enter and to transduce all HGG neurosphere culture models (n = 6), including cultures derived from adult glioblastoma, pediatric glioblastoma, and diffuse intrinsic pontine glioma. Cytotoxic effects induced by the virus have been observed in all HGG neurospheres at half maximal inhibitory concentration (IC50) doses of input virus between 1 and 10 plaque forming units per cell. H-1PV infection at this dose range was able to prevent tumorigenicity of NCH421k glioblastoma multiforme (GBM) "stem-like" cells in NOD/SCID mice. Interestingly NCH421R, an isogenic subclone with equal capacity of xenograft formation, but resistant to H-1PV infection could be isolated from the parental NCH421k culture. To reveal changes in gene expression associated with H-1PV resistance we performed a comparative gene expression analysis in these subclones. Several dysregulated genes encoding receptor proteins, endocytosis factors or regulators innate antiviral responses were identified and represent intriguing candidates for to further study molecular mechanisms of H-1PV resistance.

  12. Pediatric and Adult High-Grade Glioma Stem Cell Culture Models Are Permissive to Lytic Infection with Parvovirus H-1

    Directory of Open Access Journals (Sweden)

    Rafael Josupeit

    2016-05-01

    Full Text Available Combining virus-induced cytotoxic and immunotherapeutic effects, oncolytic virotherapy represents a promising therapeutic approach for high-grade glioma (HGG. A clinical trial has recently provided evidence for the clinical safety of the oncolytic parvovirus H-1 (H-1PV in adult glioblastoma relapse patients. The present study assesses the efficacy of H-1PV in eliminating HGG initiating cells. H-1PV was able to enter and to transduce all HGG neurosphere culture models (n = 6, including cultures derived from adult glioblastoma, pediatric glioblastoma, and diffuse intrinsic pontine glioma. Cytotoxic effects induced by the virus have been observed in all HGG neurospheres at half maximal inhibitory concentration (IC50 doses of input virus between 1 and 10 plaque forming units per cell. H-1PV infection at this dose range was able to prevent tumorigenicity of NCH421k glioblastoma multiforme (GBM “stem-like” cells in NOD/SCID mice. Interestingly NCH421R, an isogenic subclone with equal capacity of xenograft formation, but resistant to H-1PV infection could be isolated from the parental NCH421k culture. To reveal changes in gene expression associated with H-1PV resistance we performed a comparative gene expression analysis in these subclones. Several dysregulated genes encoding receptor proteins, endocytosis factors or regulators innate antiviral responses were identified and represent intriguing candidates for to further study molecular mechanisms of H-1PV resistance.

  13. Retargeting of rat parvovirus H-1PV to cancer cells through genetic engineering of the viral capsid.

    Science.gov (United States)

    Allaume, Xavier; El-Andaloussi, Nazim; Leuchs, Barbara; Bonifati, Serena; Kulkarni, Amit; Marttila, Tiina; Kaufmann, Johanna K; Nettelbeck, Dirk M; Kleinschmidt, Jürgen; Rommelaere, Jean; Marchini, Antonio

    2012-04-01

    The rat parvovirus H-1PV is a promising anticancer agent given its oncosuppressive properties and the absence of known side effects in humans. H-1PV replicates preferentially in transformed cells, but the virus can enter both normal and cancer cells. Uptake by normal cells sequesters a significant portion of the administered viral dose away from the tumor target. Hence, targeting H-1PV entry specifically to tumor cells is important to increase the efficacy of parvovirus-based treatments. In this study, we first found that sialic acid plays a key role in H-1PV entry. We then genetically engineered the H-1PV capsid to improve its affinity for human tumor cells. By analogy with the resolved crystal structure of the closely related parvovirus minute virus of mice, we developed an in silico three-dimensional (3D) model of the H-1PV wild-type capsid. Based on this model, we identified putative amino acids involved in cell membrane recognition and virus entry at the level of the 2-fold axis of symmetry of the capsid, within the so-called dimple region. In situ mutagenesis of these residues significantly reduced the binding and entry of H-1PV into permissive cells. We then engineered an entry-deficient viral capsid and inserted a cyclic RGD-4C peptide at the level of its 3-fold axis spike. This peptide binds α(v)β(3) and α(v)β(5) integrins, which are overexpressed in cancer cells and growing blood vessels. The insertion of the peptide rescued viral infectivity toward cells overexpressing α(v)β(5) integrins, resulting in the efficient killing of these cells by the reengineered virus. This work demonstrates that H-1PV can be genetically retargeted through the modification of its capsid, showing great promise for a more efficient use of this virus in cancer therapy.

  14. Distinct signalling pathways of murine histamine H1- and H4-receptors expressed at comparable levels in HEK293 cells.

    Directory of Open Access Journals (Sweden)

    Silke Beermann

    Full Text Available Histamine (HA is recognized by its target cells via four G-protein-coupled receptors, referred to as histamine H1-receptor (H1R, H2R, H3R, and H4R. Both H1R and H4R exert pro-inflammatory functions. However, their signal transduction pathways have never been analyzed in a directly comparable manner side by side. Moreover, the analysis of pharmacological properties of the murine orthologs, representing the main targets of pre-clinical research, is very important. Therefore, we engineered recombinant HEK293 cells expressing either mouse (mH1R or mH4R at similar levels and analyzed HA-induced signalling in these cells. HA induced intracellular calcium mobilization via both mH1R and mH4R, with the mH1R being much more effective. Whereas cAMP accumulation was potentiated via the mH1R, it was reduced via the mH4R. The regulation of both second messengers via the H4R, but not the H1R, was sensitive to pertussis toxin (PTX. The mitogen-activated protein kinases (MAPKs ERK 1/2 were massively activated downstream of both receptors and demonstrated a functional involvement in HA-induced EGR-1 gene expression. The p38 MAPK was moderately activated via both receptors as well, but was functionally involved in HA-induced EGR-1 gene expression only in H4R-expressing cells. Surprisingly, in this system p38 MAPK activity reduced the HA-induced gene expression. In summary, using this system which allows a direct comparison of mH1R- and mH4R-induced signalling, qualitative and quantitative differences on the levels of second messenger generation and also in terms of p38 MAPK function became evident.

  15. B7-H1 Signaling is Integrated During CD8+ T Cell Priming and Restrains Effector Differentiation

    OpenAIRE

    Gibbons, Rachel M.; Liu, Xin; Harrington, Susan M.; Krco, Christopher J.; Kwon, Eugene D.; Dong, Haidong

    2014-01-01

    A promising strategy in tumor immunotherapy is the use of activated dendritic cells as vehicles for tumor vaccines with the goal of activating anti-tumor T cell responses. Current formulations for dendritic cell-based immunotherapies have limited effects on patient survival, providing motivation for further investigation of ways to enhance dendritic cell priming of anti-tumor T cell responses. Using a brief in vitro priming model, we have found that B7-H1 expressed by activated dendritic cell...

  16. Unexpected T cell regulatory activity of anti-histone H1 autoantibody: Its mode of action in regulatory T cell-dependent and -independent manners

    Energy Technology Data Exchange (ETDEWEB)

    Takaoka, Yuki [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima (Japan); Kawamoto, Seiji, E-mail: skawa@hiroshima-u.ac.jp [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima (Japan); Katayama, Akiko [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima (Japan); Nakano, Toshiaki [Liver Transplantation Program, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan (China); Yamanaka, Yasushi; Takahashi, Miki [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima (Japan); Shimada, Yayoi; Chiang, Kuei-Chen [Kazusa Institute for Drug Discovery, Josai International University, Kisarazu (Japan); Ohmori, Naoya [Kazusa Institute for Drug Discovery, Josai International University, Kisarazu (Japan); Faculty of Nursing, Josai International University, Togane (Japan); Aki, Tsunehiro [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima (Japan); Goto, Takeshi; Sato, Shuji [Kazusa Institute for Drug Discovery, Josai International University, Kisarazu (Japan); Faculty of Nursing, Josai International University, Togane (Japan); Goto, Shigeru [Liver Transplantation Program, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan (China); Iwao Hospital, Yufuin (Japan); Chen, Chao-Long [Liver Transplantation Program, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan (China); Ono, Kazuhisa [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima (Japan)

    2013-02-08

    Highlights: ► Anti-histone H1 autoantibody (anti-H1) acts on T cells to inhibit their activation. ► Anti-H1 suppresses T cell activation in Treg cell-dependent and -independent manners. ► Suboptimal dose of anti-H1 enhances suppressor function of Treg cells. ► High dose of anti-H1 directly inhibits T cell receptor signaling. -- Abstract: Induction of anti-nuclear antibodies against DNA or histones is a hallmark of autoimmune disorders, but their actual contribution to disease predisposition remains to be clarified. We have previously reported that autoantibodies against histone H1 work as a critical graft survival factor in a rat model of tolerogeneic liver transplantation. Here we show that an immunosuppressive anti-histone H1 monoclonal antibody (anti-H1 mAb) acts directly on T cells to inhibit their activation in response to T cell receptor (TCR) ligation. Intriguingly, the T cell activation inhibitory activity of anti-H1 mAb under suboptimal dosages required regulatory T (Treg) cells, while high dose stimulation with anti-H1 mAb triggered a Treg cell-independent, direct negative regulation of T cell activation upon TCR cross-linking. In the Treg cell-dependent mode of immunosuppressive action, anti-H1 mAb did not induce the expansion of CD4{sup +}Foxp3{sup +} Treg cells, but rather potentiated their regulatory capacity. These results reveal a previously unappreciated T cell regulatory role of anti-H1 autoantibody, whose overproduction is generally thought to be pathogenic in the autoimmune settings.

  17. Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Paquette, Stéphane G. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Banner, David [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Chi, Le Thi Bao [Department of Microbiology, Hue University of Medicine and Pharmacy, Thua Thien Hue (Viet Nam); Carlo Urbani Centre, Hue University of Medicine and Pharmacy, Thua Thien Hue (Viet Nam); Leon, Alberto J. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); International Institute of Infection and Immunity, Shantou University Medical College, Shantou, Guangdong (China); Xu, Luoling; Ran, Longsi [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Huang, Stephen S.H. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Department of Immunology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Farooqui, Amber [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); International Institute of Infection and Immunity, Shantou University Medical College, Shantou, Guangdong (China); and others

    2014-01-05

    Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus–epithelial cell interaction. - Highlights: • We investigated H1N1pdm/sH1N1 infection in primary epithelial cells. • H1N1pdm directly initiated a robust inflammatory gene signature, sH1N1 did not. • H1N1pdm viral RNA triggered a stronger response than sH1N1. • H1N1pdm induces greater response due to direct virus–cell interaction. • These results have potential to impact vaccine and therapeutic development.

  18. CLO : The cell line ontology

    NARCIS (Netherlands)

    Sarntivijai, Sirarat; Lin, Yu; Xiang, Zuoshuang; Meehan, Terrence F.; Diehl, Alexander D.; Vempati, Uma D.; Schuerer, Stephan C.; Pang, Chao; Malone, James; Parkinson, Helen; Liu, Yue; Takatsuki, Terue; Saijo, Kaoru; Masuya, Hiroshi; Nakamura, Yukio; Brush, Matthew H.; Haendel, Melissa A.; Zheng, Jie; Stoeckert, Christian J.; Peters, Bjoern; Mungall, Christopher J.; Carey, Thomas E.; States, David J.; Athey, Brian D.; He, Yongqun

    2014-01-01

    Background: Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO

  19. Porcine mast cells infected with H1N1 influenza virus release histamine and inflammatory cytokines and chemokines.

    Science.gov (United States)

    Lee, In Hong; Kim, Hyun Soo; Seo, Sang Heui

    2017-04-01

    Mast cells reside in many tissues, including the lungs, and might play a role in enhancing influenza virus infections in animals. In this study, we cultured porcine mast cells from porcine bone marrow cells with IL-3 and stem cell factor to study the infectivity and activation of the 2009 pandemic H1N1 influenza virus of swine origin. Porcine mast cells were infected with H1N1 influenza virus, without the subsequent production of infectious viruses but were activated, as indicated by the release of histamines. Inflammatory cytokine- and chemokine-encoding genes, including IL-1α, IL-6, CXCL9, CXCL10, and CXCL11, were upregulated in the infected porcine mast cells. Our results suggest that mast cells could be involved in enhancing influenza-virus-mediated disease in infected animals.

  20. IFNγ and B7-H1 in the immunology of mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    @@ Mesenchymal stem cells (MSCs) are found in multiple organs in the fetus,cord blood and adult tissues [1]. However, in adults, the bone marrow is the major source of these stem cells. MSCs surround the blood vessels of bone marrow and are also in contact with the trabeculae [2].

  1. B7-H1 (PD-L1, CD274) suppresses host immunity in T-cell lymphoproliferative disorders

    OpenAIRE

    Wilcox, Ryan A.; Feldman, Andrew L.; Wada, David A.; Yang, Zhi-Zhang; Comfere, Nneka I.; Dong, Haidong; Kwon, Eugene D.; Novak, Anne J.; Markovic, Svetomir N.; Mark R Pittelkow; Witzig, Thomas E.; Ansell, Stephen M.

    2009-01-01

    Stromal elements present within the tumor microenvironment may suppress host immunity and promote the growth of malignant lymphocytes in B cell–derived non-Hodgkin lymphoma (NHL). In contrast, little is known about the microenvironment's role in T cell–derived NHL. B7-H1 (PD-L1, CD274), a member of the B7 family of costimulatory/coinhibitory ligands expressed by both malignant cells and stromal cells within the tumor microenvironment, has emerged as an important immune modulator capable of su...

  2. B7-H1 shapes T-cell–mediated brain endothelial cell dysfunction and regional encephalitogenicity in spontaneous CNS autoimmunity

    Science.gov (United States)

    Klotz, Luisa; Kuzmanov, Ivan; Hucke, Stephanie; Gross, Catharina C.; Posevitz, Vilmos; Dreykluft, Angela; Schulte-Mecklenbeck, Andreas; Janoschka, Claudia; Lindner, Maren; Herold, Martin; Schwab, Nicholas; Ludwig-Portugall, Isis; Kurts, Christian; Meuth, Sven G.; Kuhlmann, Tanja; Wiendl, Heinz

    2016-01-01

    Molecular mechanisms that determine lesion localization or phenotype variation in multiple sclerosis are mostly unidentified. Although transmigration of activated encephalitogenic T cells across the blood–brain barrier (BBB) is a crucial step in the disease pathogenesis of CNS autoimmunity, the consequences on brain endothelial barrier integrity upon interaction with such T cells and subsequent lesion formation and distribution are largely unknown. We made use of a transgenic spontaneous mouse model of CNS autoimmunity characterized by inflammatory demyelinating lesions confined to optic nerves and spinal cord (OSE mice). Genetic ablation of a single immune-regulatory molecule in this model [i.e., B7-homolog 1 (B7-H1, PD-L1)] not only significantly increased incidence of spontaneous CNS autoimmunity and aggravated disease course, especially in the later stages of disease, but also importantly resulted in encephalitogenic T-cell infiltration and lesion formation in normally unaffected brain regions, such as the cerebrum and cerebellum. Interestingly, B7-H1 ablation on myelin oligodendrocyte glycoprotein-specific CD4+ T cells, but not on antigen-presenting cells, amplified T-cell effector functions, such as IFN-γ and granzyme B production. Therefore, these T cells were rendered more capable of eliciting cell contact-dependent brain endothelial cell dysfunction and increased barrier permeability in an in vitro model of the BBB. Our findings suggest that a single immune-regulatory molecule on T cells can be ultimately responsible for localized BBB breakdown, and thus substantial changes in lesion topography in the context of CNS autoimmunity. PMID:27671636

  3. TLR-9 contributes to the antiviral innate immune sensing of rodent parvoviruses MVMp and H-1PV by normal human immune cells.

    Directory of Open Access Journals (Sweden)

    Zahari Raykov

    Full Text Available The oncotropism of Minute Virus of Mice (MVMp is partially related to the stimulation of an antiviral response mediated by type-I interferons (IFNs in normal but not in transformed mouse cells. The present work was undertaken to assess whether the oncotropism displayed against human cells by MVMp and its rat homolog H-1PV also depends on antiviral mechanisms and to identify the pattern recognition receptor (PRR involved. Despite their low proliferation rate which represents a drawback for parvovirus multiplication, we used human peripheral blood mononuclear cells (hPBMCs as normal model specifically because all known PRRs are functional in this mixed cell population and moreover because some of its subsets are among the main IFN producers upon infections in mammals. Human transformed models consisted in lines and tumor cells more or less permissive to both parvoviruses. Our results show that irrespective of their permissiveness, transformed cells do not produce IFNs nor develop an antiviral response upon parvovirus infection. However, MVMp- or H-1PV-infected hPBMCs trigger such defense mechanisms despite an absence of parvovirus replication and protein expression, pointing to the viral genome as the activating element. Substantial reduction of an inhibitory oligodeoxynucleotide (iODN of the latter IFN production identified TLR-9 as a potential PRR for parvoviruses in hPBMCs. However, neither the iODN treatment nor an antibody-induced neutralization of the IFN-triggered effects restored parvovirus multiplication in these cells as expected by their weak proliferation in culture. Finally, given that a TLR-9 activation could also not be observed in parvovirus-infected human lines reported to be endowed with a functional TLR-9 pathway (Namalwa, Raji, and HEK293-TLR9(+/+, our data suggest that transformed human cells do not sense MVMp or H-1PV either because of an absence of PRR expression or an intrinsic, or virus-driven defect in the endosomal

  4. Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells.

    Science.gov (United States)

    Ruhanen, Heini; Ushakov, Kathy; Yasukawa, Takehiro

    2011-12-01

    Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian mitochondrial DNA (mtDNA) replication, but the factors involved in lagging strand synthesis are largely uncharacterised. We investigated the effect of knockdown of the candidate proteins in cultured human cells under conditions where mtDNA appears to replicate chiefly via coupled leading and lagging strand DNA synthesis to restore the copy number of mtDNA to normal levels after transient mtDNA depletion. DNA ligase III knockdown attenuated the recovery of mtDNA copy number and appeared to cause single strand nicks in replicating mtDNA molecules, suggesting the involvement of DNA ligase III in Okazaki fragment ligation in human mitochondria. Knockdown of ribonuclease (RNase) H1 completely prevented the mtDNA copy number restoration, and replication intermediates with increased single strand nicks were readily observed. On the other hand, knockdown of neither flap endonuclease 1 (FEN1) nor DNA2 affected mtDNA replication. These findings imply that RNase H1 is indispensable for the progression of mtDNA synthesis through removing RNA primers from Okazaki fragments. In the nucleus, Okazaki fragments are ligated by DNA ligase I, and the RNase H2 is involved in Okazaki fragment processing. This study thus proposes that the mitochondrial replication system utilises distinct proteins, DNA ligase III and RNase H1, for Okazaki fragment maturation.

  5. Satisfaction and public health cost of a statewide influenza nurse triage line in response to pandemic H1N1 influenza.

    Directory of Open Access Journals (Sweden)

    Alicen B Spaulding

    Full Text Available BACKGROUND: The 2009 H1N1 pandemic strained healthcare systems. There was a need for supportive services, rapid antiviral access, and minimization of unnecessary healthcare contacts particularly face-to-face interactions. In response, the Minnesota Department of Health (MDH launched a telephone-based nurse triage line (NTL called the Minnesota FluLine coordinating all major MN healthcare systems with NTLs to form a single toll-free number triage service. Callers were evaluated for symptoms of influenza-like illness (ILI and were prescribed an antiviral if indicated, using nurse administered protocols. METHODS: To determine caller outcomes, associated healthcare seeking, and satisfaction a telephone survey of Minnesota FluLine callers was conducted using a 5% random sample of those who completed the protocol and those who did not. RESULTS: Of 6,122 callers with ILI who began the nurse protocol administered by the contract NTL, 1,221 people were contacted for the survey and 325 agreed to participate; response rate was 26%. Of those who completed the nurse protocol 73% said they would have sought healthcare without the Minnesota FluLine, 89% reported the service was moderately or very helpful, and 91% reported being satisfied or very satisfied. Of those not completing the protocol, 50% reported the service was moderately or very helpful and 50% reported being satisfied or very satisfied. 72% of qualitative responses to open-ended questions were positive regarding the MN FluLine. Cost to MDH for operating the Minnesota FluLine service was $331,226 to service 27,391 callers ($12.09/call. DISCUSSION: The Minnesota FluLine diverted patients with mild ILI symptoms away from acute care visits at low cost and had a high rate of satisfaction among callers. Early intervention likely prevented morbidity and possibly additional cases. NTLs are powerful and flexible tools for pandemic response and should be considered as an important tool for future emergency

  6. High conservation level of CD8(+) T cell immunogenic regions within an unusual H1N2 human influenza variant.

    Science.gov (United States)

    Komadina, Naomi; Quiñones-Parra, Sergio M; Kedzierska, Katherine; McCaw, James M; Kelso, Anne; Leder, Karin; McVernon, Jodie

    2016-10-01

    Current seasonal influenza vaccines require regular updates due to antigenic drift causing loss of effectiveness and therefore providing little or no protection against novel influenza A subtypes. Next generation vaccines capable of eliciting CD8(+) T cell (CTL) mediated cross-protective immunity may offer a long-term alternative strategy. However, measuring pre- and existing levels of CTL cross-protection in humans is confounded by differences in infection histories across individuals. During 2000-2003, H1N2 viruses circulated persistently in the human population for the first time and we hypothesized that the viral nucleoprotein (NP) contained novel CTL epitopes that may have contributed to the survival of the viruses. This study describes the immunogenic NP peptides of H1N1, H2N2, and H3N2 influenza viruses isolated from humans over the past century, 1918-2003, by comparing this historical dataset to reference NP peptides from H1N2 that circulated in humans during 2000-2003. Observed peptides sequences ranged from highly conserved (15%) to highly variable (12%), with variation unrelated to reported immunodominance. No unique NP peptides which were exclusive to the H1N2 viruses were noted. However, the virus had inherited the NP from a recently emerged H3N2 variant containing novel peptides, which may have assisted its persistence. Any advantage due to this novelty was subsequently lost with emergence of a newer H3N2 variant in 2003. Our approach has potential to provide insight into the population context in which influenza viruses emerge, and may help to inform immunogenic peptide selection for CTL-inducing influenza vaccines. J. Med. Virol. 88:1725-1732, 2016. © 2016 Wiley Periodicals, Inc.

  7. Pancreatic adenocarcinoma upregulated factor (PAUF) confers resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNA receptor-mediated signaling

    Energy Technology Data Exchange (ETDEWEB)

    Kaowinn, Sirichat; Cho, Il-Rae; Moon, Jeong; Jun, Seung Won; Kim, Chang Seok [BK21+, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-736 (Korea, Republic of); Kang, Ho Young [Department of Microbiology, Pusan National University, Busan 609-736 (Korea, Republic of); Kim, Manbok [Department of Medical Science, Dankook University College of Medicine, Cheonan 330-714 (Korea, Republic of); Koh, Sang Seok [Department of Biological Sciences, Dong-A University, Busan 604-714 (Korea, Republic of); Chung, Young-Hwa, E-mail: younghc@pusan.ac.kr [BK21+, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-736 (Korea, Republic of)

    2015-04-03

    Pancreatic adenocarcinoma upregulated factor (PAUF), a novel oncogene, plays a crucial role in the development of pancreatic cancer, including its metastasis and proliferation. Therefore, PAUF-expressing pancreatic cancer cells could be important targets for oncolytic virus-mediated treatment. Panc-1 cells expressing PAUF (Panc-PAUF) showed relative resistance to parvovirus H-1 infection compared with Panc-1 cells expressing an empty vector (Panc-Vec). Of interest, expression of type I IFN-α receptor (IFNAR) was higher in Panc-PAUF cells than in Panc-Vec cells. Increased expression of IFNAR in turn increased the activation of Stat1 and Tyk2 in Panc-PAUF cells compared with that in Panc-Vec cells. Suppression of Tyk2 and Stat1, which are important downstream molecules for IFN-α signaling, sensitized pancreatic cancer cells to parvovirus H-1-mediated apoptosis. Further, constitutive suppression of PAUF sensitized Bxpc3 pancreatic cancer cells to parvovirus H-1 infection. Taken together, these results suggested that PAUF conferred resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNAR-mediated signaling. - Highlights: • PAUF confers resistance against oncolytic parvovirus H-1 infection. • PAUF enhances the expression of IFNAR in Panc-1 cells. • Increased activation of Tyk2 or Stat1 by PAUF provides resistance to parvovirus H-1-mediated apoptosis. • Constitutive inhibition of PAUF enhances parvovirus H-1-mediated oncolysis of Bxpc3 pancreatic cancer cells.

  8. An analysis of peripheral blood cells in patients with influenza A (H1N1) virus infection or non-H1N1 virus infection%甲型H1N1流感病毒与非甲型H1N1流感病毒患者外周血象的对比分析

    Institute of Scientific and Technical Information of China (English)

    王新华; 邵冬华; 梁国威; 曹清云

    2010-01-01

    目的:对航天中心医院就诊的甲型H1N1流感病毒感染者与非甲型H1N1流感病毒感染者、正常对照者的外周血象进行对比分析,以期为临床的诊断、治疗以及病情监测提供有利的工具.方法:采用RT-PCR的方法对患者是否患甲型H1N1流感进行确认.采用流式细胞技术的方法,利用全血细胞计数仪对甲型H1N1组、非甲型H1N1组患者以及正常对照组外周血象进行对比分析.利用免疫比浊的方法对3组患者外周血中C反应蛋白(CRP)浓度进行比较分析.结果:甲型H1N1组患者中单核细胞百分数阳性百分率占77.1%,非甲型H1N1组患者单核细胞百分数阳性百分率占7.8%.正常对照组单核细胞百分数阳性百分率占6.7%.甲型H1N1组白细胞总数、淋巴细胞百分比以及嗜酸细胞百分比与非甲型H1N1组相比明显降低,但甲型H1N1组中性粒细胞百分比与正常对照组相比明显升高,而单核细胞百分比在甲型H1N1组中显著升高.甲型H1N1组血小板总数、血小板压积与非甲型H1N1组相比降低,而血小板分布宽度相比非甲型H1N1组数值升高,但与正常对照组相比,血小板总数以及3项参数未有明显差异.甲型H1N1组中CRP浓度与非甲型H1N1组相比差异无显著性,但与正常对照组相比明显升高.结论:甲型H1N1感染患者外周血象与一般流感存在相似之处,但它有其独特特点,在诊断过程中不应该以一般流感的外周血象以及CRP浓度特点来排除甲型H1N1流感病毒的感染.

  9. Thyroid cell lines in research on goitrogenesis.

    Science.gov (United States)

    Gerber, H; Peter, H J; Asmis, L; Studer, H

    1991-12-01

    Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes.

  10. Development and preclinical testing of HNVAC, a cell culture-based H1N1 pandemic influenza vaccine from India.

    Science.gov (United States)

    Hegde, Nagendra R; Kumar, Deepak; Rao, P Panduranga; Kumari, P Krishna; Kaushik, Yashpal; Ravikrishnan, R; Prasad, Sai D; Ella, Krishna M

    2014-06-17

    Several limitations of the use of embryonated eggs and the threat of pandemics have highlighted the need for other platforms for the production of influenza vaccines. We report the indigenous development and pre-clinical testing of an MDCK-based H1N1 pandemic influenza vaccine HNVAC from India. The cell bank and virus seed were characterized extensively. The cells were characterized by PCR, electron microscopy, and karyotyping, and found to be of female canine epithelial origin. The virus was confirmed by neutralization, haemagglutination inhibition, neuraminidase inhibition, and PCR and nucleotide sequencing. Adventitious agent testing was performed by both in vitro and in vivo studies. The in vitro studies included culturing, haemadsorption, haemagglutination, PCR and RT-PCR, whereas in vivo studies included passage in embryonated eggs and in laboratory animals. Both cell bank and virus seed were free of adventitious agents. MDCK cell lysates as well as cellular DNA did not produce tumours in newborn or adult laboratory animals. The bioprocess parameters were standardized to recover antigen with minimal levels of process-related impurities. The vaccine bulk was tested for the presence of specific antigen, and quantified by single radial immunodiffusion. Finally, non-adjuvanted and aluminium hydroxide adjuvanted vaccine formulations were found to be safe in preclinical toxicity studies in mice, rats, guinea pigs and rabbits, and immunogenic in mice and rabbits. This is the first and only cell culture-based influenza vaccine platform developed in any developing country.

  11. CD4+ T cell autoimmunity to hypocretin/orexin and cross-reactivity to a 2009 H1N1 influenza A epitope in narcolepsy

    DEFF Research Database (Denmark)

    De la Herrán-Arita, Alberto K; Kornum, Birgitte Rahbek; Mahlios, Josh

    2013-01-01

    to the 2009 H1N1 strain, pHA1275-287, with homology to HCRT56-68 and HCRT87-99. In vitro stimulation of narcolepsy CD4(+) T cells with pH1N1 proteins or pHA1275-287 increased the frequency of HCRT56-68- and HCRT87-99-reactive T cells. Our data indicate the presence of CD4(+) T cells that are reactive to HCRT...

  12. PD-L1 expression induced by the 2009 pandemic influenza A(H1N1) virus impairs the human T cell response.

    Science.gov (United States)

    Valero-Pacheco, Nuriban; Arriaga-Pizano, Lourdes; Ferat-Osorio, Eduardo; Mora-Velandia, Luz María; Pastelin-Palacios, Rodolfo; Villasís-Keever, Miguel Ángel; Alpuche-Aranda, Celia; Sánchez-Torres, Luvia Enid; Isibasi, Armando; Bonifaz, Laura; López-Macías, Constantino

    2013-01-01

    PD-L1 expression plays a critical role in the impairment of T cell responses during chronic infections; however, the expression of PD-L1 on T cells during acute viral infections, particularly during the pandemic influenza virus (A(H1N1)pdm09), and its effects on the T cell response have not been widely explored. We found that A(H1N1)pdm09 virus induced PD-L1 expression on human dendritic cells (DCs) and T cells, as well as PD-1 expression on T cells. PD-L1 expression impaired the T cell response against A(H1N1)pdm09 by promoting CD8⁺ T cell death and reducing cytokine production. Furthermore, we found increased PD-L1 expression on DCs and T cells from influenza-infected patients from the first and second 2009 pandemic waves in Mexico City. PD-L1 expression on CD8⁺ T cells correlated inversely with T cell proportions in patients infected with A(H1N1)pdm09. Therefore, PD-L1 expression on DCs and T cells could be associated with an impaired T cell response during acute infection with A(H1N1)pdm09 virus.

  13. PD-L1 Expression Induced by the 2009 Pandemic Influenza A(H1N1) Virus Impairs the Human T Cell Response

    Science.gov (United States)

    Arriaga-Pizano, Lourdes; Ferat-Osorio, Eduardo; Mora-Velandia, Luz María; Pastelin-Palacios, Rodolfo; Villasís-Keever, Miguel Ángel; Alpuche-Aranda, Celia; Sánchez-Torres, Luvia Enid; Isibasi, Armando; Bonifaz, Laura; López-Macías, Constantino

    2013-01-01

    PD-L1 expression plays a critical role in the impairment of T cell responses during chronic infections; however, the expression of PD-L1 on T cells during acute viral infections, particularly during the pandemic influenza virus (A(H1N1)pdm09), and its effects on the T cell response have not been widely explored. We found that A(H1N1)pdm09 virus induced PD-L1 expression on human dendritic cells (DCs) and T cells, as well as PD-1 expression on T cells. PD-L1 expression impaired the T cell response against A(H1N1)pdm09 by promoting CD8+ T cell death and reducing cytokine production. Furthermore, we found increased PD-L1 expression on DCs and T cells from influenza-infected patients from the first and second 2009 pandemic waves in Mexico City. PD-L1 expression on CD8+ T cells correlated inversely with T cell proportions in patients infected with A(H1N1)pdm09. Therefore, PD-L1 expression on DCs and T cells could be associated with an impaired T cell response during acute infection with A(H1N1)pdm09 virus. PMID:24187568

  14. PD-L1 Expression Induced by the 2009 Pandemic Influenza A(H1N1 Virus Impairs the Human T Cell Response

    Directory of Open Access Journals (Sweden)

    Nuriban Valero-Pacheco

    2013-01-01

    Full Text Available PD-L1 expression plays a critical role in the impairment of T cell responses during chronic infections; however, the expression of PD-L1 on T cells during acute viral infections, particularly during the pandemic influenza virus (A(H1N1pdm09, and its effects on the T cell response have not been widely explored. We found that A(H1N1pdm09 virus induced PD-L1 expression on human dendritic cells (DCs and T cells, as well as PD-1 expression on T cells. PD-L1 expression impaired the T cell response against A(H1N1pdm09 by promoting CD8+ T cell death and reducing cytokine production. Furthermore, we found increased PD-L1 expression on DCs and T cells from influenza-infected patients from the first and second 2009 pandemic waves in Mexico City. PD-L1 expression on CD8+ T cells correlated inversely with T cell proportions in patients infected with A(H1N1pdm09. Therefore, PD-L1 expression on DCs and T cells could be associated with an impaired T cell response during acute infection with A(H1N1pdm09 virus.

  15. CD206+ Cell Number Differentiates Influenza A (H1N1)pdm09 from Seasonal Influenza A Virus in Fatal Cases

    Science.gov (United States)

    Rodriguez-Ramirez, Heidi G.; Salinas-Carmona, Mario C.; Barboza-Quintana, Oralia; Melo-de la Garza, Americo; Ceceñas-Falcon, Luis Angel; Rangel-Martinez, Lilia M.; Rosas-Taraco, Adrian G.

    2014-01-01

    In 2009, a new influenza A (H1N1) virus affected many persons around the world. There is an urgent need for finding biomarkers to distinguish between influenza A (H1N1)pdm09 and seasonal influenza virus. We investigated these possible biomarkers in the lung of fatal cases of confirmed influenza A (H1N1)pdm09. Cytokines (inflammatory and anti-inflammatory) and cellular markers (macrophages and lymphocytes subpopulation markers) were analyzed in lung tissue from both influenza A (H1N1)pdm09 and seasonal influenza virus. High levels of IL-17, IFN-γ, and TNF-α positive cells were identical in lung tissue from the influenza A (H1N1)pdm09 and seasonal cases when compared with healthy lung tissue (P < 0.05). Increased IL-4+ cells, and CD4+ and CD14+ cells were also found in high levels in both influenza A (H1N1)pdm09 and seasonal influenza virus (P < 0.05). Low levels of CD206+ cells (marker of alternatively activated macrophages marker in lung) were found in influenza A (H1N1)pdm09 when compared with seasonal influenza virus (P < 0.05), and the ratio of CD206/CD14+ cells was 2.5-fold higher in seasonal and noninfluenza group compared with influenza A (H1N1)pdm09 (P < 0.05). In conclusion, CD206+ cells differentiate between influenza A (H1N1)pdm09 and seasonal influenza virus in lung tissue of fatal cases. PMID:25614715

  16. CD206+ Cell Number Differentiates Influenza A (H1N1pdm09 from Seasonal Influenza A Virus in Fatal Cases

    Directory of Open Access Journals (Sweden)

    Heidi G. Rodriguez-Ramirez

    2014-01-01

    Full Text Available In 2009, a new influenza A (H1N1 virus affected many persons around the world. There is an urgent need for finding biomarkers to distinguish between influenza A (H1N1pdm09 and seasonal influenza virus. We investigated these possible biomarkers in the lung of fatal cases of confirmed influenza A (H1N1pdm09. Cytokines (inflammatory and anti-inflammatory and cellular markers (macrophages and lymphocytes subpopulation markers were analyzed in lung tissue from both influenza A (H1N1pdm09 and seasonal influenza virus. High levels of IL-17, IFN-γ, and TNF-α positive cells were identical in lung tissue from the influenza A (H1N1pdm09 and seasonal cases when compared with healthy lung tissue (P<0.05. Increased IL-4+ cells, and CD4+ and CD14+ cells were also found in high levels in both influenza A (H1N1pdm09 and seasonal influenza virus (P<0.05. Low levels of CD206+ cells (marker of alternatively activated macrophages marker in lung were found in influenza A (H1N1pdm09 when compared with seasonal influenza virus (P<0.05, and the ratio of CD206/CD14+ cells was 2.5-fold higher in seasonal and noninfluenza group compared with influenza A (H1N1pdm09 (P<0.05. In conclusion, CD206+ cells differentiate between influenza A (H1N1pdm09 and seasonal influenza virus in lung tissue of fatal cases.

  17. Alteration of protein levels during influenza virus H1N1 infection in host cells: a proteomic survey of host and virus reveals differential dynamics.

    Directory of Open Access Journals (Sweden)

    Susann Kummer

    Full Text Available We studied the dynamics of the proteome of influenza virus A/PR/8/34 (H1N1 infected Madin-Darby canine kidney cells up to 12 hours post infection by mass spectrometry based quantitative proteomics using the approach of stable isotope labeling by amino acids in cell culture (SILAC. We identified 1311 cell proteins and, apart from the proton channel M2, all major virus proteins. Based on their abundance two groups of virus proteins could be distinguished being in line with the function of the proteins in genesis and formation of new virions. Further, the data indicate a correlation between the amount of proteins synthesized and their previously determined copy number inside the viral particle. We employed bioinformatic approaches such as functional clustering, gene ontology, and pathway (KEGG enrichment tests to uncover co-regulated cellular protein sets, assigned the individual subsets to their biological function, and determined their interrelation within the progression of viral infection. For the first time we are able to describe dynamic changes of the cellular and, of note, the viral proteome in a time dependent manner simultaneously. Through cluster analysis, time dependent patterns of protein abundances revealed highly dynamic up- and/or down-regulation processes. Taken together our study provides strong evidence that virus infection has a major impact on the cell status at the protein level.

  18. Determinants of vaccine immunogenicity in HIV-infected pregnant women: analysis of B and T cell responses to pandemic H1N1 monovalent vaccine.

    Directory of Open Access Journals (Sweden)

    Adriana Weinberg

    Full Text Available Influenza infections have high frequency and morbidity in HIV-infected pregnant women, underscoring the importance of vaccine-conferred protection. To identify the factors that determine vaccine immunogenicity in this group, we characterized the relationship of B- and T-cell responses to pandemic H1N1 (pH1N1 vaccine with HIV-associated immunologic and virologic characteristics. pH1N1 and seasonal-H1N1 (sH1N1 antibodies were measured in 119 HIV-infected pregnant women after two double-strength pH1N1 vaccine doses. pH1N1-IgG and IgA B-cell FluoroSpot, pH1N1- and sH1N1-interferon γ (IFNγ and granzyme B (GrB T-cell FluoroSpot, and flow cytometric characterization of B- and T-cell subsets were performed in 57 subjects. pH1N1-antibodies increased after vaccination, but less than previously described in healthy adults. pH1N1-IgG memory B cells (Bmem increased, IFNγ-effector T-cells (Teff decreased, and IgA Bmem and GrB Teff did not change. pH1N1-antibodies and Teff were significantly correlated with each other and with sH1N1-HAI and Teff, respectively, before and after vaccination. pH1N1-antibody responses to the vaccine significantly increased with high proportions of CD4+, low CD8+ and low CD8+HLADR+CD38+ activated (Tact cells. pH1N1-IgG Bmem responses increased with high proportions of CD19+CD27+CD21- activated B cells (Bact, high CD8+CD39+ regulatory T cells (Treg, and low CD19+CD27-CD21- exhausted B cells (Bexhaust. IFNγ-Teff responses increased with low HIV plasma RNA, CD8+HLADR+CD38+ Tact, CD4+FoxP3+ Treg and CD19+IL10+ Breg. In conclusion, pre-existing antibody and Teff responses to sH1N1 were associated with increased responses to pH1N1 vaccination in HIV-infected pregnant women suggesting an important role for heterosubtypic immunologic memory. High CD4+% T cells were associated with increased, whereas high HIV replication, Tact and Bexhaust were associated with decreased vaccine immunogenicity. High Treg increased antibody responses but

  19. Post-translational modifications of linker histone H1 variants in mammals

    Science.gov (United States)

    Starkova, T. Yu; Polyanichko, A. M.; Artamonova, T. O.; Khodorkovskii, M. A.; Kostyleva, E. I.; Chikhirzhina, E. V.; Tomilin, A. N.

    2017-02-01

    The covalent modifications of the linker histone H1 and the core histones are thought to play an important role in the control of chromatin functioning. Histone H1 variants from K562 cell line (hH1), mouse (mH1) and calf (cH1) thymi were studied by matrix-activated laser desorption/ionization fourier transform ion cyclotron resonance mass-spectroscopy (MALDI-FT-ICR-MS). The proteomics analysis revealed novel post-translational modifications of the histone H1, such as meK34-mH1.4, meK35-cH1.1, meK35-mH1.1, meK75-hH1.2, meK75-hH1.3, acK26-hH1.4, acK26-hH1.3 and acK17-hH1.1. The comparison of the hH1, mH1 and cH1 proteins has demonstrated that the types and positions of the post-translational modifications of the globular domains of the H1.2–H1.4 variants are very conservative. However, the post-translational modifications of the N- and C-terminal tails of H1.2, H1.3 and H1.4 are different. The differences of post-translational modifications in the N- and C-terminal tails of H1.2, H1.3 and H1.4 likely lead to the differences in DNA-H1 and H1-protein interactions.

  20. Influence of the oncolytic parvovirus H-1, CTLA-4 antibody tremelimumab and cytostatic drugs on the human immune system in a human in vitro model of colorectal cancer cells

    Directory of Open Access Journals (Sweden)

    Heinrich B

    2013-08-01

    Full Text Available Bernd Heinrich,* Katrin Goepfert,* Maike Delic, Peter R Galle, Markus MoehlerUniversity Medical Center of the Johannes Gutenberg University Mainz, 1st Department of Internal Medicine, Langenbeckstrasse, Mainz, Germany *These authors contributed equally to this workIntroduction: Tumor-directed and immune-system-stimulating therapies are of special interest in cancer treatment. Here, we demonstrate the potential of parvovirus H-1 (H-1PV to efficiently kill colorectal cancer cells and induce immunogenicity of colorectal tumors by inducing maturation of dendritic cells (DCs alone and also in combination with cytostatic drugs in vitro. Using our cell culture model, we have additionally investigated the effects of anti-CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4 receptor antibody tremelimumab on this process.Materials and methods: Colon carcinoma cell lines were treated with different concentrations of cytostatic drugs or tremelimumab or were infected with H-1PV in different multiplicities of infection (MOIs, and viability was determined using MTT assays. Expression of CTLA-4 in colon carcinoma cell lines was measured by FACScan™. For the coculture model, we isolated monocytes using adherence, and differentiation into immature DCs (iDCs was stimulated using interleukin-4 and granulocyte-macrophage colony-stimulating factor. Maturation of iDCs into mature DCs (mDCs was induced by a cytokine cocktail. SW480 colon carcinoma cells were infected with H-1PV or treated with cytostatic drugs. Drug treated and H-1PV-infected SW480 colon carcinoma cells were cocultured with iDCs and expression of maturation markers was measured using FACScan™. Cytokine measurements were performed using enzyme-linked immunosorbent assay.Results: Colon carcinoma cells SW480 were potently infected and killed by H-1PV. CTLA-4 expression in SW480 cells increased after infection with H-1PV and also after treatment with cytostatic drugs. Tremelimumab had no influence on

  1. MUC1* ligand, NM23-H1, is a novel growth factor that maintains human stem cells in a more naive state.

    Directory of Open Access Journals (Sweden)

    Benoit J Smagghe

    Full Text Available We report that a single growth factor, NM23-H1, enables serial passaging of both human ES and iPS cells in the absence of feeder cells, their conditioned media or bFGF in a fully defined xeno-free media on a novel defined, xeno-free surface. Stem cells cultured in this system show a gene expression pattern indicative of a more "naïve" state than stem cells grown in bFGF-based media. NM23-H1 and MUC1* growth factor receptor cooperate to control stem cell self-replication. By manipulating the multimerization state of NM23-H1, we override the stem cell's inherent programming that turns off pluripotency and trick the cells into continuously replicating as pluripotent stem cells. Dimeric NM23-H1 binds to and dimerizes the extra cellular domain of the MUC1* transmembrane receptor which stimulates growth and promotes pluripotency. Inhibition of the NM23-H1/MUC1* interaction accelerates differentiation and causes a spike in miR-145 expression which signals a cell's exit from pluripotency.

  2. Power-laws and the use of pluripotent stem cell lines

    NARCIS (Netherlands)

    Schuldt, Bernhard M; Guhr, Anke; Lenz, Michael; Kobold, Sabine; MacArthur, Ben D; Schuppert, Andreas; Löser, Peter; Müller, Franz-Josef

    2013-01-01

    It is widely accepted that the (now reversed) Bush administration's decision to restrict federal funding for human embryonic stem cell (hESC) research to a few "eligible" hESC lines is responsible for the sustained preferential use of a small subset of hESC lines (principally the H1 and H9 lines) in

  3. Histone H3 Methyltransferase Suv39h1 Prevents Myogenic Terminal Differentiation by Repressing MEF2 Activity in Muscle Cells

    Directory of Open Access Journals (Sweden)

    Wei Jin

    2016-11-01

    Full Text Available The myogenic regulatory factors (MRFs and myocyte enhancer factor 2 (MEF2 transcription factors have been extensively studied as key transcription factors that regulate myogenic gene expression. However, few reports on the molecular mechanism that modulates chromatin remodeling during skeletal muscle differentiation are available. We reported here that the expression of the H3-K9 methyltransferase Suv39h1 was decreased during myoblast differentiation. Ectopic expression of Suv39h1 could inhibit myoblast differentiation, increasing H3-K9 methylation levels, whereas knockdown of Suv39h1 stimulated myoblast differentiation. Furthermore, Suv39h1 interacted with MEF2C directly and inhibited MEF2 transcription activity in a dose-dependent manner. Together, our studies revealed a molecular mechanism wherein Suv39h1 modulated myogenic gene expression and activation during skeletal muscle differentiation.

  4. Silencing B7-H1 enhances the anti-tumor effect of bladder cancer antigen-loaded dendritic cell vaccine in vitro

    Directory of Open Access Journals (Sweden)

    Wang S

    2014-08-01

    Full Text Available Shuo Wang,1 Yonghua Wang,1 Jing Liu,2 Shixiu Shao,1 Xianjun Li,1 Jiannan Gao,1 Haitao Niu,1 Xinsheng Wang1 1Department of Urology, 2Department of Pediatrics, The Affiliated Hospital of Qingdao University, Qingdao, People's Republic of China Objective: The aim of this study was to examine whether short hairpin RNA (shRNA expressing lentiviral particles targeting B7-H1 infection could result in B7-H1 knockdown on dendritic cells (DCs and to investigate whether B7-H1 silencing could augment the immune function of DCs and further elicit a more potent anti-tumor immune effect against bladder cancer cells in vitro. Methods: Monocyte-derived DCs, which were generated from peripheral blood mononuclear cells, were infected by a recombinant lentivirus containing shRNA sequence aimed at B7-H1. After that, the infected DCs were pulsed by tumor antigens and used to stimulate cytotoxic T lymphocytes-based anti-tumor effect in vitro. Results: The lentivirus-mediated shRNA delivery method efficiently and effectively silenced B7-H1 in DCs. Furthermore, the B7-H1 silencing enhanced the stimulatory capacity and the secretion of interleukin-12, but down-regulated interleukin-10 secretion. And more importantly, the anti-tumor effect of bladder cancer antigen-loaded DC vaccine in vitro was also potentially augmented. Conclusion: This study suggests that a combination of B7-H1 knockdown and target antigen delivery could augment anti-tumor effects in vitro, which potentially provides a novel strategy in the immunotherapy of bladder cancer. Keywords: B7-H1, bladder cancer, dendritic cell, vaccine, immunotherapy

  5. Pancreatic adenocarcinoma upregulated factor (PAUF) confers resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNA receptor-mediated signaling.

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    Kaowinn, Sirichat; Cho, Il-Rae; Moon, Jeong; Jun, Seung Won; Kim, Chang Seok; Kang, Ho Young; Kim, Manbok; Koh, Sang Seok; Chung, Young-Hwa

    2015-04-01

    Pancreatic adenocarcinoma upregulated factor (PAUF), a novel oncogene, plays a crucial role in the development of pancreatic cancer, including its metastasis and proliferation. Therefore, PAUF-expressing pancreatic cancer cells could be important targets for oncolytic virus-mediated treatment. Panc-1 cells expressing PAUF (Panc-PAUF) showed relative resistance to parvovirus H-1 infection compared with Panc-1 cells expressing an empty vector (Panc-Vec). Of interest, expression of type I IFN-α receptor (IFNAR) was higher in Panc-PAUF cells than in Panc-Vec cells. Increased expression of IFNAR in turn increased the activation of Stat1 and Tyk2 in Panc-PAUF cells compared with that in Panc-Vec cells. Suppression of Tyk2 and Stat1, which are important downstream molecules for IFN-α signaling, sensitized pancreatic cancer cells to parvovirus H-1-mediated apoptosis. Further, constitutive suppression of PAUF sensitized Bxpc3 pancreatic cancer cells to parvovirus H-1 infection. Taken together, these results suggested that PAUF conferred resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNAR-mediated signaling.

  6. Characterization of functional antibody and memory B-cell responses to pH1N1 monovalent vaccine in HIV-infected children and youth.

    Directory of Open Access Journals (Sweden)

    Donna J Curtis

    Full Text Available We investigated immune determinants of antibody responses and B-cell memory to pH1N1 vaccine in HIV-infected children.Ninety subjects 4 to <25 years of age received two double doses of pH1N1 vaccine. Serum and cells were frozen at baseline, after each vaccination, and at 28 weeks post-immunization. Hemagglutination inhibition (HAI titers, avidity indices (AI, B-cell subsets, and pH1N1 IgG and IgA antigen secreting cells (ASC were measured at baseline and after each vaccination. Neutralizing antibodies and pH1N1-specific Th1, Th2 and Tfh cytokines were measured at baseline and post-dose 1.At entry, 26 (29% subjects had pH1N1 protective HAI titers (≥1:40. pH1N1-specific HAI, neutralizing titers, AI, IgG ASC, IL-2 and IL-4 increased in response to vaccination (p<0.05, but IgA ASC, IL-5, IL-13, IL-21, IFNγ and B-cell subsets did not change. Subjects with baseline HAI ≥1:40 had significantly greater increases in IgG ASC and AI after immunization compared with those with HAI <1:40. Neutralizing titers and AI after vaccination increased with older age. High pH1N1 HAI responses were associated with increased IgG ASC, IFNγ, IL-2, microneutralizion titers, and AI. Microneutralization titers after vaccination increased with high IgG ASC and IL-2 responses. IgG ASC also increased with high IFNγ responses. CD4% and viral load did not predict the immune responses post-vaccination, but the B-cell distribution did. Notably, vaccine immunogenicity increased with high CD19+CD21+CD27+% resting memory, high CD19+CD10+CD27+% immature activated, low CD19+CD21-CD27-CD20-% tissue-like, low CD19+CD21-CD27-CD20-% transitional and low CD19+CD38+HLADR+% activated B-cell subsets.HIV-infected children on HAART mount a broad B-cell memory response to pH1N1 vaccine, which was higher for subjects with baseline HAI≥1:40 and increased with age, presumably due to prior exposure to pH1N1 or to other influenza vaccination/infection. The response to the vaccine was dependent

  7. Autophagy is involved in regulating the immune response of dendritic cells to influenza A (H1N1) pdm09 infection.

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    Zang, Farong; Chen, Yinghu; Lin, Zhendong; Cai, Zhijian; Yu, Lei; Xu, Feng; Wang, Jiaoli; Zhu, Weiguo; Lu, Huoquan

    2016-05-01

    Autophagy can mediate antiviral immunity. However, it remains unknown whether autophagy regulates the immune response of dendritic cells (DCs) to influenza A (H1N1) pdm09 infection. In this study, we found that infection with the H1N1 virus induced DC autophagy in an endocytosis-dependent manner. Compared with autophagy-deficient Beclin-1(+/-) mice, we found that bone-marrow-derived DCs from wild-type mice (WT BMDCs) presented a more mature phenotype on H1N1 infection. Wild-type BMDCs secreted higher levels of interleukin-6 (IL-6), tumour necrosis factor- α (TNF-α), interferon-β (IFN-β), IL-12p70 and IFN-γ than did Beclin-1(+/-) BMDCs. In contrast to Beclin-1(+/-) BMDCs, H1N1-infected WT BMDCs exhibited increased activation of extracellular signal-regulated kinase, Jun N-terminal kinase, p38, and nuclear factor-κB as well as IFN regulatory factor 7 nuclear translocation. Blockade of autophagosomal and lysosomal fusion by bafilomycin A1 decreased the co-localization of H1N1 viruses, autophagosomes and lysosomes as well as the secretion of IL-6, TNF-α and IFN-β in H1N1-infected BMDCs. In contrast to Beclin-1(+/-) BMDCs, H1N1-infected WT BMDCs were more efficient in inducing allogeneic CD4(+) T-cell proliferation and driving T helper type 1, 2 and 17 cell differentiation while inhibiting CD4(+) Foxp3(+) regulatory T-cell differentiation. Moreover, WT BMDCs were more efficient at cross-presenting the ovalbumin antigen to CD8(+) T cells. We consistently found that Beclin-1(+/-) BMDCs were inferior in their inhibition of H1N1 virus replication and their induction of H1N1-specific CD4(+) and CD8(+) T-cell responses, which produced lower levels of IL-6, TNF-α and IFN-β in vivo. Our data indicate that autophagy is important in the regulation of the DC immune response to H1N1 infection, thereby extending our understanding of host immune responses to the virus.

  8. A critical role of IFNγ in priming MSC-mediated suppression of T cell proliferation through up-regulation of B7-H1

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Bone-marrow-derived mesenchymal stem cells (MSCs) have been shown to possess immunosuppressive properties, e.g., by inhibiting T cell proliferation. Activated T cells can also enhance the immunosuppression ability of MSCs. The precise mechanisms underlying MSC-mediated immunosuppression remain largely undefined, although both cell-cell contact and soluble factors have been implicated; nor is it clear how the immunosuppressive property of MSCs is modulated by T cells. Using MSCs isolated from mouse bone marrow, we show here that interferon gamma (IFNγ), a well-known proinflammatory cytokine produced by activated T cells, plays an important role in priming the immunosuppressive property of MSCs. Mechanistically, IFNγ acts directly on MSCs and leads to up-regulation of B7-H1, an inhibitory surface molecule in these stem cells. MSCs primed by activated T cells derived from IFNγ-/- mouse exhibited dramatically reduced ability to suppress T cell proliferation, a defect that can be rescued by supplying exogenous IFNy. Moreover, siRNA-mediated knockdown of B7-H1 in MSCs abolished immunosuppression by these cells. Taken together, our results suggest that IFNy plays a critical role in triggering the immunosuppresion by MSCs through upregulating B7-H1 in these cells, and provide evidence supporting the cell-cell contact mechanism in MSC-mediated immunosuppression.

  9. Molluscan cells in culture: primary cell cultures and cell lines.

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    Yoshino, T P; Bickham, U; Bayne, C J

    2013-06-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome.

  10. Helicobacter Pylori Promote B7-H1 Expression by Suppressing miR-152 and miR-200b in Gastric Cancer Cells

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    Xie, Gengchen; Li, Wei; Li, Ruidong; Wu, Ke; Zhao, Ende; Zhang, Yu; Zhang, Peng; Shi, Liang; Wang, Di; Yin, Yuping; Deng, Rui; Tao, Kaixiong

    2017-01-01

    The most common cause of gastric cancer is infection with helicobacter pylori (HP), but the associated molecular mechanism is not well understood. In the present study, we found a marked increase in the expression of B7-H1, a member of the B7 co-stimulatory family of molecules that bind to programmed death-1 (PD-1) and play a critical immunoregulatory role in the cell-mediated immune response, in HP-positive gastric cancer tissue. Infection of cultured gastric cancer cells with HP promoted B7-H1 expression and inhibited miR-152 and miR-200b expression. We further demonstrated that these two miRNAs targeted B7-H1 mRNA and suppressed B7-H1 expression in gastric cancer cells. Finally, B7-H1 expression was found to correlate with miR-152 and miR-200b levels in gastric tumor tissues from human patients. Our findings suggest a novel mechanism by which HP infection promotes gastric cancer and also suggest potential targets, i.e., miR-152 and miR-200b, for the prevention and treatment of gastric cancer. PMID:28056089

  11. Irradiation with narrowband-ultraviolet B suppresses phorbol ester-induced up-regulation of H1 receptor mRNA in HeLa cells.

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    Kitamura, Yoshiaki; Mizuguchi, Hiroyuki; Okamoto, Kentaro; Kitayama, Mika; Fujii, Tatsuya; Fujioka, Akira; Matsushita, Toshio; Mukai, Takashi; Kubo, Yoshiaki; Kubo, Nobuo; Fukui, Hiroyuki; Takeda, Noriaki

    2016-01-01

    Conclusion These findings suggest that low dose irradiation with 310 nm NB-UVB specifically suppressed the up-regulation of H1R gene expression without inducing apoptosis and that UVB of shorter or longer wavelength than 310 nm NB-UVB had no such effects. Objective To develop a narrowband-ultraviolet B(NB-UVB) phototherapy for allergic rhinitis, this study investigated the effects of irradiation with NB-UVB at wavelength of 310 nm on phorbol-12-myristate-13-acetate (PMA)-induced up-regulation of histamine H1 receptor (H1R) mRNA in HeLa cells. Methods The mRNA levels of H1R in HeLa cells were measured using real-time RT-PCR. Apoptosis were evaluated with DNA fragmentation assay. Results PMA induced a significant increase in H1R mRNA expression in HeLa cells. Irradiation with 305 nm UVB and 310 nm NB-UVB, but not with 315 nm UVB at doses of 200 and 300 mJ/cm(2) significantly suppressed PMA-induced up-regulation of H1R mRNA. At a dose of 200 mJ/cm(2), irradiation with 305 nm UVB, but not with 310 nm NB-UVB, induced apoptosis, although exposure of the cells to both 305 and 310 nm UVB induced apoptosis at a dose of 300 mJ/cm(2) after PMA treatment in HeLa cells. Conversely, irradiation with 315 nm UVB at doses of 200 and 300 mJ/cm(2) did not induce apoptosis.

  12. PI3K-delta mediates double-stranded RNA-induced upregulation of B7-H1 in BEAS-2B airway epithelial cells

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    Kan-o, Keiko [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Matsumoto, Koichiro, E-mail: koichi@kokyu.med.kyushu-u.ac.jp [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Asai-Tajiri, Yukari; Fukuyama, Satoru; Hamano, Saaka; Seki, Nanae; Nakanishi, Yoichi [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Inoue, Hiromasa [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Department of Pulmonary Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan)

    2013-05-31

    Highlights: •Double-stranded RNA upregulates B7-H1 on BEAS-2B airway epithelial cells. •The upregulation of B7-H1 is attenuated by inhibition of PI3Kδ isoform. •PI3Kδ-mediated upregulation of B7-H1 is independent of NF-κB activation. •Inhibition of PI3Kδ may prevent persistent viral infection induced by B7-H1. -- Abstract: Airway viral infection disturbs the health-related quality of life. B7-H1 (also known as PD-L1) is a coinhibitory molecule associated with the escape of viruses from the mucosal immunity, leading to persistent infection. Most respiratory viruses generate double-stranded (ds) RNA during replication. The stimulation of cultured airway epithelial cells with an analog of viral dsRNA, polyinosinic-polycytidylic acid (poly IC) upregulates the expression of B7-H1 via activation of the nuclear factor κB(NF-κB). The mechanism of upregulation was investigated in association with phosphatidylinositol 3-kinases (PI3Ks). Poly IC-induced upregulation of B7-H1 was profoundly suppressed by a pan-PI3K inhibitor and partially by an inhibitor or a small interfering (si)RNA for PI3Kδ in BEAS-2B cells. Similar results were observed in the respiratory syncytial virus-infected cells. The expression of p110δ was detected by Western blot and suppressed by pretreatment with PI3Kδ siRNA. The activation of PI3Kδ is typically induced by oxidative stress. The generation of reactive oxygen species was increased by poly IC. Poly IC-induced upregulation of B7-H1 was attenuated by N-acetyl-L-cysteine, an antioxidant, or by oxypurinol, an inhibitor of xanthine oxidase. Poly IC-induced activation of NF-κB was suppressed by a pan-PI3K inhibitor but not by a PI3Kδ inhibitor. These results suggest that PI3Kδ mediates dsRNA-induced upregulation of B7-H1 without affecting the activation of NF-κB.

  13. Conservation and diversity of influenza A H1N1 HLA-restricted T cell epitope candidates for epitope-based vaccines.

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    Paul Thiamjoo Tan

    Full Text Available BACKGROUND: The immune-related evolution of influenza viruses is exceedingly complex and current vaccines against influenza must be reformulated for each influenza season because of the high degree of antigenic drift among circulating influenza strains. Delay in vaccine production is a serious problem in responding to a pandemic situation, such as that of the current H1N1 strain. Immune escape is generally attributed to reduced antibody recognition of the viral hemagglutinin and neuraminidase proteins whose rate of mutation is much greater than that of the internal non-structural proteins. As a possible alternative, vaccines directed at T cell epitope domains of internal influenza proteins, that are less susceptible to antigenic variation, have been investigated. METHODOLOGY/PRINCIPAL FINDINGS: HLA transgenic mouse strains expressing HLA class I A*0201, A*2402, and B*0702, and class II DRB1*1501, DRB1*0301 and DRB1*0401 were immunized with 196 influenza H1N1 peptides that contained residues of highly conserved proteome sequences of the human H1N1, H3N2, H1N2, H5N1, and avian influenza A strains. Fifty-four (54 peptides that elicited 63 HLA-restricted peptide-specific T cell epitope responses were identified by IFN-gamma ELISpot assay. The 54 peptides were compared to the 2007-2009 human H1N1 sequences for selection of sequences in the design of a new candidate H1N1 vaccine, specifically targeted to highly-conserved HLA-restricted T cell epitopes. CONCLUSIONS/SIGNIFICANCE: Seventeen (17 T cell epitopes in PB1, PB2, and M1 were selected as vaccine targets based on sequence conservation over the past 30 years, high functional avidity, non-identity to human peptides, clustered localization, and promiscuity to multiple HLA alleles. These candidate vaccine antigen sequences may be applicable to any avian or human influenza A virus.

  14. Preventive Activity against Influenza (H1N1 Virus by Intranasally Delivered RNA-Hydrolyzing Antibody in Respiratory Epithelial Cells of Mice

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    Seungchan Cho

    2015-09-01

    Full Text Available The antiviral effect of a catalytic RNA-hydrolyzing antibody, 3D8 scFv, for intranasal administration against avian influenza virus (H1N1 was described. The recombinant 3D8 scFv protein prevented BALB/c mice against H1N1 influenza virus infection by degradation of the viral RNA genome through its intrinsic RNA-hydrolyzing activity. Intranasal administration of 3D8 scFv (50 μg/day for five days prior to infection demonstrated an antiviral activity (70% survival against H1N1 infection. The antiviral ability of 3D8 scFv to penetrate into epithelial cells from bronchial cavity via the respiratory mucosal layer was confirmed by immunohistochemistry, qRT-PCR, and histopathological examination. The antiviral activity of 3D8 scFv against H1N1 virus infection was not due to host immune cytokines or chemokines, but rather to direct antiviral RNA-hydrolyzing activity of 3D8 scFv against the viral RNA genome. Taken together, our results suggest that the RNase activity of 3D8 scFv, coupled with its ability to penetrate epithelial cells through the respiratory mucosal layer, directly prevents H1N1 virus infection in a mouse model system.

  15. AID activity in B cells strongly correlates with polyclonal antibody affinity maturation in-vivo following pandemic 2009-H1N1 vaccination in humans.

    Science.gov (United States)

    Khurana, Surender; Frasca, Daniela; Blomberg, Bonnie; Golding, Hana

    2012-09-01

    The role of Activation-Induced Cytidine Deaminase (AID) in somatic hypermutation and polyclonal antibody affinity maturation has not been shown for polyclonal responses in humans. We investigated whether AID induction in human B cells following H1N1pdm09 vaccination correlated with in-vivo antibody affinity maturation against hemagglutinin domains in plasma of young and elderly individuals. AID was measured by qPCR in B cells from individuals of different ages immunized with the H1N1pdm09 influenza vaccine. Polyclonal antibody affinity in human plasma for the HA1 and HA2 domains of the H1N1pdm09 hemagglutinin was measured by antibody-antigen complex dissociation rates using real time kinetics in Surface Plasmon Resonance. Results show an age-related decrease in AID induction in B cells following H1N1pdm09 vaccination. Levels of AID mRNA before vaccination and fold-increase of AID mRNA expression after H1N1pdm09 vaccination directly correlated with increase in polyclonal antibody affinity to the HA1 globular domain (but not to the conserved HA2 stalk). In the younger population, significant affinity maturation to the HA1 globular domain was observed, which associated with initial levels of AID and fold-increase in AID after vaccination. In some older individuals (>65 yr), higher affinity to the HA1 domain was observed before vaccination and H1N1pdm09 vaccination resulted in minimal change in antibody affinity, which correlated with low AID induction in this age group. These findings demonstrate for the first time a strong correlation between AID induction and in-vivo antibody affinity maturation in humans. The ability to generate high affinity antibodies could have significant impact on the elucidation of age-specific antibody responses following vaccination and eventual clinical efficacy and disease outcome.

  16. Increase in IFNγ(-IL-2(+ cells in recent human CD4 T cell responses to 2009 pandemic H1N1 influenza.

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    Jason M Weaver

    Full Text Available Human CD4 T cell recall responses to influenza virus are strongly biased towards Type 1 cytokines, producing IFNγ, IL-2 and TNFα. We have now examined the effector phenotypes of CD4 T cells in more detail, particularly focusing on differences between recent versus long-term, multiply-boosted responses. Peptides spanning the proteome of temporally distinct influenza viruses were distributed into pools enriched for cross-reactivity to different influenza strains, and used to stimulate antigen-specific CD4 T cells representing recent or long-term memory. In the general population, peptides unique to the long-circulating influenza A/New Caledonia/20/99 (H1N1 induced Th1-like responses biased toward the expression of IFNγ(+TNFα(+ CD4 T cells. In contrast, peptide pools enriched for non-cross-reactive peptides of the pandemic influenza A/California/04/09 (H1N1 induced more IFNγ(-IL-2(+TNFα(+ T cells, similar to the IFNγ(-IL-2(+ non-polarized, primed precursor T cells (Thpp that are a predominant response to protein vaccination. These results were confirmed in a second study that compared samples taken before the 2009 pandemic to samples taken one month after PCR-confirmed A/California/04/09 infection. There were striking increases in influenza-specific TNFα(+, IFNγ(+, and IL-2(+ cells in the post-infection samples. Importantly, peptides enriched for non-cross-reactive A/California/04/09 specificities induced a higher proportion of Thpp-like IFNγ(-IL-2(+TNFα(+ CD4 T cells than peptide pools cross-reactive with previous influenza strains, which induced more Th1 (IFNγ(+TNFα(+ responses. These IFNγ(-IL-2(+TNFα(+ CD4 T cells may be an important target population for vaccination regimens, as these cells are induced upon infection, may have high proliferative potential, and may play a role in providing future effector cells during subsequent infections.

  17. Seroprotection of HIV-infected subjects after influenza A(H1N1) vaccination is directly associated with baseline frequency of naive T cells.

    Science.gov (United States)

    Ramirez, Lorenzo A; Daniel, Alexander; Frank, Ian; Tebas, Pablo; Boyer, Jean D

    2014-08-15

    Human immunodeficiency virus type 1 (HIV-1)-infected individuals, despite receipt of antiretroviral therapy (ART), often have impaired vaccine responses. We examined the role that immune activation and cellular phenotypes play in influenza A(H1N1) vaccine responsiveness in HIV-infected subjects receiving ART. Subjects received the H1N1 vaccine (15-µg dose; Novartis), and antibody titers at baseline and after immunization were evaluated. Subjects were classified as responders if, by week 3, seroprotection guidelines were met. Responders had higher percentages of baseline naive T cells and lower percentages of terminally differentiated T cells, compared with nonresponders. Additionally, the naive CD4(+) T-cell percentage and age were negatively correlated. Preservation of naive T-cell populations by starting therapy early could impact vaccine responses against influenza virus and other pathogens, especially as this population ages.

  18. Effect of sesamin against cytokine production from influenza type A H1N1-induced peripheral blood mononuclear cells: computational and experimental studies.

    Science.gov (United States)

    Fanhchaksai, Kanda; Kodchakorn, Kanchanok; Pothacharoen, Peraphan; Kongtawelert, Prachya

    2016-01-01

    In 2009, swine flu (H1N1) had spread significantly to levels that threatened pandemic influenza. There have been many treatments that have arisen for patients since the WHO first reported the disease. Although some progress in controlling influenza has taken place during the last few years, the disease is not yet under control. The development of new and less expensive anti-influenza drugs is still needed. Here, we show that sesamin from the seeds of the Thai medicinal plant Sesamum indicum has anti-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) induced by 2009 influenza virus type A H1N1. In this study, the combinatorial screening method combined with the computational approach was applied to investigate the new molecular binding structures of sesamin against the 2009 influenza virus type A H1N1 (p09N1) crystallized structure. Experimental methods were applied to propose the mechanisms of sesamin against cytokine production from H1N1-induced human PBMC model. The molecular dynamics simulation of sesamin binding with the p09N1 crystallized structure showed new molecular binding structures at ARG118, ILE222, ARG224, and TYR406, and it has been proposed that sesamin could potentially be used to produce anti-H1N1 compounds. Furthermore, the mechanisms of sesamin against cytokine production from influenza type A H1N1-induced PBMCs by ELISA and signaling transduction showed that sesamin exhibits the ability to inhibit proinflammatory cytokines, IL-1β and TNF-α, and to enhance the activity of the immune cell cytokine IL-2 via downregulating the phosphorylated JNK, p38, and ERK1/2 MAPK signaling pathways. This information might very well be useful in the prevention and treatment of immune-induced inflammatory disorders.

  19. Pharmacological and functional characterisation of the wild-type and site-directed mutants of the human H1 histamine receptor stably expressed in CHO cells.

    Science.gov (United States)

    Moguilevsky, N; Varsalona, F; Guillaume, J P; Noyer, M; Gillard, M; Daliers, J; Henichart, J P; Bollen, A

    1995-01-01

    A cDNA clone for the human histamine H1 receptor was isolated from a lung cDNA library and stably expressed in CHO cells. The recombinant receptor protein present in the cell membranes, displayed the functional and binding characteristics of histamine H1 receptors. Mutation of Ser155 to Ala in the fourth transmembrane domain did not significantly change the affinity of the receptor for histamine and H1 antagonists. However, mutation of the fifth transmembrane Asn198 to Ala resulted in a dramatic decrease of the affinity for histamine binding, and for the histamine-induced polyphosphoinositides breakdown, whereas the affinity towards antagonists was not significantly modified. In addition, mutation of another fifth transmembrane amino acid, Thr194 to Ala also diminished, but to a lesser extent, the affinity for histamine. These data led us to propose a molecular model for histamine interaction with the human H1 receptor. In this model, the amide moiety of Asn198 and the hydroxyl group of Thr194 are involved in hydrogen bonding with the nitrogen atoms of the imidazole ring of histamine. Moreover, mutation of Thr194 to Ala demonstrated that this residue is responsible for the discrimination between enantiomers of cetirizine.

  20. Influenza H5N1 and H1N1 virus replication and innate immune responses in bronchial epithelial cells are influenced by the state of differentiation.

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    Renee W Y Chan

    Full Text Available Influenza H5N1 virus continues to be enzootic in poultry and transmits zoonotically to humans. Although a swine-origin H1N1 virus has emerged to become pandemic, its virulence for humans remains modest in comparison to that seen in zoonotic H5N1 disease. As human respiratory epithelium is the primary target cells for influenza viruses, elucidating the viral tropism and host innate immune responses of influenza H5N1 virus in human bronchial epithelium may help to understand the pathogenesis. Here we established primary culture of undifferentiated and well differentiated normal human bronchial epithelial (NHBE cells and infected with highly pathogenic influenza H5N1 virus (A/Vietnam/3046/2004 and a seasonal influenza H1N1 virus (A/Hong Kong/54/1998, the viral replication kinetics and cytokine and chemokine responses were compared by qPCR and ELISA. We found that the in vitro culture of the well differentiated NHBE cells acquired the physiological properties of normal human bronchi tissue which express high level of alpha2-6-linked sialic acid receptors and human airway trypsin-like (HAT protease, in contrast to the low expression in the non-differentiated NHBE cells. When compared to H1N1 virus, the H5N1 virus replicated more efficiently and induced a stronger type I interferon response in the undifferentiated NHBE cells. In contrast, in well differentiated cultures, H5N1 virus replication was less efficient and elicited a lower interferon-beta response in comparison with H1N1 virus. Our data suggest that the differentiation of bronchial epithelial cells has a major influence in cells' permissiveness to human H1N1 and avian H5N1 viruses and the host innate immune responses. The reduced virus replication efficiency partially accounts for the lower interferon-beta responses in influenza H5N1 virus infected well differentiated NHBE cells. Since influenza infection in the bronchial epithelium will lead to tissue damage and associate with the

  1. Generation of iPSC line epiHUVEC from human umbilical vein endothelial cells

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    Peggy Matz

    2015-11-01

    Full Text Available Human umbilical vein endothelial cells (HUVECs were used to generate the iPSC line epiHUVEC employing a combination of three episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and KLF4. Pluripotency was confirmed both in vivo and in vitro. The transcriptome profile of epiHUVEC and the human embryonic stem cell lineH1 have a Pearson correlation of 0.899.

  2. Generation of iPSC lines from primary human chorionic villi cells

    Directory of Open Access Journals (Sweden)

    Björn Lichtner

    2015-11-01

    Full Text Available Primary human chorionic villi (CV cells were used to generate the iPSC line by retroviral transduction of the four Yamanaka-factors OCT4, SOX2, KLF4 and c-MYC. Pluripotency was confirmed both in vivo and in vitro. The transcriptomes of the CV-derived iPSC lines and the human embryonic stem cell lines—H1 and H9 have a Pearson correlation of 0.929 and 0.943 respectively.

  3. Systems-level comparison of host responses induced by pandemic and seasonal influenza A H1N1 viruses in primary human type I-like alveolar epithelial cells in vitro

    Directory of Open Access Journals (Sweden)

    Guan Yi

    2010-10-01

    Full Text Available Abstract Background Pandemic influenza H1N1 (pdmH1N1 virus causes mild disease in humans but occasionally leads to severe complications and even death, especially in those who are pregnant or have underlying disease. Cytokine responses induced by pdmH1N1 viruses in vitro are comparable to other seasonal influenza viruses suggesting the cytokine dysregulation as seen in H5N1 infection is not a feature of the pdmH1N1 virus. However a comprehensive gene expression profile of pdmH1N1 in relevant primary human cells in vitro has not been reported. Type I alveolar epithelial cells are a key target cell in pdmH1N1 pneumonia. Methods We carried out a comprehensive gene expression profiling using the Affymetrix microarray platform to compare the transcriptomes of primary human alveolar type I-like alveolar epithelial cells infected with pdmH1N1 or seasonal H1N1 virus. Results Overall, we found that most of the genes that induced by the pdmH1N1 were similarly regulated in response to seasonal H1N1 infection with respect to both trend and extent of gene expression. These commonly responsive genes were largely related to the interferon (IFN response. Expression of the type III IFN IL29 was more prominent than the type I IFN IFNβ and a similar pattern of expression of both IFN genes was seen in pdmH1N1 and seasonal H1N1 infection. Genes that were significantly down-regulated in response to seasonal H1N1 but not in response to pdmH1N1 included the zinc finger proteins and small nucleolar RNAs. Gene Ontology (GO and pathway over-representation analysis suggested that these genes were associated with DNA binding and transcription/translation related functions. Conclusions Both seasonal H1N1 and pdmH1N1 trigger similar host responses including IFN-based antiviral responses and cytokine responses. Unlike the avian H5N1 virus, pdmH1N1 virus does not have an intrinsic capacity for cytokine dysregulation. The differences between pdmH1N1 and seasonal H1N1 viruses

  4. Transfection with extracellularly UV-damaged DNA induces human and rat cells to express a mutator phenotype towards parvovirus H-1

    Energy Technology Data Exchange (ETDEWEB)

    Dinsart, C.; Cornelis, J.J.; Klein, B.; van der Eb, A.J.; Rommelaere, J.

    1984-02-01

    Human and rat cells transfected with UV-irradiated linear double-stranded DNA from calf thymus displayed a mutator activity. This phenotype was identified by growing a lytic thermosensitive single-stranded DNA virus (parvovirus H-1) in those cells and determining viral reversion frequencies. Likewise, exogenous UV-irradiated closed circular DNAs, either double-stranded (simian virus 40) or single-stranded (phi X174), enhanced the ability of recipient cells to mutate parvovirus H-1. The magnitude of mutator activity expression increased along with the number of UV lesions present in the inoculated DNA up to a saturation level. Unirradiated DNA displayed little inducing capacity, irrespective of whether it was single or double stranded. Deprivation of a functional replication origin did not impede UV-irradiated simian virus 40 DNA from providing rat and human cells with a mutator function. Our data suggest that in mammalian cells a trans-acting mutagenic signal might be generated from UV-irradiated DNA without the necessity for damaged DNA to replicate.

  5. Vaccination for 2009 pandemic H1N1 influenza A did not induce conserved epitope-specific memory CD8 T cell responses in HIV+ northern Thai children.

    Science.gov (United States)

    Chawansuntati, Kriangkrai; Aurpibul, Linda; Wipasa, Jiraprapa

    2015-09-11

    The influenza virus causes severe illness in susceptible populations, including children and people living with human immunodeficiency virus (HIV). Here, we investigated cell-mediated immune responses (CMI) against influenza CD8 T cell conserved epitopes in HIV-infected (HIV+) northern Thai children following the 2009 pandemic H1N1 influenza A vaccination. Sixty HIV+ children were vaccinated with two doses of the 2009 pandemic influenza vaccine and their CD8T cell responses were assessed. We found no significant differences in the increase of cytokines-producing and CD107a-expressing CD8+ T cells or CD8+ memory T cells in response to pooled conserved epitopes stimulation in vitro between children with different serologic responses to the vaccine at all time points of the study. Our results suggest that the 2009 pandemic H1N1 vaccine did not induce the conserved epitope-specific immune responses in HIV+ children. Vaccine design and vaccination strategy against influenza in these populations warrant further studies.

  6. Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

    Science.gov (United States)

    Frandsen, Stine Krog; McNeil, Anna K; Novak, Ivana; McNeil, Paul L; Gehl, Julie

    2016-08-01

    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment.

  7. Entrapment of H1N1 Influenza Virus Derived Conserved Peptides in PLGA Nanoparticles Enhances T Cell Response and Vaccine Efficacy in Pigs.

    Directory of Open Access Journals (Sweden)

    Jagadish Hiremath

    Full Text Available Pigs are believed to be one of the important sources of emerging human and swine influenza viruses (SwIV. Influenza virus conserved peptides have the potential to elicit cross-protective immune response, but without the help of potent adjuvant and delivery system they are poorly immunogenic. Biodegradable polylactic-co-glycolic acid (PLGA nanoparticle (PLGA-NP based vaccine delivery system enhances cross-presentation of antigens by the professional antigen presenting cells. In this study, Norovirus P particle containing SwIV M2e (extracellular domain of the matrix protein 2 chimera and highly conserved two each of H1N1 peptides of pandemic 2009 and classical human influenza viruses were entrapped in PLGA-NPs. Influenza antibody-free pigs were vaccinated with PLGA-NPs peptides cocktail vaccine twice with or without an adjuvant, Mycobacterium vaccae whole cell lysate, intranasally as mist. Vaccinated pigs were challenged with a virulent heterologous zoonotic SwIV H1N1, and one week later euthanized and the lung samples were analyzed for the specific immune response and viral load. Clinically, pigs vaccinated with PLGA-NP peptides vaccine had no fever and flu symptoms, and the replicating challenged SwIV was undetectable in the bronchoalveolar lavage fluid. Immunologically, PLGA-NP peptides vaccination (without adjuvant significantly increased the frequency of antigen-specific IFNγ secreting CD4 and CD8 T cells response in the lung lymphocytes, despite not boosting the antibody response both at pre- and post-challenge. In summary, our data indicated that nanoparticle-mediated delivery of conserved H1N1 influenza peptides induced the virus specific T cell response in the lungs and reduced the challenged heterologous virus load in the airways of pigs.

  8. Higher Viral Load and Prolonged Viral Shedding Period is Associated with Impaired Th17 Cell Response in Patients with H1N1 Influenza A

    Institute of Scientific and Technical Information of China (English)

    Gui-lin; Yang; Ying-xia; Liu; Mu-tong; Fang; Wei-long; Liu; Xin-chun; Chen; John; Nunnari; Jing-jing; Xie; Ming-feng; Liao; Ming-xia; Zhang; Guo-bao; Li; Pei-ze; Zhang; Yi; Guan; Bo-ping; Zhou

    2012-01-01

    Objective To explore whether age,disease severity,cytokines and lymphocytes in H1N1 influenza A patients correlate with viral load and clearance.Methods Total of 70 mild and 16 severe patients infected with H1N1 influenza A virus were enrolled in this study.Results It was found that the patients under 14 years old and severe patients displayed significantly higher viral loads and prolonged viral shedding periods compared with the patients over 14 years old and mild patients,respectively(P < 0.05).Moreover,the patients under 14 years old and severe patients displayed significantly lower Th17 cell frequency than the patients over 14 years old and mild patients(P < 0.01).The viral shedding period inversely correlated with the frequency of IL-17+IFN-γ-CD4+ T cells.Additionally,the decreased concentration of serum TGF-β correlated with the decreased frequency of IL-17+IFN-γ-CD4+ T cells.Conclusions Both younger and severe patients are associated with higher viral loads and longer viral shedding periods,which may partially be attributed to the impaired Th17 cell response.

  9. 重型甲型H1N1流感患者外周血免疫细胞变化及其临床意义%Measurement of peripheral blood immune cells in severe influenza A (H1N1) patients and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    黄美先; 朱婧; 潘韵峰; 冷建杭

    2010-01-01

    Objective To observe the changes of blood lymphocyte in severe influenza A (H1N1) patients, and to investigate the immunopathogenesis of the influenza A( H1N1 ) patients and the clinical value of lymphocyte detection in influenza A (H1N1) diagnosis. Methods The changes of blood lymphocyte subs,ets (T cells, B cells, NK cells) were determined by the flow cytometer analysis in41 patients with serious and critical influenza A (H1N1) and 41 patients with mild influenza A(H1N1). Results The counts of total T cells, CD4+ T cells,CD8+ T cells, NK cells all significantly decreased in acute phase of serious and critical patients compared with those in mild patients (total T cells, CD8+T cells,NK cells, P<0.01;CD4+T cells, P<0.05). The counts of above cells in critical influenza A ( H1N1 ) patients were less than those in serious patients(P<0.01). As the conditions improved in serious and critical patients, the counts of total T cells, CD8+T cells and NK cells were still less than those in mild patients(total T cells, CD8+T cells, P<0.05; NK cells, P < 0.01 ). The counts of B cells were higher than that in mild individuals ( P<0.05). Conclus The patients with serious and critical influenza A (H1N1) are markedly impaired with cellular immunity.%目的 通过动态观察重型甲型H1N1流感(重症及危重症)患者外周血淋巴细胞的数量变化,探讨患者的免疫病理机制及淋巴细胞亚群检测在甲型H1N1流感诊断中的临床意义.方法 采用流式细胞技术检测2009年10-12月在我院住院治疗的41例重症、危重症甲型H1N1流感患者和同期41例轻症患者的外周血T、B和NK细胞数量.结果 在发病初期,重症及危重症患者外周血总T细胞、CD8+T细胞、NK细胞较轻症甲型H1N1流感患者显著降低(P<0.01),CD4+T细胞较轻症患者降低(P<0.05);危重症患者总T细胞、CD4+T细胞、CD8+T细胞、NK细胞低于重症患者(P<0.01).恢复期重症及危重症甲型H1N1

  10. Fully human broadly neutralizing monoclonal antibodies against influenza A viruses generated from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Weibin [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Chen, Aizhong [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Miao, Yi [Shanghai Xuhui Central Hospital, Shanghai 200031 (China); Xia, Shengli [Center for Disease Control and Prevention of Henan Province, Zhengzhou 450016 (China); Ling, Zhiyang; Xu, Ke; Wang, Tongyan [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Xu, Ying; Cui, Jun; Wu, Hongqiang; Hu, Guiyu; Tian, Lin; Wang, Lingling [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Shu, Yuelong [Chinese Center for Disease Control and Prevention, Beijing 102206 (China); Ma, Xiaowei [Hualan Biological Bacterin Company, Xinxiang 453003 (China); Xu, Bianli; Zhang, Jin [Center for Disease Control and Prevention of Henan Province, Zhengzhou 450016 (China); Lin, Xiaojun, E-mail: linxiaojun@hualan.com [Hualan Biological Bacterin Company, Xinxiang 453003 (China); Bian, Chao, E-mail: cbian@sibs.ac.cn [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Sun, Bing, E-mail: bsun@sibs.ac.cn [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)

    2013-01-20

    Whether the 2009 pandemic H1N1 influenza vaccine can induce heterosubtypic cross-protective anti-hemagglutinin (HA) neutralizing antibodies is an important issue. We obtained a panel of fully human monoclonal antibodies from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient. Most of the monoclonal antibodies targeted the HA protein but not the HA1 fragment. Among the analyzed antibodies, seven mAbs exhibited neutralizing activity against several influenza A viruses of different subtypes. The conserved linear epitope targeted by the neutralizing mAbs (FIEGGWTGMVDGWYGYHH) is part of the fusion peptide on HA2. Our work suggests that a heterosubtypic neutralizing antibody response primarily targeting the HA stem region exists in recipients of the 2009 pandemic H1N1 influenza vaccine. The HA stem region contains various conserved neutralizing epitopes with the fusion peptide as an important one. This work may aid in the design of a universal influenza A virus vaccine.

  11. Roles of histamine on the expression of aldehyde dehydrogenase 1 in endometrioid adenocarcinoma cell line.

    Science.gov (United States)

    Wang, Yi; Jiang, Yang; Ikeda, Jun-Ichiro; Tian, Tian; Sato, Atsushi; Ohtsu, Hiroshi; Morii, Eiichi

    2014-10-01

    Cancer-initiating cells (CICs) are a limited number of cells that are essential for maintenance, recurrence, and metastasis of tumors. Aldehyde dehydrogenase 1 (ALDH1) has been recognized as a marker of CICs. We previously reported that ALDH1-high cases of uterine endometrioid adenocarcinoma showed poor prognosis, and that ALDH1 high population was more tumorigenic, invasive, and resistant to apoptosis than ALDH1 low population. Histamine plays a critical role in cancer cell proliferation, migration, and invasion. Here, we examined the effect of histamine on ALDH1 expression in endometrioid adenocarcinoma cell line. The addition of histamine increased ALDH1 high population, which was consistent with the result that histamine enhanced the invasive ability and the resistance to anticancer drug. Among 4 types of histamine receptors, histamine H1 and H2 receptor (H1R and H2R) were expressed in endometrioid adenocarcinoma cell line. The addition of H1R agonist but not H2R agonist increased ALDH1. The antagonist H1R but not H2R inhibited the effect of histamine on ALDH1 expression. These results indicated that histamine increased the expression of ALDH1 via H1R but not H2R. These findings may provide the evidence for exploring a new strategy to suppress CICs by inhibiting ALDH1 expression with histamine.

  12. H-1 and N-15 resonance assignment of the second fibronectin type III module of the neural cell adhesion molecule

    DEFF Research Database (Denmark)

    Kiselyov, Vladislav V; Berezin, Vladimir; Bock, Elisabeth;

    2008-01-01

    We report here the NMR assignment of the second fibronectin type III module of the neural cell adhesion molecule (NCAM). This module has previously been shown to interact with the fibroblast growth factor receptor (FGFR), and the FGFR-binding site was mapped by NMR to the FG-loop region...... of the module. The FG-loop region also contains a putative nucleotide-binding motif, which was shown by NMR to interact with ATP. Furthermore, ATP was demonstrated to inhibit binding of the second F3 module of NCAM to FGFR....

  13. False leukemia-lymphoma cell lines: an update on over 500 cell lines.

    Science.gov (United States)

    Drexler, H G; Dirks, W G; Matsuo, Y; MacLeod, R A F

    2003-02-01

    Human leukemia-lymphoma (LL) cell lines represent an extremely important resource for research in a variety of fields and disciplines. As the cell lines are used as in vitro model systems in lieu of primary cell material, it is crucial that the cells in the culture flasks faithfully correspond to the purported objects of study. Obviously, proper authentication of cell line derivation and precise characterization are indispensable requirements to use as model systems. A number of studies has shown an unacceptable level of LL cell lines to be false. We present here the results of authenticating a comprehensively large sample (n = 550) of LL cell lines mainly by DNA fingerprinting and cytogenetic evaluation. Surprisingly, near-identical incidences (ca 15%) of false cell lines were observed among cell lines obtained directly from original investigators (59/395: 14.9%) and from secondary sources (23/155: 14.8%) implying that most cross-contamination is perpetrated by originators, presumably during establishment. By comparing our data with those published, we were further able to subclassify the false cell lines as (1) virtual: cross-contaminated with and unretrievably overgrown by other cell lines during initiation, never enjoying independent existence; (2) misidentified: cross-contaminated subsequent to establishment so that an original prototype may still exist; or (3) misclassified: unwittingly established from an unintended (often normal) cell type. Prolific classic leukemia cell lines were found to account for the majority of cross-contaminations, eg CCRF-CEM, HL-60, JURKAT, K-562 and U-937. We discuss the impact of cross-contaminations on scientific research, the reluctance of scientists to address the problem, and consider possible solutions. These findings provide a rationale for mandating the procurement of reputably sourced LL cell lines and their regular authentication thereafter.

  14. Over-expression of nm23-H1 in HeLa cells provides cells with higher resistance to oxidative stress possibly due to raising intracellular p53 and GPX1

    Institute of Scientific and Technical Information of China (English)

    Run AN; Yong-lie CHU; Chan TIAN; Xiao-xia DAI; Jing-hong CHEN; Qi SHI; Jun HAN; Xiao-ping DONG

    2008-01-01

    Aim: To determine whether the antitumor factor nm23 is related with antioxi-dation. Methods: Full-length human nm23-Hl was cloned into a mammalian-expressing vector and transiently introduced into HeLa cells. Results: A remark-ably low level of reactive oxygen species (ROS) was detected in the cells over-expressing nm23-Hl. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trypan blue assays found that the cells transfected with a nm23-H1-expressing plasmid had higher viability and stronger resistance to oxidative stress. Immunoprecipitation tests revealed that endogenous nm23-H1 formed a protein complex with p53. Furthermore, the intracellular levels of p53 and p53-regulated gene GPXI were obviously increased in the cells overexpressing nm23-H1. The downregulation of p53 in the cells overexpressing nm23-H1 resulted in a higher cellular ROS level and lower cell viability. Conclusion: The findings suggest that nm23-H1 may act as a cellular protector against oxidative stress, possibly triggering the p53-related antioxidative pathway.

  15. Virus Discovery Using Tick Cell Lines

    Science.gov (United States)

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

    While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks’ genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area. PMID:27679414

  16. Antiviral activity of Basidiomycete mycelia against influenza type A(serotype H1N1) and herpes simplex virus type 2 in cell culture

    Institute of Scientific and Technical Information of China (English)

    Tetiana; Krupodorova; Svetlana; Rybalko; Victor; Barshteyn

    2014-01-01

    In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.

  17. Antiviral activity of Basidiomycete mycelia against influenza type A (serotype H1N1) and herpes simplex virus type 2 in cell culture.

    Science.gov (United States)

    Krupodorova, Tetiana; Rybalko, Svetlana; Barshteyn, Victor

    2014-10-01

    In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A (serotype H1N1) and herpes simplex virus type 2 (HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47 (H1N1) in MDCK cells reducing the infectious titer by 2.0-6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species-Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes-this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index (324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes (amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.

  18. B7-H1在结直肠癌血管内皮细胞与内皮细胞中的差异性表达及机制%Differential expression of B7-H1 between colorectal carcinoma vascular endothelial cells and endothelial cells and the mechanism

    Institute of Scientific and Technical Information of China (English)

    施强; 郑勇斌; 罗海平; 刘克杰; 曹峰瑜; 丁瑜; 童仕伦

    2013-01-01

    目的 探讨共刺激分子B7-H1在结直肠癌血管内皮细胞(CCVEC)与内皮细胞(EC)中的差异性表达及机制.方法 应用免疫组织化学、实时定量聚合酶链反应(Real-time PCR)及Western blot等检测CCVEC与EC的B7-H1差异性表达,并通过白细胞介素(IL)-10、干扰素(IFN)-γ和双因子联合(BFC)的阳性与阴性干预来探讨其机制.差异性表达3组(CCVEC、EC和共培养成纤维细胞组),阳性干预4组(IL-10、IFN-γ、BFC、CCVEC组),阴性干预4组(IL-10中和组、IFN-γ中和组、BFC中和组、CCVEC组),干预前后分别检测EC的B7-H1表达.结果 与EC的阴性表达不同,CCVECB7-H1阳性细胞表达率为(75.00±17.41)%,中位染色评分为3.13;阳性干预IL-10、IFN-γ与BFC刺激EC后,其B7-H1表达率分别为(29.58±4.86)%、(32.08±5.08)%和(34.79±4.40)%,评分为1.46、1.58和1.71(P >0.05),但与CCVEC差异有统计学意义(P<0.05);阴性干预IL-10、IFN-γ与BFC中和后,EC B7-H1表达率分别为(36.21±4.05)%、(31.04±3.56)%和(7.02±2.31)%,评分为1.89、1.61、0.62(P <0.05).且B7-H1 mRNA与蛋白表达比较也证实了该结果.结论 B7-H1为区别EC与CCVEC的分子标志物之一,其表达与肿瘤微环境相关,IL-10和IFN-γ为微环境中的重要相关作用因子.%Objective To investigate the differential expression of costimulatory molecules B7-H1 between colorectal carcinoma vascular endothelial cells (CCVEC) and endothelial cells (EC) and the mechanism.Methods Immunohistochemistry,real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting were applied to detect the differential expression of B7-H1 between CCVEC and human umbilical vein endothelial cells (HUVEC).Intervened positively with interleukin (IL)-10 or interferon (IFN)-γ or Bi-factor combined (BFC) and negative handle with anti-IL-10 neutralization antibody or anti-IFN-γ neutralization antibody or BFC neutralization antibodies to investigate the differential

  19. Changes and analysis of peripheral white blood cells and lymphocyte subsets for patients with pandemic influenza A virus (H1N1) infection%甲型H1N1流感患者外周血白细胞和淋巴细胞亚群变化分析

    Institute of Scientific and Technical Information of China (English)

    陈威巍; 赵敏; 毛远丽; 谢杨新; 张云辉; 冯艳青; 李伯安; 李波; 洪炜; 刘佳; 马洪滨

    2010-01-01

    目的 探讨甲型H1N1流感患者白细胞和淋巴细胞亚群的变化特点,为甲型H1N1流感的诊断、治疗和预后判断提供实验室依据.方法 采用全血细胞分析和流式细胞分析法分别检测59例甲型H1N1流感患者急性期和恢复期的外周血白细胞及淋巴细胞亚群,与43名健康人和24例普通甲型流感患者比较.结果 甲型H1N1流感轻症患者白细胞总数在急性期显著下降,重症患者白细胞总数下降不显著,而中性粒细胞显著升高;与普通甲型流感相似,所有H1N1流感患者淋巴细胞总数、CD3、CD4、CD8百分比和绝对值在急性期大幅度下降,恢复期迅速回升;而重症患者NK和NKT细胞绝对值在急性期下降幅度超过20%.结论 甲型H1N1流感患者白细胞和淋巴细胞亚群变化与普通甲流相似;急性期NK、NKT细胞绝对值的大幅降低可以提示病情重症化倾向.%Objective To investigate the characters and changes of peripheral white blood cells and lymphocyte subsets of patients with pandemic influenza A virus (H1N1 ) infection and to provide evidences for diagnosis, treatment and prognosis of influenza A ( H1N1 ) infection. Methods Peripheral white blood cell parameters and the percentages of lymphocyte subsets in acute and recovery phases of 59 cases of influenza A virus (H1N1) infectious patients (42 mild cases and 17 severe cases)were investigated and analyzed, and compared respectively with those of 43 cases of healthy adults as control (HC) and 24 cases of general influenza A virus (no-H1N1) infectious using whole blood cell analysis and flow cytometry.Results Peripheral white blood cell counts of mild cases decreased greatly but those of severe cases did not decrease significantly; the neutrophils of severe cases increased significantly in acute phase; similar to general influenza A virus (no-H1N1) infectious, the peripheral lymphocytes, CD3, CD4, CD8 and B cells of all patients with influenza A virus (H1N1) infection

  20. TGF-β regulates LARG and GEF-H1 during EMT to affect stiffening response to force and cell invasion.

    Science.gov (United States)

    Osborne, Lukas D; Li, George Z; How, Tam; O'Brien, E Tim; Blobe, Gerard C; Superfine, Richard; Mythreye, Karthikeyan

    2014-11-05

    Recent studies implicate a role for cell mechanics in cancer progression. The epithelial-to-mesenchymal transition (EMT) regulates the detachment of cancer cells from the epithelium and facilitates their invasion into stromal tissue. Although classic EMT hallmarks include loss of cell-cell adhesions, morphology changes, and increased invasion capacity, little is known about the associated mechanical changes. Previously, force application on integrins has been shown to initiate cytoskeletal rearrangements that result in increased cell stiffness and a stiffening response. Here we demonstrate that transforming growth factor β (TGF-β)-induced EMT results in decreased stiffness and loss of the normal stiffening response to force applied on integrins. We find that suppression of the RhoA guanine nucleotide exchange factors (GEFs) LARG and GEF-H1 through TGF-β/ALK5-enhanced proteasomal degradation mediates these changes in cell mechanics and affects EMT-associated invasion. Taken together, our results reveal a functional connection between attenuated stiffness and stiffening response and the increased invasion capacity acquired after TGF-β-induced EMT.

  1. Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines.

    Science.gov (United States)

    Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

    2009-01-01

    The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines.

  2. Specificities and genomic distribution of somatic mammalian histone H1 subtypes.

    Science.gov (United States)

    Millán-Ariño, Lluís; Izquierdo-Bouldstridge, Andrea; Jordan, Albert

    2016-03-01

    Histone H1 is a structural component of chromatin that may have a role in the regulation of chromatin dynamics. Unlike core histones, the linker histone H1 family is evolutionarily diverse and many organisms have multiple H1 variants or subtypes, distinguishable between germ-line and somatic cells. In mammals, the H1 family includes seven somatic H1 variants with a prevalence that varies between cell types and over the course of differentiation, H1.1 to H1.5 being expressed in a replication-dependent manner, whereas H1.0 and H1X are replication-independent. Until recently, it has not been known whether the different variants had specific roles in the regulation of nuclear processes or were differentially distributed across the genome. To address this, an increasing effort has been made to investigate divergent features among H1 variants, regarding their structure, expression patterns, chromatin dynamics, post-translational modifications and genome-wide distribution. Although H1 subtypes seem to have redundant functions, several reports point to the idea that they are also differently involved in specific cellular processes. Initial studies investigating the genomic distribution of H1 variants have started to suggest that despite a wide overlap, different variants may be enriched or preferentially located at different chromatin types, but this may depend on the cell type, the relative abundance of the variants, the differentiation state of the cell, or whether cells are derived from a neoplastic process. Understanding the heterogeneity of the histone H1 family is crucial to elucidate their role in chromatin organization, gene expression regulation and other cellular processes.

  3. Orally administered Salacia reticulata extract reduces H1N1 influenza clinical symptoms in murine lung tissues putatively due to enhanced natural killer cell activity

    Directory of Open Access Journals (Sweden)

    Gustavo Adolfo Romero-Pérez

    2016-03-01

    Full Text Available Influenza is a major cause of respiratory tract infection. Although most cases do not require further hospitalization, influenza periodically causes epidemics in humans that can potentially infect and kill millions of people. To countermeasure this threat, new vaccines need to be developed annually to match emerging influenza viral strains with increased resistance to existing vaccines. Thus, there is a need for finding and developing new anti-influenza viral agents as alternatives to current treatments. Here, we tested the antiviral effects of an extract from the stems and roots of Salacia reticulata (SSRE, a plant rich in phytochemicals such as salacinol, kotalanol and catechins, on H1N1 influenza virus-infected mice. Following oral administration of 0.6 mg/day of SSRE, the incidence of coughing decreased in 80% of mice, and only one case of severe pulmonary inflammation was detected. Moreover, when compared with mice given Lactobacillus casei JCM1134, a strain previously shown to help increase in vitro NK cell activity, SSRE-administered mice showed greater and equal NK cell activity in splenocytes and pulmonary cells, respectively, at high effector cell:target cell ratios. Next, to test whether or not SSRE would exert protective effects against influenza in the absence of gut microbiota, mice were given antibiotics before being inoculated influenza virus and subsequently administered SSRE. SSRE administration induced an increase in NK cell activity in splenocytes and pulmonary cells at levels similar to those detected in mice not treated with antibiotics. Based on our results, it can be concluded that phytochemicals in the SSRE exerted protective effects against influenza infection putatively via modulation of the immune response including enhancement of NK cell activity, although some protective effects were not necessarily through modulation of gut microbiota. Further investigation is necessary to elucidate the molecular mechanisms

  4. Orally Administered Salacia reticulata Extract Reduces H1N1 Influenza Clinical Symptoms in Murine Lung Tissues Putatively Due to Enhanced Natural Killer Cell Activity

    Science.gov (United States)

    Romero-Pérez, Gustavo A.; Egashira, Masayo; Harada, Yuri; Tsuruta, Takeshi; Oda, Yuriko; Ueda, Fumitaka; Tsukahara, Takamitsu; Tsukamoto, Yasuhiro; Inoue, Ryo

    2016-01-01

    Influenza is a major cause of respiratory tract infection. Although most cases do not require further hospitalization, influenza periodically causes epidemics in humans that can potentially infect and kill millions of people. To countermeasure this threat, new vaccines need to be developed annually to match emerging influenza viral strains with increased resistance to existing vaccines. Thus, there is a need for finding and developing new anti-influenza viral agents as alternatives to current treatments. Here, we tested the antiviral effects of an extract from the stems and roots of Salacia reticulata (SSRE), a plant rich in phytochemicals, such as salacinol, kotalanol, and catechins, on H1N1 influenza virus-infected mice. Following oral administration of 0.6 mg/day of SSRE, the incidence of coughing decreased in 80% of mice, and only one case of severe pulmonary inflammation was detected. Moreover, when compared with mice given Lactobacillus casei JCM1134, a strain previously shown to help increase in vitro natural killer (NK) cell activity, SSRE-administered mice showed greater and equal NK cell activity in splenocytes and pulmonary cells, respectively, at high effector cell:target cell ratios. Next, to test whether or not SSRE would exert protective effects against influenza in the absence of gut microbiota, mice were given antibiotics before being inoculated influenza virus and subsequently administered SSRE. SSRE administration induced an increase in NK cell activity in splenocytes and pulmonary cells at levels similar to those detected in mice not treated with antibiotics. Based on our results, it can be concluded that phytochemicals in the SSRE exerted protective effects against influenza infection putatively via modulation of the immune response, including enhancement of NK cell activity, although some protective effects were not necessarily through modulation of gut microbiota. Further investigation is necessary to elucidate the molecular mechanisms

  5. Adjuvant effects of invariant NKT cell ligand potentiates the innate and adaptive immunity to an inactivated H1N1 swine influenza virus vaccine in pigs.

    Science.gov (United States)

    Dwivedi, Varun; Manickam, Cordelia; Dhakal, Santosh; Binjawadagi, Basavaraj; Ouyang, Kang; Hiremath, Jagadish; Khatri, Mahesh; Hague, Jacquelyn Gervay; Lee, Chang Won; Renukaradhya, Gourapura J

    2016-04-15

    Pigs are considered as the source of some of the emerging human flu viruses. Inactivated swine influenza virus (SwIV) vaccine has been in use in the US swine herds, but it failed to control the flu outbreaks. The main reason has been attributed to lack of induction of strong local mucosal immunity in the respiratory tract. Invariant natural killer T (iNKT) cell is a unique T cell subset, and activation of iNKT cell using its ligand α-Galactosylceramide (α-GalCer) has been shown to potentiate the cross-protective immunity to inactivated influenza virus vaccine candidates in mice. Recently, we discovered iNKT cell in pig and demonstrated its activation using α-GalCer. In this study, we evaluated the efficacy of an inactivated H1N1 SwIV coadministered with α-GalCer intranasally against a homologous viral challenge. Our results demonstrated the potent adjuvant effects of α-GalCer in potentiating both innate and adaptive immune responses to SwIV Ags in the lungs of pigs, which resulted in reduction in the lung viral load by 3 logs compared to without adjuvant. Immunologically, in the lungs of pigs vaccinated with α-GalCer an increased virus specific IgA response, IFN-α secretion and NK cell-cytotoxicity was observed. In addition, iNKT cell-stimulation enhanced the secretion of Th1 cytokines (IFN-γ and IL-12) and reduced the production of immunosuppressive cytokines (IL-10 and TGF-β) in the lungs of pigs⋅ In conclusion, we demonstrated for the first time iNKT cell adjuvant effects in pigs to SwIV Ags through augmenting the innate and adaptive immune responses in the respiratory tract.

  6. Radiation sensitivity of Merkel cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

    1995-07-30

    Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

  7. Stem cell characteristics in prostate cancer cell lines.

    NARCIS (Netherlands)

    Pfeiffer, M.J.; Schalken, J.A.

    2010-01-01

    BACKGROUND: Recent studies indicate the presence of a small, stem-like cell population in several human cancers that is crucial for the tumour (re)population. OBJECTIVE: Six established prostate cancer (PCa) cell lines-DU145, DuCaP, LAPC-4, 22Rv1, LNCaP, and PC-3-were examined for their stem cell pr

  8. Chicken interferon alpha pretreatment reduces virus replication of pandemic H1N1 and H5N9 avian influenza viruses in lung cell cultures from different avian species

    Directory of Open Access Journals (Sweden)

    Yang Hanchun

    2011-09-01

    Full Text Available Abstract Background Type I interferons, including interferon alpha (IFN-α, represent one of the first lines of innate immune defense against influenza virus infection. Following natural infection of chickens with avian influenza virus (AIV, transcription of IFN-α is quickly up regulated along with multiple other immune-related genes. Chicken IFN-α up regulates a number of important anti-viral response genes and has been demonstrated to be an important cytokine to establish anti-viral immunity. However, the mechanisms by which interferon inhibit virus replication in avian species remains unknown as does the biological activity of chicken interferon in other avian species. Methods In these studies, we assessed the protective potential of exogenous chicken IFN-α applied to chicken, duck, and turkey primary lung cell cultures prior to infection with the pandemic H1N1 virus (A/turkey/Virginia/SEP-4/2009 and an established avian H5N9 virus (A/turkey/Wisconsin/1968. Growth kinetics and induction of select immune response genes, including IFN-α and myxovirus-resistance gene I (Mx, as well as proinflammatory cytokines (IL-1β and IL-6, were measured in response to chicken IFN-α and viral infection over time. Results Results demonstrate that pretreatment with chicken IFN-α before AIV infection significantly reduced virus replication in both chicken-and turkey-origin lung cells and to a lesser degree the duck-origin cells. Virus growth was reduced by approximately 200-fold in chicken and turkey cells and 30-fold in duck cells after 48 hours of incubation. Interferon treatment also significantly decreased the interferon and proinflammatory response during viral infection. In general, infection with the H1N1 virus resulted in an attenuated interferon and proinflammatory response in these cell lines, compared to the H5N9 virus. Conclusions Taken together, these studies show that chicken IFN-α reduces virus replication, lower host innate immune

  9. nm23-H1基因和增殖细胞核抗原在肝癌组织中的表达及其与癌细胞DNA含量的关系%Relationship between nm23-H1, proliferating cell nuclear antigen expression and DNA content of human hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    赵新; 丛文铭; 谭璐; 吴孟超

    2001-01-01

    目的 探讨转移抑制基因nm23-H1在肝癌组织中的表达和癌细胞增殖及DNA含量间的关系。方法 应用免疫组织化学染色法检测56例肝癌组织标本中nm23-H1基因和增殖细胞核抗原(PCNA)的表达,应用图像分析系统测定癌细胞DNA含量,分析与肝癌病理生物学行为之间的关系。结果 nm23-H1表达阳性率无包膜组肝癌(29.6%)比包膜完整(64.3%)和包膜突破组肝癌(66.7%)明显减低(P<0.05)。DNA指数(DI)与肝癌包膜情况、组织类型、组织分级也有显著相关性(P<0.05)。nm23-H1表达阴性的肝癌P CNA标记指数(LI)高于nm23-H1阳性者(P<0.05);PCNA标记指数高增殖组肝癌D I值(2.30±0.90)较低增殖组肝癌DI值(1.86±0.7)明显增高(P<0.05)。结论 nm23-H1表达与肝癌包膜形成具有一定关系,并与肝癌增殖活性相关。DNA含量测定结合PCNA免疫组织化学染色可较为准确的反映肝癌浸润侵袭特征和增殖活性。%Objective To evaluate the relationship be tween expressions of nm23-H1, proliferating cell nuclear antigen (PCNA) in hepa tocellular carcinoma and DNA content of cancer cells. Methods The expression of nm23-H1 and PCNA were detected in 56 cases of HCC by us ing immunohistochemistry technique and DNA content of cancer cells were analyzed by DNA imaging analyses system as well in order to find their relationship with biopathologic characters of HCC. Results The expression levels of nm23-H1 in HCC without encapsulation (29.6%) were significantly reduc ed when compared with that with encapsulation (64.3%) or with incomplete capsula tion (66.7%,P<0.05). The increased DNA content of HCC was correlated with n on-encapsulation, compact type and high grade of the tumor (P<0.05). The labeling indexes of PCNA in the group with negative nm23-H1 expression of HCC w ere significantly higher than those in the group with positive nm23-H1 expressi on of HCC (P<0.05). The DI in

  10. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection

    OpenAIRE

    2011-01-01

    The 2009 pandemic H1N1 influenza pandemic demonstrated the global health threat of reassortant influenza strains. Herein, we report a detailed analysis of plasmablast and monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike antibodies elicited by annual influenza vaccinations, most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglutinin (HA) stalk and head domain of multiple influenza strains. T...

  11. Preparing photochromic nanofibers and animal cells using a photochromic compound of 1',3',3'-trimethyl-6-nitrospiro (2H-1-benzopyran-2,2'-indoline).

    Science.gov (United States)

    Li, Xiaoqiang; Lin, Lin; Kanjwal, Muzafar A; Chronakis, Ioannis S; Liu, Shuiping; Chen, Yanmo

    2012-01-01

    In this work, the photochromic compound 1',3',3'-trimethyl-6-nitrospiro (2H-1-benzopyran-2,2'-indoline) (NOSP) was synthesized by a two step process. The photochromic properties of NOSP were investigated by ultraviolet-visible (UV-Vis) spectrophotometry. The results showed that NOSP was very sensitive to UV irradiation with absorption peaks at about 336 nm and 567 nm. Our hypothesis was that both photochromic nanofibers and photochromic living animal cells could be obtained by combining them with NOSP. To test the hypothesis, photochromic nanofibers were fabricated by electrospinning from various mixed solutions of NOSP and polymers (including a synthetic polymer of poly(methyl methacrylate) and a natural polymer of gelatin); NOSP/ethanol solution was dissolved in culture medium to stain pig iliac endothelial cells (PIEC) and endow them with photochromic capability. Polymer nanofibers from electrospinning were characterized by water contact angle measurements, ultraviolet-visible (UV-Vis) spectrophotometry and fluorescence microscopy. Morphology of photochromic PIEC was observed by fluorescence microscopy after being irradiated. It was shown that nanofibers from electrospun polymers and NOSP-treated PIEC had photochromic properties. The bio-toxicity of the photochromic compound was also evaluated and it was shown that ~50% of PIEC remained viable for at least 20 min. The photochromic compound NOSP could be a potentially powerful tool for development of multi-functional nanofibers and biological applications.

  12. Microarray analysis of MicroRNA expression in peripheral blood mononuclear cells of critically ill patients with influenza A (H1N1)

    Science.gov (United States)

    2013-01-01

    Background With concerns about the disastrous health and economic consequences caused by the influenza pandemic, comprehensively understanding the global host response to influenza virus infection is urgent. The role of microRNA (miRNA) has recently been highlighted in pathogen-host interactions. However, the precise role of miRNAs in the pathogenesis of influenza virus infection in humans, especially in critically ill patients is still unclear. Methods We identified cellular miRNAs involved in the host response to influenza virus infection by performing comprehensive miRNA profiling in peripheral blood mononuclear cells (PBMCs) from critically ill patients with swine-origin influenza pandemic H1N1 (2009) virus infection via miRNA microarray and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assays. Receiver operator characteristic (ROC) curve analysis was conducted and area under the ROC curve (AUC) was calculated to evaluate the diagnostic accuracy of severe H1N1 influenza virus infection. Furthermore, an integrative network of miRNA-mediated host-influenza virus protein interactions was constructed by integrating the predicted and validated miRNA-gene interaction data with influenza virus and host-protein-protein interaction information using Cytoscape software. Moreover, several hub genes in the network were selected and validated by qRT-PCR. Results Forty-one significantly differentially expressed miRNAs were found by miRNA microarray; nine were selected and validated by qRT-PCR. QRT-PCR assay and ROC curve analyses revealed that miR-31, miR-29a and miR-148a all had significant potential diagnostic value for critically ill patients infected with H1N1 influenza virus, which yielded AUC of 0.9510, 0.8951 and 0.8811, respectively. We subsequently constructed an integrative network of miRNA-mediated host-influenza virus protein interactions, wherein we found that miRNAs are involved in regulating important pathways, such as mitogen

  13. Effect of Quercetin on Caspase-3 of A549 Cell Induced by Influenza Virus H1N1%槲皮素对甲型H1N1流感病毒诱导的A549细胞凋亡效应酶Caspase-3的影响

    Institute of Scientific and Technical Information of China (English)

    万巧凤; 吴莉; 杨美玲; 马锐; 梁军; 顾立刚

    2011-01-01

    Objective To investigate the influence of quercetin on Caspase-3 of lung epithelial tumour A549 infected with H1N1. Methods MTT method was adopted to determine H1N1 virulence, quercetin cytotoxicity, inhibitory effect of quercetin on A549 cytopathic with H1N1 cause. H1N1 of 100 TCID50 was used to infect A549 for 2 h and then change quercetin fluid containing 10 mg/L to cultivate. After 4, 12, 24, 48 h, cells were collected to extract total protein. By adopting the Western-blot method and applying Image-Pro Plus, the grayscale value of Caspase-3 and fl-actin were determined. The gray ratio was relative expression quantity of Caspase-3. Results The TCIDso of H1N1 to A549 was IO-4175, the highest non-toxic concentration of quercetin to A549 (Tco) was 40 mg/L, the least effective concentration (MEC) of quercetin inhibition to A549 cytopathic with H1N1 cause was 10 mg/L. After the infection of H1N1, quercetin significantly inhibited Caspase-3 expression within 4-48 h, showing that quercetin plays an obvious antiapoptotic role. Conclusion Quercetin can play antiviral infectious functions by inhibiting the content or activity of Caspase-3.%目的 观察银杏叶主要活性成分槲皮素对甲型H1N1流感病毒感染的人肺上皮瘤细胞A549凋亡效应酶Caspase-3的影响.方法 采用MTT法测定H1N1毒力、槲皮素细胞毒性作用、槲皮素对H1N1致A549细胞病变的抑制作用.然后用100 TCID50甲型H1N1感染A549 2 h后换用含10 mg/L槲皮素维持液继续培养,于感染后4、12、24、48 h收集细胞,提取细胞总蛋白,采用Western blot方法,应用Image-Pro Plus测定Caspase-3与β-actin灰度值,两者灰度比值为Caspase-3的相对表达量.结果 甲型H1N1 A549的TCID50为10-4.75,槲皮素A549的最大无毒浓度为40 mg/L,槲皮素抑制甲型H1N1致A549细胞病变的最小有效浓度为10 mg/L.在甲型H1N1感染A549细胞后4~ 48 h内,槲皮素可显著抑制Caspase-3蛋白表达,具有明显的抗凋亡作用.结论

  14. Cellular radiosensitivity of small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Krarup, M; Poulsen, H S; Spang-Thomsen, M

    1997-01-01

    PURPOSE: The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based...

  15. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

    Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

    2009-01-01

    compare susceptibility between cell lines and between lineages within a laboratory and between laboratories (Inter-laboratory Proficiency Test). The objective being that the most sensitive cell line and lineages are routinely selected for diagnostic purposes.In comparing cell lines, we simulated "non......-cell-culture-adapted" virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at - 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...

  16. Induced pluripotent stem cell lines derived from human somatic cells.

    Science.gov (United States)

    Yu, Junying; Vodyanik, Maxim A; Smuga-Otto, Kim; Antosiewicz-Bourget, Jessica; Frane, Jennifer L; Tian, Shulan; Nie, Jeff; Jonsdottir, Gudrun A; Ruotti, Victor; Stewart, Ron; Slukvin, Igor I; Thomson, James A

    2007-12-21

    Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

  17. Derivation and characterization of Chinese human embryonic stem cell line with high potential to differentiate into pancreatic and hepatic cells

    Institute of Scientific and Technical Information of China (English)

    SHI Cheng; SHEN Huan; JIANG Wei; SONG Zhi-hua; WANG Cheng-yan; WEI Li-hui

    2011-01-01

    Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background,which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use,especially in China due to its large population. This study contributes to isolating new Chinese human embryonic stem cell lines with clarified directly differentiation ability.Methods Donated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer (IVF-ET) center were collected to establish human embryonic stem cells lines with informed consent. The classic growth factors of basic fibroblast growth factor (bFGF) and recombinant human leukaemia inhibitory factor (hLIF) for culturing embryonic stem cells were used to capture the stem cells from the plated embryos. Mechanical and enzymetic methods were used to propogate the newly established human embryonic stem cells line. The new cell line was checked for pluripotent characteristics with detecting the expression of stemness genes and observing spontaneous differentiation both in vitro and in vivo. Finally similar step-wise protocols from definitive endoderm to target specific cells were used to check the cell line's ability to directly differentiate into pancreatic and hepatic cells.Results We generated a new Chinese human embryonic stem cells line,CH1. This cell line showed the same characteristics as other reported Chinese human embryonic stem cells lines:normal morphology,karyotype and pluripotency in vitro and in vivo. The CH1 cells could be directly differentiated towards pancreatic and hepatic cells with equal efficiency compared to the H1 cell line.Conclusions This newly established Chinese cell line,CH1,which is pluripotent and has high potential to differentiate into pancreatic and hepatic cells,will provide

  18. Stable expression of human H1-histamine-receptor cDNA in Chinese hamster ovary cells. Pharmacological characterisation of the protein, tissue distribution of messenger RNA and chromosomal localisation of the gene.

    Science.gov (United States)

    Moguilevsky, N; Varsalona, F; Noyer, M; Gillard, M; Guillaume, J P; Garcia, L; Szpirer, C; Szpirer, J; Bollen, A

    1994-09-01

    A cDNA clone for the histamine H1 receptor was isolated from a human lung cDNA library; it encoded a protein of 487 amino acids which showed characteristic features of G-protein-coupled receptors. The percentages of identity of the deduced amino acid sequence with bovine, rat and guinea pig H1 histamine receptors were 82.6%, 79.4% and 73.3%, respectively, whereas these percentages decreased to 74.6%, 66% and 56.7% for the amino acid sequence of the third intracellular loop. The human H1-receptor cDNA was transfected into Chinese hamster ovary cells (CHO) via an eukaryotic expression vector; the receptor protein present on cell membranes specifically bound [3H]mepyramine with a Kd of 3.7 nM. The binding was displaced by H1-histamine-receptor antagonists and histamine. Northern blot analysis indicated the presence of two histamine H1 receptor mRNAs of 3.5 kb and 4.1 kb in various human tissues and an additional mRNA of 4.8 kb restricted to the human brain. Finally, by means of somatic cell hybrids segregating either human or rat chromosomes, the gene for histamine H1 receptor was found to reside on human chromosome 3 and rat chromosome 4.

  19. NY-ESO-1-specific circulating CD4+ T cells in ovarian cancer patients are prevalently T(H)1 type cells undetectable in the CD25+ FOXP3+ Treg compartment.

    Science.gov (United States)

    Redjimi, Nassima; Duperrier-Amouriaux, Karine; Raimbaud, Isabelle; Luescher, Immanuel; Dojcinovic, Danijel; Classe, Jean-Marc; Berton-Rigaud, Dominique; Frenel, Jean-Sébastien; Bourbouloux, Emmanuelle; Valmori, Danila; Ayyoub, Maha

    2011-01-01

    Spontaneous CD4(+) T-cell responses to the tumor-specific antigen NY-ESO-1 (ESO) are frequently found in patients with epithelial ovarian cancer (EOC). If these responses are of effector or/and Treg type, however, has remained unclear. Here, we have used functional approaches together with recently developed MHC class II/ESO tetramers to assess the frequency, phenotype and function of ESO-specific cells in circulating lymphocytes from EOC patients. We found that circulating ESO-specific CD4(+) T cells in EOC patients with spontaneous immune responses to the antigen are prevalently T(H)1 type cells secreting IFN-γ but no IL-17 or IL-10 and are not suppressive. We detected tetramer(+) cells ex vivo, at an average frequency of 1:25,000 memory cells, that is, significantly lower than in patients immunized with an ESO vaccine. ESO tetramer(+) cells were mostly effector memory cells at advanced stages of differentiation and were not detected in circulating CD25(+)FOXP3(+)Treg. Thus, spontaneous CD4(+) T-cell responses to ESO in cancer patients are prevalently of T(H)1 type and not Treg. Their relatively low frequency and advanced differentiation stage, however, may limit their efficacy, that may be boosted by immunogenic ESO vaccines.

  20. 无血清微载体培养MDCK细胞和甲型流感病毒H1N1的条件优化%Optimizing the serum- free micro- carrier culture condition of MDCK cells and influenza Virus H1N1

    Institute of Scientific and Technical Information of China (English)

    张严予; 马磊; 沈霏; 周文晶; 周玉波; 王微; 姜述德; 李卫东; 廖国阳

    2011-01-01

    The article is about the optimum culture conditions for micro - carrier cultivating MDCK cells and influenza virus H1N1 proliferation in Serum - Free Medium, which is the basement for the cell influenza vaccine in the future.We cultivated MDCK cell in VP - SFM,detected the effect of MDCK cell growth with different concentration of cell inoculation, then cultivated influenza virus H1N1 in MDCK cell and also detected the effect of different pH value,TPCK -Trypsin content,MOI,whether or not adding TPCK -Trypsin after inoculating influenza virus and different virus harvest time.When the cell quantity is 1.0 x 10 mL-1 ,MDCK cell can grow for 5-6 days,which cell status are in good condition.After inoculating influenza virus H1N1 to MDCK cells,when pH value is between 7.2—7.4 ,TPCK - Trypsin content is 1.0 μg/mL, MOI is 1.0, and virus harvest time is 72 h, the hemagglutination titer is up to 512.In the selected suitable conditions, we could use microcarrier culture method for cultivation of MDCK cells and influenza virus H1N1 in Serum -Free Medium on a large scale in the future.%血清微载体培养MDCK细胞,并接种流感病毒H1N1,优化培养条件,为细胞流感疫苗的工艺研发奠定基础.采用不同的细胞接种量在50mL无血清微载体搅拌瓶中培养MDCK细胞,并接种甲型流感病毒H1N1,检测不同pH值和TPCK-胰酶含量的病毒培养液,不同病毒接种量,补加TPCK-胰酶,以及不同收毒时间对血凝效价的影响.以1.0×105 mL-1的MDCK细胞数量接种到无血清微载体上,病毒培养液pH值为7.2~7.4范围内,TPCK-胰酶质量浓度为1.0μg/mL,接种后不补加胰酶,病毒接种量MOI=1.0,并在72 h收获,最高血凝效价达到512.由此获得了在无血清为载体上培养MDCK细胞和甲型流感病毒H1N1的适宜条件.

  1. 77 FR 5489 - Identification of Human Cell Lines Project

    Science.gov (United States)

    2012-02-03

    ... Genetics Group Web site at http://www.nist.gov/mml/biochemical/genetics/index.cfm . Once the total number... methods for human cell line authentication the identity of a cell line need no longer be in doubt. NIST...

  2. Expression of Cyclooxygenase-2 in Ovarian Cancer Cell Lines

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To investigate the expression of cyclooxygenase-2 (COX-2) in ovarian cancer cell lines,RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a basis for the chemoprevention of ovarian cancer.

  3. Transcription analysis of TIMP-1 and NM23-h1 genes in glioma cell invasion Análise transcricional dos genes TIMP-1 e NM23-H1 na invasão celular em astrocitoma difuso e glioblastoma multiforme

    Directory of Open Access Journals (Sweden)

    José Augusto Nasser

    2006-09-01

    Full Text Available PURPOSE: To evaluate using transcription analysis the presence and importance of two genes: NM23-H1 and TIMP-1 on control of tumor cell invasion in diffuse astrocytomas (WHO II and glioblastoma multiforme (WHO IV. METHOD: Northern blot analysis of NM23-H1 and TIMP-1 was performed. Eight diffuse astrocytomas and 19 glioblastomas (WHO IV were analyzed to determine if TIMP-1 and NM23-H1 were candidates to inhibition of tumor cell invasion quantitated RNA levels. The samples were collected directly from operating room. Total cellular RNA was extracted from frozen tissue samples using guanidinium-isothiocyanate and cesium chloride gradients. Total RNA (10 mg per sample from tumor tissue were size fractionated through 1% agarose-formaldehyde gel and transferred to nylon filters and then hybridized to 32P-labeled DNA probes and placed for autoradiography. Levels of specific RNAs were determined by computer-assisted laser densitometry. Blot filters were sequentially hybridized to nm23 and TIMP-1 probes in addition to GAPDH, as a control. Statistical analyses were carried out according to t-test for equality of means. RESULTS: NM23-H1 were detected in each sample, however it did not correlate with malignancy and invasiveness. On the other side TIMP-1 gene expression showed a clear correlation between low expression and invasiveness. CONCLUSION: The data suggest that TIMP-1 is an inhibitor of high grade gliomas invasion. NM23-H1 was present in the entire gliomas sample, but it did not vary in diffuse astrocytomas and glioblastomas.OBJETIVO: Comparar através da análise da expressão dos níveis de RNA, a presença e a relevância dos genes NM23-H1 e TIMP-1 no controle da invasão celular tumoral dentro do tecido cerebral normal em: astrocitoma difuso (OMS II e glioblastoma multiforme (OMS:IV. MÉTODO: Análise em "Northern blot" dos genes NM23-H1 e TIMP-1. Oito astrocitomas fibrilares difusos (OMS II e 19 glioblastomas multiformes foram analisados para

  4. Radiosensitivity of Human Melanoma Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Bergoc, R. M.; Medina, V.; Cricco, G.; Mohamed, N.; Martin, G.; Nunez, M.; Croci, M.; Crescenti, E. J.; Rivera, E. S.

    2004-07-01

    Cutaneous melanoma is a skin cancer resulting from the malign transformation of skin-pigment cells, the melanocytes. The radiotherapy, alone or in combination with other treatment, is an important therapy for this disease. the objective of this paper was to determine in vitro the radiosensitivity of two human melanoma cell lines with different metastatic capability: WM35 and MI/15, and to study the effect of drugs on radiobiological parameters. The Survival Curves were adjusted to the mathematical Linear-quadratic model using GrapsPad Prism software. Cells were seeded in RPMI medium (3000-3500 cells/flask), in triplicate and irradiated 24 h later. The irradiation was performed using an IBL 437C H Type equipment (189 TBq, 7.7 Gy/min) calibrated with a TLD 700 dosimeter. The range of Doses covered from 0 to 10 Gy and the colonies formed were counted at day 7th post-irradiation. Results obtained were: for WM35, {alpha}=0.37{+-}0.07 Gy''-1 and {beta}=0.06{+-}0.02 Gy''-2, for M1/15m {alpha}=0.47{+-}0.03 Gy''-1 and {beta}=0.06{+-}0.01 Gy''-2. The {alpha}/{beta} values WM35: {alpha}/{beta} values WM35: {alpha}/{beta}=6.07 Gy and M1/15: {alpha}/{beta}{sub 7}.33 Gy were similar, independently of their metastatic capabillity and indicate that both lines exhibit high radioresistance. Microscopic observation of irradiated cells showed multinuclear cells with few morphologic changes non-compatible with apoptosis. By means of specific fluorescent dyes and flow cytometry analysis we determined the intracellular levels of the radicals superoxide and hydrogen peroxide and their modulation in response to ionizing radiation. The results showed a marked decreased in H{sub 2}O{sub 2} intracellular levels with a simultaneous increase in superoxide that will be part of a mechanism responsible for induction of cell radioresistance. This response triggered by irradiated cells could not be abrogated by different treatments like histamine or the

  5. High proportions of regulatory B and T cells are associated with decreased cellular responses to pH1N1 influenza vaccine in HIV-infected children and youth (IMPAACT P1088).

    Science.gov (United States)

    Weinberg, Adriana; Muresan, Petronella; Fenton, Terence; Richardson, Kelly; Dominguez, Teresa; Bloom, Anthony; Petzold, Elizabeth; Anthony, Patricia; Cunningham, Coleen K; Spector, Stephen A; Nachman, Sharon; Siberry, George K; Handelsman, Edward; Flynn, Patricia M

    2013-05-01

    HIV-infected individuals have poor responses to inactivated influenza vaccines. To evaluate the potential role of regulatory T (Treg) and B cells (Breg), we analyzed their correlation with humoral and cell-mediated immune (CMI) responses to pandemic influenza (pH1N1) monovalent vaccine in HIV-infected children and youth. Seventy-four HIV-infected, 4- to 25-y old participants in a 2-dose pH1N1 vaccine study had circulating and pH1N1-stimulated Treg and Breg measured by flow cytometry at baseline, post-dose 1 and post-dose 2. Concomitantly, CMI was measured by ELISPOT and flow cytometry; and antibodies by hemagglutination inhibition (HAI). At baseline, most of the participants had pH1N1-specific IFNγ ELISPOT responses, whose magnitude positively correlated with the baseline pH1N1, but not with seasonal H1N1 HAI titers. pH1N1-specific IFNγ ELISPOT responses did not change post-dose 1 and significantly decreased post-dose 2. In contrast, circulating CD4+CD25+% and CD4+FOXP3+% Treg increased after vaccination. The decrease in IFNγ ELISPOT results was marginally associated with higher pH1N1-specific CD19+FOXP3+ and CD4+TGFβ+% Breg and Treg, respectively. In contrast, increases in HAI titers post-dose 1 were associated with significantly higher circulating CD19+CD25+% post-dose 1, whereas increases in IFNγ ELISPOT results post-dose 1 were associated with higher circulating CD4+/C8+CD25+FOXP3+%. In conclusion, in HIV-infected children and youth, influenza-specific Treg and Breg may contribute to poor responses to vaccination. However, robust humoral and CMI responses to vaccination may result in increased circulating Treg and/or Breg, establishing a feed-back mechanism.

  6. Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.

    Science.gov (United States)

    Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

    2014-01-01

    Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

  7. B7-H1基因修饰的调节性DC对小鼠甲状腺相关性眼病的治疗作用%Inhibition effect of B7-H1 gene-modified regulatory dendritic cells on thyroid -associated ophthalmopathy in mice

    Institute of Scientific and Technical Information of China (English)

    陈华新; 邵伯棕; 陈宣辰; 周维明; 张意

    2014-01-01

    目的:构建表达小鼠B7-H1基因的腺病毒载体,转染修饰树突状细胞,并研究该细胞对小鼠甲状腺相关性眼病( thyroid-associated ophthalmopathy,TAO)的治疗效应。方法:设计并构建小鼠B7-H1的腺病毒表达载体,转染小鼠骨髓来源的树突状细胞,检测该树突状细胞的表型和功能,鉴定其对免疫应答的负向调控能力,采用实验动物模型观察B7-H1基因修饰的树突状细胞在体内治疗TAO的效果。  结果:成功构建出具有良好B7-H1表达效力的腺病毒载体,病毒滴度为1.8伊109 PFU/mL,转染腺病毒的小鼠骨髓来源的树突状细胞表现出调节性树突状细胞性能,能够负向抑制免疫应答;在动物模型中使用该型树突状细胞可以有效控制甲状腺眼病的发生发展。  结论:成功构建了表达小鼠B7-H1基因的腺病毒表达载体,转染了该载体的树突状细胞具有调节性树突状细胞的性能,抑制正向免疫应答,能够有效抑制甲状腺眼病的发生发展,揭示基因修饰的树突状细胞可能成为治疗甲状腺眼病的潜在生物制剂。%AIM:To construct adenovirus vector expressing mice B7-H1 gene, transfect dendritic cells ( DCs ) , and to study the therapeutic effect of modified DC on thyroid-associated ophthalmopathy ( TAO) in mice. METHODS: We designed and constructed B7-H1 gene adenovirus expression vector, and transfected DCs from mouse bone marrow, tested the phenotype and function of modified DCs, identificated its negative regulation to immune responses. The modified DCs were infected the sicked mice. And then the immunotherapeutic effect of modified DCs to TAO were tested. RESULTS: B7 - H1 gene adenovirus vector was constructed and transfected DCs from bone marrow. The titer of the recombinant adenovirus was 1. 8í109 PFU/mL. B7-H1 gene modified DCs characteristics of regulatory DCs, could inhibit positive immune responses. The inhibition proceeding of TAO into mice

  8. H1抗组胺药%H1-antihistamines

    Institute of Scientific and Technical Information of China (English)

    张罗; 韩德民

    2013-01-01

    While histamine plays an important role in the pathogenesis of allergic diseases, such as allergic rhinitis, H1-antihistamines, which have been using in the treatment of allergic diseases for more than 70 years, are considered as the cornerstone of the medication of allergic diseases. In this review, we discuss the history of histamine studies and anti - histamine discovery, the histamine receptors, as well as the mechanisms and the safety of H1-antihistamines.

  9. EXPRESSION OF Fas LIGAND IN HUMAN COLON CANCER CELL LINES

    Institute of Scientific and Technical Information of China (English)

    张建军; 丁尔迅; 王强; 陈学云; 付志仁

    2001-01-01

    To investigate the expression of Fas ligand in human colon carcinoma cell lines. Methods: A total of six human colon cancer cell lines were examined for the expression of Fas ligand mRNA and cell surface protein by using RT-PCR and flow cytometry respectively. Results: The results showed that Fas ligand mRNA was expressed in all of the six cancer cell lines and Fas ligand cell surface protein was expressed in part of them. Conclusion: These data suggest that Fas ligand was expressed, at least in part, in human colon cancer cell lines and might facilitate to escape from immune surveillance of the host.

  10. Biological characteristics of cell lines of human dental alveolus

    Institute of Scientific and Technical Information of China (English)

    陈世璋; 黄靖香; 孙明学; 赵斌

    2003-01-01

    Objective To investigate the biological characteristics of cell lines of healthy and diseased human dental alveoli. Methods Primary cell lines from either healthy or diseased human dental alveoli were obtained. Two cell lines, H-258 and H-171 derived from healthy and diseased human tissues respectively, were selected for morphological study and research on their growth and aging, using cell counting, and histochemical and immunohistochemical staining. Results Primary cell lines were successfully established from innormal dental alveoli. After freezing and thawing for three times, cell growth was continued and no morphological alterations were observed. The doubling time was 53.4 hours and mean division index (MDI) was 4‰. Cells were kept normal after twenty generations with no obvious reduction of doubling time and MDI. Of twenty-six primary cell lines derived from healthy human dental alveoli, only three cell lines achieved generation. After freezing and thawing for twice, cultured cells were still alive at a decreased growth speed, with doubling time of 85.9 hours and MDI of 3‰. Both cell lines, H-171 and H-258, shared the characteristics of osteoblast. Conclusions Primary cell lines of diseased human dental alveoli show greater growth potential. All cell lines of dental alveoli share characteristics of osteoblast. The technique we developed may be put into practice for the treatment of abnormal dental alveoli.

  11. Molluscan cells in culture: primary cell cultures and cell lines

    OpenAIRE

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as bi...

  12. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.

    1981-05-01

    The in vitro radiobiologic survival values (anti n, D/sub 0/) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL60 promyelocytic leukemia; K562 erythroleukemia; 45 acute lymphocytic leukemia; and 176 acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

  13. Cloning of aminopeptidase Npromoter and its activity in hematopoietic cell and different tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Aminopeptidase N (APN) promoter region was cloned and sequenced from peripheral blood mononuclear cells. The recombinant reporter construct containing the promoter and luciferase gene, designated pXP1-APNLuc, was introduced into myeloblastic cell line, T lymphocyte cell line and various tumor cell lines. Luciferase assay showed that APN upstream promoter is myeloid-specific for high expression in myeloblastic cell line and much lower expres sion in T lymphocyte cell line. The promoter activity was relatively high in lung adenoma cell line compared with other tumor cell lines including hepatoma cell line, tong cancer cell line and esophageal cancer cell line in which the promoter activity significantly diminished or was almost undetectable. The characteristics of APN promoter may pro vide a new strategy for specific myeloprotection while tumor patients are being treated with chemotherapy and/or radio therapy.

  14. Analysis list: Suv39h1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Suv39h1 Pluripotent stem cell + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Suv...39h1.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Suv39h1.5.tsv http://dbarchive.bi...osciencedbc.jp/kyushu-u/mm9/target/Suv39h1.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Suv39h

  15. MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

    NARCIS (Netherlands)

    KOLODZIEJ, H; HABERLAND, C; WOERDENBAG, HJ; KONINGS, AWT

    1995-01-01

    In the present study the cytotoxicity of 16 proanthocyanidins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values ranging from 18 to >200 mu m following continuous

  16. Epitope of titin A-band-specific monoclonal antibody Tit1 5 H1.1 is highly conserved in several Fn3 domains of the titin molecule. Centriole staining in human, mouse and zebrafish cells

    Directory of Open Access Journals (Sweden)

    Mikelsaar Aavo-Valdur

    2012-09-01

    Full Text Available Abstract Background Previously we have reported on the development of a new mouse anti-titin monoclonal antibody, named MAb Titl 5 H1.1, using the synthetic peptide N-AVNKYGIGEPLESDSVVAK-C which corresponds to an amino acid sequence in the A-region of the titin molecule as immunogen. In the human skeletal muscles, MAb Titl 5 H1.1 reacts specifically with titin in the A-band of the sarcomere and in different non-muscle cell types with nucleus and cytoplasm, including centrioles. In this report we have studied the evolutionary aspects of the binding of MAb Tit1 5 H1.1 with its target antigen (titin. Results We have specified the epitope area of MAb Tit1 5 H1.1 by subpeptide mapping to the hexapeptide N-AVNKYG-C. According to protein databases this amino acid sequence is located in the COOH-terminus of several different Fn3 domains of the A-region of titin molecule in many organisms, such as human being, mouse, rabbit, zebrafish (Danio rerio, and even in sea squirt (Ciona intestinalis. Our immunohisto- and cytochemical studies with MAb Tit1 5 H1.1 in human, mouse and zebrafish tissues and cell cultures showed a striated staining pattern in muscle cells and also staining of centrioles, cytoplasm and nuclei in non-muscle cells. Conclusions The data confirm that titin can play, in addition to the known roles in striated muscle cells also an important role in non-muscle cells as a centriole associated protein. This phenomenon is highly conserved in the evolution and is related to Fn3 domains of the titin molecule. Using titin A-band-specific monoclonal antibody MAb Tit1 5 H1.1 it was possible to locate titin in the sarcomeres of skeletal muscle cells and in the centrioles, cytoplasm and nuclei of non-muscle cells in phylogenetically so distant organisms as Homo sapiens, Mus musculus and zebrafish (Danio rerio.

  17. POTENTIAL CELL LINE TOXICITY OF ENVIRONMENTAL NANOPARTICLES

    Directory of Open Access Journals (Sweden)

    Mohan Durga

    2012-01-01

    Full Text Available In India, the unprecedented growth rate and urbanization along with the rapid increase in motor vehicle activity and industrialization are contributing to high levels of urban air pollution. The population is mainly exposed to high air pollution concentrations, where motor vehicle emissions constitute the main source of fine and ultrafine particles. Motor exhaust emissions is a mixture of gases and Particulate Matter (PM. Diesel and petrol fuels in vehicles produce combustion-derived particles as a result of combustion. Vehicle exhaust particles are the main constituents of environmental nanoparticles. In the present investigation, environmental nanoparticles such as Diesel Exhaust Particles (DEP and Petrol Exhaust Particles (PEP were collected from on-road vehicles using a specially designed collection chamber. The surface morphology of the collected particles was analyzed through Transmission Electron Microscope (TEM, and the elemental mapping was performed through EDAX analysis. Results indicated the presence of nanometer-size particles in both the categories of vehicle exhaust. These small-size particles of respirable range can enter the respiratory tract of humans and get deposited in the lungs and cause various effects inside the human body. The aim of this study is to assess the cytotoxicity of the collected Diesel Exhaust Nanoparticles (DENPs and Petrol Exhaust Nanoparticles (PENPs. Cytotoxicity endpoint, such as IC50 (50% Inhibitory Concentration, was determined after a 24-h exposure. Results of this study indicated that all five cell lines were sensitive to these vehicle exhaust nanoparticles at varying levels.

  18. Derivation and Utilization of Functional CD8(+) Dendritic Cell Lines.

    Science.gov (United States)

    Pigni, Matteo; Ashok, Devika; Acha-Orbea, Hans

    2016-01-01

    It is notoriously difficult to obtain large quantities of non-activated dendritic cells ex vivo. For this reason, we produced and characterized a mouse model expressing the large T oncogene under the CD11c promoter (Mushi mice), in which CD8α(+) dendritic cells transform after 4 months. We derived a variety of stable cell lines from these primary lines. These cell lines reproducibly share with freshly isolated dendritic cells most surface markers, mRNA and protein expression, and all tested biological functions. Cell lines can be derived from various strains and knockout mice and can be easily transduced with lentiviruses. In this article, we describe the derivation, culture, and lentiviral transduction of these dendritic cell lines.

  19. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan

    2011-01-01

    incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size...... except MeSeA. Speciation analysis showed that MeSeA was completely transformed during the incubations, while metabolic conversion of the other Se compounds was limited. Production of volatile dimethyl diselenide was observed for MeSeA and MeSeCys. MeSeA, MeSeCys and selenite showed noticeable protein......The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...

  20. Inhibitory effect of parvovirus H—1 on the formation of colonies of human hepatoma cell line in vitro and its tumors in nude mice

    Institute of Scientific and Technical Information of China (English)

    YANSHANGJUN; CHENGWUMA; 等

    1994-01-01

    The inhibitory effect of parvovirus H-1 on the colonyforming ability.in vitro of QGY-7703,a cultured human hepatoma cell line,and on the formation and growth of its tumors in nude mice was studied.With higher multiplicity of infection(MOI) of H-1 given,survival of the QGY-7703 cells was found to be decreased.H-1 DNA amplification level at 30h postinfection(p.i.) was detected to be 7.4 times higher than that at 2h by dispersed cells assay,while the cells were delayed to enter into S phase.Plaques were formed in the indicator cells(new-born human kidney cell line,NBK) by progeny H-1 virus particles released from the infected QGY-7703 cells by infectious cell center assay.The formation of tumors in nude mice by QGY-7703 cells which were injected s c at 2h postinfection was observed to by prevented in 2 proups with given MOI 25 and 50.The tumor growth of MOI 10 group occurred at a lower exponential rate than that of control,after a 20d latent period.It was evident that parvovirus H-1 exhibited a direct inhibitory effect on the formation and growth of human hepatoma cells in vivo as well as in vitro.

  1. Heterogeneity of the MDCK cell line and its applicability for influenza virus research.

    Directory of Open Access Journals (Sweden)

    Vladimir Y Lugovtsev

    Full Text Available Single-cell clones have been established from the MDCK cell line, characterized for their morphology and evaluated for their suitability for influenza virus research. Three discrete cell morphotypes were identified using light microscopy. Besides morphological features, the cell types can be distinguished by the level of expression of surface glycans recognized by peanut agglutinin (PNA. All clones were susceptible to infection by influenza viruses of different subtypes of influenza A virus (H1N1, H1N1pdm09, H3N2, H5N1 and influenza B virus, and all possessed on their surface terminally sialylated glycans with both types of glycosidic linkage (α2-3 and α2-6. The Type-1 cell lines were able to support a multicycle replication of influenza A and B viruses without help of an exogenous trypsin. In contrast, cell lines exhibiting Type-2 morphology were unable to support multicycle replication of influenza A viruses without trypsin supplementation. Western blot analysis of the hemagglutinin of H1N1 strains demonstrated that Type-2 cells were deficient in production of proteolytically activated hemagglutinin (no cleavage between HA1/HA2 was observed. HA1/HA2 cleavage of influenza B viruses in the Type-2 cells was also significantly impaired, but not completely abrogated, producing sufficient amount of activated HA to support efficient virus replication without trypsin. In contrast, all clones of Type-1 cells were able to produce proteolytically activated hemagglutinin of influenza A and B viruses. However, the growth kinetics and plaque size of influenza A viruses varied significantly in different clones. Influenza B virus also showed different plaque size, with the biggest plaque formation in the Type-2 cells, although the growth kinetics and peak infectivity titers were similar in all clones. Taken together, the study demonstrates that the population of original MDCK cells is represented by various types of cells that differ in their capacities to

  2. Authentication of the R06E Fruit Bat Cell Line

    Directory of Open Access Journals (Sweden)

    Ingo Jordan

    2012-05-01

    Full Text Available Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in colonies of the Egyptian rousette (Rousettus aegyptiacus. To facilitate molecular studies on virus-reservoir host interactions and isolation of viruses from environmental samples, we established cell lines from primary cells of this animal. The cell lines were given to several laboratories until we realized that a contamination with Vero cells in one of the cultures had occurred. Here we describe a general diagnostic procedure for identification of cross-species contamination with the focus on Vero and Rousettus cell lines, and summarize newly discovered properties of the cell lines that may pertain to pathogen discovery.

  3. Effect of leptin on cell proliferation and apoptosis of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29

    Institute of Scientific and Technical Information of China (English)

    Chang-Wen Yu; Bi-Sheng Zhu

    2016-01-01

    Objective:To explore effect of leptin on cell proliferation and apoptosis of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29.Methods: MTT and flow cytometry were adopted for detecting the effect of exogenous leptin on cell cycle of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29.Results: Leptin with mass concentration (0 ng/mL, 5 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL) could stimulate the growth of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29; exogenous leptin with mass concentration (5 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL) could inhibit cell growth of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29 after 72 h; among which, inhibiting effects of cell line-SGC7901 and cell line-HT-29 were the most significant under the effect of exogenous leptin with mass concentration-200 ng/mL.Conclusion:Within a certain concentration and action time, exogenous leptin can stimulate the growth of gastric adenocarcinoma cell line and colon cancer cell line, and then promot the tumor cell proliferation and/or inhibit the tumor cell apoptosis.

  4. Cancer and inflammation studies using zebrafish cell lines

    NARCIS (Netherlands)

    He, Shuning

    2010-01-01

    As the zebrafish, Danio rerio, has been increasingly used as an animal model for biomedical research, we aimed to establish zebrafish cell line models for inflammation and cancer studies in this thesis. Several zebrafish cell lines were characterized and their genetic and physiological properties we

  5. Derivation of the human embryonic stem cell line RCM1

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  6. Trichloroethylene toxicity in a human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Thevenin, E.; McMillian, J. [Medical Univ. of Charleston South Carolina, SC (United States)

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  7. Beryllium-stimulated apoptosis in macrophage cell lines.

    Science.gov (United States)

    Sawyer, R T; Fadok, V A; Kittle, L A; Maier, L A; Newman, L S

    2000-08-21

    In vitro stimulation of bronchoalveolar lavage cells from patients with chronic beryllium disease (CBD) induces the production of TNF-alpha. We tested the hypothesis that beryllium (Be)-stimulated TNF-alpha might induce apoptosis in mouse and human macrophage cell lines. These cell lines were selected because they produce a range of Be-stimulated TNF-alpha. The mouse macrophage cell line H36.12j produces high levels of Be-stimulated TNF-alpha. The mouse macrophage cell line P388D.1 produces low, constitutive, levels of TNF-alpha and does not up-regulate Be-stimulated TNF-alpha production. The DEOHS-1 human CBD macrophage cell line does not produce constitutive or Be-stimulated TNF-alpha. Apoptosis was determined by microscopic observation of propidium iodide stained fragmented nuclei in unstimulated and BeSO(4)-stimulated macrophage cell lines. BeSO(4) induced apoptosis in all macrophage cell lines tested. Beryllium-stimulated apoptosis was dose-responsive and maximal after 24 h of exposure to 100 microM BeSO(4). In contrast, unstimulated and Al(2)(SO(4))(3)-stimulated macrophage cell lines did not undergo apoptosis. The general caspase inhibitor BD-fmk inhibited Be-stimulated macrophage cell line apoptosis at concentrations above 50 microM. Our data show that Be-stimulated macrophage cell line apoptosis was caspase-dependent and not solely dependent on Be-stimulated TNF-alpha levels. We speculate that the release of Be-antigen from apoptotic macrophages may serve to re-introduce Be material back into the lung microenvironment, make it available for uptake by new macrophages, and thereby amplify Be-stimulated cytokine production, promoting ongoing inflammation and granuloma maintenance in CBD.

  8. Regulated expression of erythropoietin by two human hepatoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  9. Human embryonic stem cell lines derived from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhen Fu FANG; Fan JIN; Hui GAI; Ying CHEN; Li WU; Ai Lian LIU; Bin CHEN; Hui Zhen SHENG

    2005-01-01

    Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF,Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.

  10. Pharmacokinetics of intravitreal glial cell line-derived neurotrophic factor: experimental studies in pigs

    DEFF Research Database (Denmark)

    Ejstrup, Rasmus; Kiilgaard, J F; Tucker, B A;

    2010-01-01

    The purpose of this study was to establish the intravitreal (ITV) pharmacokinetics of glial cell line-derived neurotrophic factor (GDNF) and observe possible complications after ITV injection. Twenty Danish landrace pigs and 34 eyes were included in the study; 30 were injected with 100 ng of GDNF......, two controls were injected without GDNF, and two received no injection. At post-injection time points of 1, 2, 3, 6 hours (h), 1, 2, 4 or 7 days (d) eyes were enucleated and the ITV concentration of GDNF (cGDNF) was determined by enzyme-linked immunosorbent assay, and activity was tested using...... a retinal ganglion cell line (RGC5) bioassay. Indirect ophthalmoscopy, intraocular pressure assessment, and fundus photography were performed before enucleation. There was initial variability in the cGDNF, but after 24h GDNF was cleared in a monoexponential fashion with a half-life of 37 h (CL 33-43 h...

  11. Motoneuron differentiation of immortalized human spinal cord cell lines.

    Science.gov (United States)

    Li, R; Thode, S; Zhou, J; Richard, N; Pardinas, J; Rao, M S; Sah, D W

    2000-02-01

    Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABA(+) interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury.

  12. Histone H1 Limits DNA Methylation in Neurospora crassa.

    Science.gov (United States)

    Seymour, Michael; Ji, Lexiang; Santos, Alex M; Kamei, Masayuki; Sasaki, Takahiko; Basenko, Evelina Y; Schmitz, Robert J; Zhang, Xiaoyu; Lewis, Zachary A

    2016-07-07

    Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-3XFLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq) revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stoichiometry of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa, we performed micrococcal nuclease digestion in the wild-type and the [Formula: see text]hH1 strain followed by sequencing (MNase-seq). Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation by whole-genome bisulfite sequencing (MethylC-seq) revealed a modest but global increase in DNA methylation in the [Formula: see text]hH1 mutant. Together, these data suggest that H1 acts as a nonspecific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa.

  13. Histone H1 Limits DNA Methylation in Neurospora crassa

    Directory of Open Access Journals (Sweden)

    Michael Seymour

    2016-07-01

    Full Text Available Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-3XFLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stoichiometry of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa, we performed micrococcal nuclease digestion in the wild-type and the ΔhH1 strain followed by sequencing (MNase-seq. Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation by whole-genome bisulfite sequencing (MethylC-seq revealed a modest but global increase in DNA methylation in the ΔhH1 mutant. Together, these data suggest that H1 acts as a nonspecific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa.

  14. H-1 Upgrades (4BW/4BN) (H-1 Upgrades)

    Science.gov (United States)

    2015-12-01

    Nautical Miles R&M - Reliability and Maintainability RM - Reference Model TV-1 - Technical Standards Profile Univ . - Universal H-1 Upgrades December 2015...Speed (kts) 165 165 135 139 139 Payload (Hot Day) (lbs) 3500 lbs 3500 lbs 2500 lbs 6 Wing Stations 4 Universal Under Wing Stations 3429 3429 Weapon...Stations Universal Mounts 6 6 4 4 4 Precision Guided Munitions 16 16 12 16 16 Maneuverability/Agility (G’s) -0.5 to +2.5 -0.5 to +2.5 -0.5 to +2.5 -0.5 to

  15. Derivation of human embryonic stem cell line Genea022

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea022 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea022 was demonstrated with 84% of cells expressed Nanog, 98% Oct4, 55% Tra1–60 and 97% SSEA4, gave a Pluritest Pluripotency score of 42.95, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  16. Further characterization of the first seminoma cell line TCam-2.

    Science.gov (United States)

    de Jong, Jeroen; Stoop, Hans; Gillis, Ad J M; Hersmus, Remko; van Gurp, Ruud J H L M; van de Geijn, Gert-Jan M; van Drunen, Ellen; Beverloo, H Berna; Schneider, Dominik T; Sherlock, Jon K; Baeten, John; Kitazawa, Sohei; van Zoelen, E Joop; van Roozendaal, Kees; Oosterhuis, J Wolter; Looijenga, Leendert H J

    2008-03-01

    Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs.

  17. Establishment of Germ-line Competent C57BL/6J Embryonic Stem Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Gui-jun YAN; Zheng GU; Jian WANG; Jia-ke TSO

    2004-01-01

    Objective To establish C57BL/6J embryonic stem (ES) cell lines with potential germline contribution Methods ES cells were isolated from blastocyst inner cell mass of C57BL/6J mice, and cultured for 15 passages, and then injected into blastococels of lCR mice blastocysts to establish chimeric mice.Results Three ES cell lines (mC57ESl,mC57ES3, mC57ES7) derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker "stage-specific embryonic antigen-1" and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC5 7ES1 cells consis tently differentiated into derivatives of all three embryonic germ layers. When mC57ES1cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained.One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.

  18. Cytotoxinic Mechanism of Hydroxyapatite Nanoparticles on Human Hepatoma Cell Lines

    Institute of Scientific and Technical Information of China (English)

    CAO Xian-ying; QI Zhi-tao; DAI Hong-lian; YAN Yu-hua; LI Shi-pu

    2003-01-01

    Stable and single-dispersed HAP nanoparticles were synthesized with chemical method assisted by ultrasonic treatment.HAP nanoparticles were surveyed by AFM and Zataplus.The effect on the Bel-7402 human hepatoma cell lines treated with HAP nanoparticles was investigated by the MTT methods and observation of morphology,and the mechanism was studied in changes of cell cycle and ultrastructure.The result shows that inhibition of HAP nanoparticles on the Bel-7402 human hepatoma cell lines is obviously in vitro.HAP nanoparticles the entered cancer cytoplasm,and cell proliferation is stopped at G1 phase of cell cycle,thus,cancer cells die directly.

  19. Deriving cell lines from zebrafish embryos and tumors.

    Science.gov (United States)

    Choorapoikayil, Suma; Overvoorde, John; den Hertog, Jeroen

    2013-09-01

    Over the last two decades the zebrafish has emerged as a powerful model organism in science. The experimental accessibility, the broad range of zebrafish mutants, and the highly conserved genetic and biochemical pathways between zebrafish and mammals lifted zebrafish to become one of the most attractive vertebrate models to study gene function and to model human diseases. Zebrafish cell lines are highly attractive to investigate cell biology and zebrafish cell lines complement the experimental tools that are available already. We established a straightforward method to culture cells from a single zebrafish embryo or a single tumor. Here we describe the generation of fibroblast-like cell lines from wild-type and ptenb(-/-) embryos and an endothelial-like cell line from a tumor of an adult ptena(+/-)ptenb(-/-) zebrafish. This protocol can easily be adapted to establish stable cell lines from any mutant or transgenic zebrafish line and the average time to obtain a pro-stable cell line is 3-5 months.

  20. Recombinant protein production from stable mammalian cell lines and pools.

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced.

  1. Establishment of human embryonic stem cell line from gamete donors

    Institute of Scientific and Technical Information of China (English)

    LI Tao; ZHOU Can-quan; MAI Qing-yun; ZHUANG Guang-lun

    2005-01-01

    Background Human embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study. Methods Three oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed.Results Four ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line (cHES-1) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types.Conclusions HES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widespread impact on biomedical research.

  2. Susceptibility of various cell lines to Neospora caninum tachyzoites cultivation

    Directory of Open Access Journals (Sweden)

    Khordadmehr, M.,

    2014-05-01

    Full Text Available Neospora caninum is a coccidian protozoan parasite which is a major cause of bovine abortions and neonatal mortality in cattle, sheep, goat and horse. Occasionally, cultured cells are used for isolation and multiplication of the agent in vitro with several purposes. In this study the tachyzoite yields of N. caninum were compared in various cell cultures as the host cell lines. Among the cell cultures tested, two presented good susceptibility to the agent: cell lines Vero and MA-104. SW742 and TLI (in vitro suspension culture of lymphoid cells infected with Theileria lestoquardi showed moderate sensitivity. No viable tachyzoite were detected in the culture of MDCK and McCoy cell lines. These results demonstrate that MA-104 and SW742 cells present adequate susceptibility to N. caninum compared to Vero cells, which have been largely used to multiply the parasite in vitro. Moreover, these have easy manipulation, fast multiplication and relatively low nutritional requirements. In addition, the result of this study showed that TLI cell line as a suspension cell culture is susceptible to Nc-1 tachyzoites infection and could be used as an alternative host cell line for tachyzoites culture in vitro studies.

  3. SUV39H1 interacts with HTLV-1 Tax and abrogates Tax transactivation of HTLV-1 LTR

    Directory of Open Access Journals (Sweden)

    Tanaka Yuetsu

    2006-01-01

    Full Text Available Abstract Background Tax is the oncoprotein of HTLV-1 which deregulates signal transduction pathways, transcription of genes and cell cycle regulation of host cells. Transacting function of Tax is mainly mediated by its protein-protein interactions with host cellular factors. As to Tax-mediated regulation of gene expression of HTLV-1 and cellular genes, Tax was shown to regulate histone acetylation through its physical interaction with histone acetylases and deacetylases. However, functional interaction of Tax with histone methyltransferases (HMTase has not been studied. Here we examined the ability of Tax to interact with a histone methyltransferase SUV39H1 that methylates histone H3 lysine 9 (H3K9 and represses transcription of genes, and studied the functional effects of the interaction on HTLV-1 gene expression. Results Tax was shown to interact with SUV39H1 in vitro, and the interaction is largely dependent on the C-terminal half of SUV39H1 containing the SET domain. Tax does not affect the methyltransferase activity of SUV39H1 but tethers SUV39H1 to a Tax containing complex in the nuclei. In reporter gene assays, co-expression of SUV39H1 represses Tax transactivation of HTLV-1 LTR promoter activity, which was dependent on the methyltransferase activity of SUV39H1. Furthermore, SUV39H1 expression is induced along with Tax in JPX9 cells. Chromatin immunoprecipitation (ChIP analysis shows localization of SUV39H1 on the LTR after Tax induction, but not in the absence of Tax induction, in JPX9 transformants retaining HTLV-1-Luc plasmid. Immunoblotting shows higher levels of SUV39H1 expression in HTLV-1 transformed and latently infected cell lines. Conclusion Our study revealed for the first time the interaction between Tax and SUV39H1 and apparent tethering of SUV39H1 by Tax to the HTLV-1 LTR. It is speculated that Tax-mediated tethering of SUV39H1 to the LTR and induction of the repressive histone modification on the chromatin through H3 K9

  4. Three phases of CD8 T cell response in the lung following H1N1 influenza infection and sphingosine 1 phosphate agonist therapy.

    Directory of Open Access Journals (Sweden)

    Melanie P Matheu

    Full Text Available Influenza-induced lung edema and inflammation are exacerbated by a positive feedback loop of cytokine and chemokine production termed a 'cytokine storm', a hallmark of increased influenza-related morbidity and mortality. Upon infection, an immune response is rapidly initiated in the lungs and draining lymph node, leading to expansion of virus-specific effector cells. Using two-photon microscopy, we imaged the dynamics of dendritic cells (DC and virus-specific eGFP(+CD8(+ T cells in the lungs and draining mediastinal lymph nodes during the first two weeks following influenza infection. Three distinct phases of T cell and CD11c(+ DC behavior were revealed: 1 Priming, facilitated by the arrival of lung DCs in the lymph node and characterized by antigen recognition and expansion of antigen-specific CD8(+ T cells; asymmetric T cell division in contact with DCs was frequently observed. 2 Clearance, during which DCs re-populate the lung and T cells leave the draining lymph node and re-enter the lung tissue where enlarged, motile T cells come into contact with DCs and form long-lived interactions. 3 Maintenance, characterized by T-cell scanning of the lung tissue and dissociation from local antigen presenting cells; the T cells spend less time in association with DCs and migrate rapidly on collagen. A single dose of a sphingosine-1-phosphate receptor agonist, AAL-R, sufficient to suppress influenza-induced cytokine-storm, altered T cell and DC behavior during influenza clearance, delaying T cell division, cellular infiltration in the lung, and suppressing T-DC interactions in the lung. Our results provide a detailed description of T cell and DC choreography and dynamics in the lymph node and the lung during influenza infection. In addition, we suggest that phase lags in T cell and DC dynamics induced by targeting S1P receptors in vivo may attenuate the intensity of the immune response and can be manipulated for therapeutic benefit.

  5. EMP INDUCES APOPTOSIS IN HUMAN LUNG CARCINOMA CELL LINE GLC-82

    Institute of Scientific and Technical Information of China (English)

    曹晓哲; 赵梅兰; 王德文; 董波

    2002-01-01

    Objective: To study the effect of electromagnetic pulse (EMP) on apoptosis of human lung carcinoma cell line GLC-82. Methods: The injury changes in GLC-82 cells after irradiated by EMP (electric field intensity was 60 kV/m with 5 pulses/2 min) were analyzed by cytometry, MTT chronometry and flow cytometry. The immuno- histochemical SP staining was used to determine the expressions of bcl-2 protein and p53 protein. The stained positive cells were analyzed by CMIAS-II image analysis system at a magnification 400. All data were analyzed by SPSS8.0 software. Results: EMP could obviously inhibited lung carcinoma cell line GLC-82 proliferation and increased the number of non-adherent cells. The absorbance value (A570) of MTT decreased immediately, at 0 h, 1 h and 6 h after the GLC-82 cells irradiated by EMP as compared with control group. The highest apoptosis rate was found to reach 13.38% by flow cytometry at 6 h after EMP irradiation. Down-regulation of bcl-2 expression and up-regulation of bax expression were induced by EMP. Conclusion: EMP promoted apoptosis of GLC-82 cells. At same time, EMP can down-regulate bcl-2 expression and up-regulate p53 expression in GLC-82 cells. The bcl-2 and the p53 protein may involve the apoptotic process.

  6. JKT-1 is not a human seminoma cell line.

    Science.gov (United States)

    de Jong, Jeroen; Stoop, Hans; Gillis, Ad J M; van Gurp, Ruud J H L M; van Drunen, Ellen; Beverloo, H Berna; Lau, Yun-Fai Chris; Schneider, Dominik T; Sherlock, Jon K; Baeten, John; Hatakeyama, Shingo; Ohyama, Chikara; Oosterhuis, J Wolter; Looijenga, Leendert H J

    2007-08-01

    The JKT-1 cell line has been used in multiple independent studies as a representative model of human testicular seminoma. However, no cell line for this specific tumour type has been independently confirmed previously; and therefore, the seminomatous origin of JKT-1 must be proven. The genetic constitution of the JKT-1 cells was determined using flow cytometry and spectral karyotyping, as well as array comparative genomic hybridization and fluorescent in situ hybridization. Marker profiling, predominantly based on differentially expressed proteins during normal germ cell development, was performed by immunohistochemistry and Western blot analyses. Moreover, genome wide affymetrix mRNA expression and profiling of 157 microRNAs was performed, and the status of genomic imprinting was determined. A germ cell origin of the JKT-1 cells was in line with genomic imprinting status and marker profile (including positive staining for several cancer-testis antigens). However, the supposed primary tumour, from which the cell line was derived, being indeed a classical seminoma, was molecularly proven not to be the origin of the cell line. The characteristic chromosomal anomalies of seminoma, e.g. gain of the short arm of chromosome 12, as well as the informative marker profile (positive staining for OCT3/4, NANOG, among others) were absent in the various JKT-1 cell lines investigated, irrespective of where the cells were cultured. All results indicate that the JKT-1 cell line is not representative of human seminoma. Although it can originate from an early germ cell, a non-germ cell derivation cannot be excluded.

  7. Investigation of radiosensitivity gene signatures in cancer cell lines.

    Directory of Open Access Journals (Sweden)

    John S Hall

    Full Text Available Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n = 16] and head and neck [n = 11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2 by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2 median was investigated using Affymetrix GeneChip Exon 1.0ST (cervix or U133A Plus2 (head and neck arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4% were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI, and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins.

  8. Development of a cell line from Echinococcus granulosus germinal layer.

    Science.gov (United States)

    Albani, Clara María; Cumino, Andrea Carina; Elissondo, María Celina; Denegri, Guillermo María

    2013-10-01

    In vitro culture of parasitic helminths provides an important tool to study cell regeneration and physiology, as well as for molecular biology and genetic engineering studies. In the present study, we established in vitro propagation of cells from Echinococcus granulosus germinal cyst layer. E. granulosus germinal cells grew beyond 100 passages and showed no signs of reduced proliferation capacity. Microscopic analysis revealed that cells grew both attached to the substrate and in suspension, forming three-dimensional structures like mammalian stem cell aggregates. Examination of the chromosome number of attached germinal cells showed a high degree of heteroploidy, suggesting the occurrence of transformation during culture. Monolayer cells survived cryopreservation and were able to proliferate after thawing. Based on the characteristics displayed by E. granulosus germinal cells, we establish a cell line from the E. granulosus germinal layer. Furthermore, we propose that this cell line could be useful for drug screening and for obtaining parasite material.

  9. Establishment of Jurkat tet-on cell line

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Tet-control system is developed to tightly control target gene expression in mammalian cells by using the regulatory elements of tetracycline-repressor of the transposor Tn10 from E.Coli.We have transfected reverse tetracycline-controlled transactivator gene (rtTA) into genome of Jurkat cells and established two Jurkat tet-on cell lines.Induction of luciferase reporter activity with doxycycline,a tetracycline derivative,is dose-dependent with a peak value of 32-fold increment.Establishment of Jurkat tet-on cell lines greatly facilitates quantitative studies on target gene functions in the cells.

  10. Establishment and characterization of rat portal myofibroblast cell lines.

    Directory of Open Access Journals (Sweden)

    Michel Fausther

    Full Text Available The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC and portal fibroblasts (PF. In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5'-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myofibroblasts and their contribution to the progression of liver fibrosis.

  11. Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.

    Science.gov (United States)

    Habibi, Laleh; Shokrgozar, Mohammad Ali; Tabrizi, Mina; Modarressi, Mohammad Hossein; Akrami, Seyed Mohammad

    2014-01-01

    L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time.

  12. Apoptotic effect of noscapine in breast cancer cell lines.

    Science.gov (United States)

    Quisbert-Valenzuela, Edwin O; Calaf, Gloria M

    2016-06-01

    Cancer is a public health problem in the world and breast cancer is the most frequently cancer in women. Approximately 15% of the breast cancers are triple-negative. Apoptosis regulates normal growth, homeostasis, development, embryogenesis and appropriate strategy to treat cancer. Bax is a protein pro-apoptotic enhancer of apoptosis in contrast to Bcl-2 with antiapoptotic properties. Initiator caspase-9 and caspase-8 are features of intrinsic and extrinsic apoptosis pathway, respectively. NF-κB is a transcription factor known to be involved in the initiation and progression of breast cancer. Noscapine, an alkaloid derived from opium is used as antitussive and showed antitumor properties that induced apoptosis in cancer cell lines. The aim of the present study was to determine the apoptotic effect of noscapine in breast cancer cell lines compared to breast normal cell line. Three cell lines were used: i) a control breast cell line MCF-10F; ii) a luminal-like adenocarcinoma triple-positive breast cell line MCF-7; iii) breast cancer triple-negative cell line MDA-MB-231. Our results showed that noscapine had lower toxicity in normal cells and was an effective anticancer agent that induced apoptosis in breast cancer cells because it increases Bax gene and protein expression in three cell lines, while decreases Bcl-xL gene expression, and Bcl-2 protein expression decreased in breast cancer cell lines. Therefore, Bax/Bcl-2 ratio increased in the three cell lines. This drug increased caspase-9 gene expression in breast cancer cell lines and caspase-8 gene expression increased in MCF-10F and MDA-MB-231. Furthermore, it increased cleavage of caspase-8, suggesting that noscapine-induced apoptosis is probably due to the involvement of extrinsic and intrinsic apoptosis pathways. Antiapoptotic gene and protein expression diminished and proapoptotic gene and protein expression increased noscapine-induced expression, probably due to decrease in NF-κB gene and protein expression

  13. Differential effects of bisphosphonates on breast cancer cell lines

    NARCIS (Netherlands)

    Verdijk, R.; Franke, H.R.; Wolbers, F.; Vermes, I.

    2007-01-01

    Bisphosphonates may induce direct anti-tumor effects in breast cancers cells in virtro. In this study, six bisphosphonates were administered to three breast caner cell lines. Cell proliferation was measured by quantification of th expressio of Cyclin D1 mRNA. Apoptosis was determined by flow cytome

  14. Investigation of the selenium metabolism in cancer cell lines.

    Science.gov (United States)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan; Andresen, Lars; Skov, Søren; Gammelgaard, Bente

    2011-02-01

    The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 μM were incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size exclusion chromatography and ICP-MS detection. The selenium compounds exhibited large differences in their ability to induce cell death in the three cell lines and the susceptibilities of the cell lines were different. Full recovery of selenium in the cellular fractions was observed for all Se compounds except MeSeA. Speciation analysis showed that MeSeA was completely transformed during the incubations, while metabolic conversion of the other Se compounds was limited. Production of volatile dimethyl diselenide was observed for MeSeA and MeSeCys. MeSeA, MeSeCys and selenite showed noticeable protein binding. Correlations between cell death induction and the Se compounds transformations could not be demonstrated.

  15. Global Conservation of Protein Status between Cell Lines and Xenografts

    Directory of Open Access Journals (Sweden)

    Julian Biau

    2016-08-01

    Full Text Available Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery.

  16. Phenotypes and karyotypes of human malignant mesothelioma cell lines.

    Directory of Open Access Journals (Sweden)

    Vandana Relan

    Full Text Available BACKGROUND: Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma. METHODS: Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs. RESULTS: Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30-72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5-17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions. CONCLUSION: These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of

  17. Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2006-11-01

    Full Text Available Abstract Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM. Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs, and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative

  18. Determinants of intrinsic radiosensitivity of mammalian cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Radford, I.R. [Peter MacCallum Cancer Institute, East Melbourne, VIC (Australia). Research Division

    1998-12-31

    Differences in the radiosensitivity of normal and cancerous cells could arise in various ways. Although there is no compelling data to support the view, the currently prevailing opinion is that differences in radiosensitivity are related to differences in some aspect of enzymatic DNA repair. A test of the importance of possible differences in enzymatic DNA repair in determining relative radiosensitivity would be to compare lethality in cells containing equivalent numbers of DNA lesions. Six cell lines were used in these studies: two Chinese hamster (CHO and V79) and a monkey (Vero) fibroblast-like line, a mouse melanoma line (B16-F1), and a rat (RUC-2) and a human (SQ-20B) carcinoma line. This group of cell lines displays a wide range of sensitivities to external beam low-LET radiation, ranging from the relatively radiosensitive B16-F1 and Vero lines through to the highly radioresistant RUC-2 line. However, it is important to note that none of the lines has a demonstrated defect in enzymatic DNA repair and that all appear to die by necrosis following a lethal radiation insult. Despite having significantly different radiosensitivities, CHO and V79 cells showed comparable responses to DNA-associated {sup 125}I-decays with D{sub o} values of around 65. More surprisingly, the radiosensitive B16-F1 line and the radioresistant RUC-2 line both had responses with D{sub o} values of around 133 {sup 125}I-decays. The factor of two difference between the D{sub o} values for these two pairs of cell lines is probably attributable to CHO and V79 cells being pseudo-diploid whereas B 16-F1 and RUC2 appear to have derived from tetraploid cells. The generality of the above result, for DNA lesions of different quality, was tested by comparing the sensitivities of CHO and V79 cells to DNA-associated {sup 3}H-decays. Again, consistent with the {sup 125}I-decay data, there was no significant difference in the D{sub o} values for these lines. Our {sup 3}H- and {sup 125}I-decay data are

  19. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy...... are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  20. Cold storage and cryopreservation of tick cell lines

    Directory of Open Access Journals (Sweden)

    Lallinger Gertrud

    2010-04-01

    Full Text Available Abstract Background Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short- and long-term low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from Rhipicephalus (Boophilus decoloratus, Rhipicephalus (Boophilus microplus, Ixodes ricinus and Ixodes scapularis. For short-term cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG as cryoprotectant was compared with dimethylsulfoxide (DMSO supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears. Results Cold storage at 6°C for up to 30 days was successful in preserving R. (B. microplus, R. (B. decoloratus, I. ricinus and I. scapularis cell lines; lines from the latter three species could be easily re-cultivated after 45 days under refrigeration. While cell lines from all four tick species cryopreserved with 6% DMSO were successfully resuscitated, the R. (B. decoloratus cells did not survive freezing in SPG and of the other three species, only the R. (B. microplus cells resumed growth during the observation period. Conclusions This constitutes the first report on successful short-term refrigeration of cells derived from R. (B. decoloratus, R. (B. microplus, and I. ricinus, and use of SPG as an alternative to DMSO for cryopreservation, thus making an important contribution to more reliable and convenient tick cell culture maintenance.

  1. An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.

    Science.gov (United States)

    Muller, Ryan Y; Hammond, Ming C; Rio, Donald C; Lee, Yeon J

    2015-12-01

    The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.

  2. Establishment of a new bovine leukosis virus producing cell line.

    Science.gov (United States)

    Beier, D; Riebe, R; Blankenstein, P; Starick, E; Bondzio, A; Marquardt, O

    2004-11-01

    Due to the prevalence of different bovine leukosis virus (BLV) species in the cattle population in Europe, problems may arise in the serological diagnosis of BLV infections. In addition, earlier investigations demonstrated that contamination of the BLV antigen-producing cell culture systems by bovine viral diarrhea virus (BVDV) may give rise to misinterpretation of serological test results after BVDV vaccination of cattle. By co-cultivation of peripheral leukocytes of a BLV-infected cow with a permanent sheep kidney cell line, a new BLV-producing cell line named PO714 was established. This line carries a BLV provirus of the Belgian species and has been tested to be free of a variety of possibly contaminating viruses and mycoplasms. Investigations of a panel of well-characterised sera by agar gel immunodiffusion (AGID) and capture ELISA (cELISA) tests using antigen prepared from this new cell line in comparison with antigen of the well-known cell line FLK/BLV yielded comparable results. False positive results caused by BVDV cross-reactions could be eliminated when tests were carried out with antigen derived from the new cell line.

  3. Expressional patterns of chaperones in ten human tumor cell lines

    Directory of Open Access Journals (Sweden)

    Slavc Irene

    2004-12-01

    Full Text Available Abstract Background Chaperones (CH play an important role in tumor biology but no systematic work on expressional patterns has been reported so far. The aim of the study was therefore to present an analytical method for the concomitant determination of several CH in human tumor cell lines, to generate expressional patterns in the individual cell lines and to search for tumor and non-tumor cell line specific CH expression. Human tumor cell lines of neuroblastoma, colorectal and adenocarcinoma of the ovary, osteosarcoma, rhabdomyosarcoma, malignant melanoma, lung, cervical and breast cancer, promyelocytic leukaemia were homogenised, proteins were separated on two-dimensional gel electrophoresis with in-gel digestion of proteins and MALDI-TOF/TOF analysis was carried out for the identification of CH. Results A series of CH was identified including the main CH groups as HSP90/HATPas_C, HSP70, Cpn60_TCP1, DnaJ, Thioredoxin, TPR, Pro_isomerase, HSP20, ERP29_C, KE2, Prefoldin, DUF704, BAG, GrpE and DcpS. Conclusions The ten individual tumor cell lines showed different expression patterns, which are important for the design of CH studies in tumor cell lines. The results can serve as a reference map and form the basis of a concomitant determination of CH by a protein chemical rather than an immunochemical method, independent of antibody availability or specificity.

  4. Metronidazole decreases viability of DLD-1 colorectal cancer cell line.

    Science.gov (United States)

    Sadowska, Anna; Krętowski, Rafał; Szynaka, Beata; Cechowska-Pasko, Marzanna; Car, Halina

    2013-10-01

    The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test.

  5. Pandemic 2009 Influenza A (H1N1 virus infection in cancer and hematopoietic stem cell transplant recipients; a multicenter observational study. [v1; ref status: indexed, http://f1000r.es/4bi

    Directory of Open Access Journals (Sweden)

    Maria Cecilia Dignani

    2014-09-01

    Full Text Available Background: During March 2009 a novel Influenza A virus emerged in Mexico. We describe the clinical picture of the pandemic Influenza A (H1N1 Influenza in cancer patients during the 2009 influenza season. Methods: Twelve centers participated in a multicenter retrospective observational study of cancer patients with confirmed infection with the 2009 H1N1 Influenza A virus (influenza-like illness or pneumonia plus positive PCR for the 2009 H1N1 Influenza A virus  in respiratory secretions. Clinical data were obtained by retrospective chart review and analyzed.  Results: From May to August 2009, data of 65 patients were collected. Median age was 51 years, 57 % of the patients were female. Most patients (47 had onco-hematological cancers and 18 had solid tumors. Cancer treatment mainly consisted of chemotherapy (46, or stem cell transplantation (SCT (16. Only 19 of 64 patients had received the 2009 seasonal Influenza vaccine. Clinical presentation included pneumonia (43 and upper respiratory tract infection (22. Forty five of 58 ambulatory patients were admitted. Mechanical ventilation was required in 12 patients (18%. Treatment included oseltamivir monotherapy or in combination with amantadine for a median of 7 days. The global 30-day mortality rate was 18%. All 12 deaths were among the non-vaccinated patients. No deaths were observed among the 19 vaccinated patients. Oxygen saturation <96% at presentation was a predictor of mortality (OR 19.5; 95%CI: 2.28 to 165.9. Conclusions: In our cancer patient population, the pandemic 2009 Influenza A (H1N1 virus was associated with high incidence of pneumonia (66%, and 30-day mortality (18.5%. Saturation <96% was significantly associated with death. No deaths were observed among vaccinated patients.

  6. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.

    1981-05-01

    The in vitro radiobiologic survival values (n, D0) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL50 (n . 1.3, D0 . 117 rad(1.17 Gy)), promyelocytic leukemia; K562 (n . 1.4, D0 . 165 rad(1.65 Gy)), erythroleukemia; 45 (n . 1.1, D0 . 147 rad(1.47 Gy)), acute lymphocyte leukemia; and 176 (n . 4.0, D0 . 76 rad(0.76 Gy)), acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

  7. Effect of failures and repairs on multiple cell production lines

    Energy Technology Data Exchange (ETDEWEB)

    Legato, P.; Bobbio, A.; Roberti, L.

    1989-01-01

    This paper examines a production line composed of multiple stages, or cells, which are passed in sequential order to arrive to the final product. Two possible coordination disciplines are considered, namely: the classical tandem arrangement of sequential working centers with input buffer and the kanban scheme, considered the Japanese shop floor realization of the Just-In-Time (JIT) manifacturing approach. The production line is modelled and analysed by means of Stochastic Petri Nets (SPN). Finally an analysis is made of the possibility that the working cells can incur failure/repair cycles perturbing the production flow of the line and thus reduce performance indices.

  8. Efficacy of ribavirin against malignant glioma cell lines

    Science.gov (United States)

    OGINO, AKIYOSHI; SANO, EMIKO; OCHIAI, YUSHI; YAMAMURO, SHUN; TASHIRO, SHINYA; YACHI, KAZUNARI; OHTA, TAKASHI; FUKUSHIMA, TAKAO; OKAMOTO, YUTAKA; TSUMOTO, KOUHEI; UEDA, TAKUYA; YOSHINO, ATSUO; KATAYAMA, YOICHI

    2014-01-01

    Ribavirin (1-β-D-ribofuranosy-1,2,4-triazole-3-carboxamide) has been widely administered as an antiviral agent against RNA and DNA viruses. Ribavirin, in combination with interferon, has predominantly been applied in the treatment of the hepatitis C virus infection and its potential antitumor efficacy has recently become a point of interest. The aim of the present study was to evaluate the effect of ribavirin on the growth of malignant glioma cells, to identify novel predictive genes in malignant glioma cells (by analyzing gene expression profiles) and to assess the influence of ribavirin on the cell cycle of malignant glioma cells. The present study evaluated the antitumor efficacy of ribavirin against various malignant glioma cell lines (A-172, AM-38, T98G, U-87MG, U-138MG, U-251MG and YH-13). After culturing the cells in ribavirin-containing culture medium (final concentration, 0–1,000 μM) for 72 h, the viable proliferated cells were harvested and counted. The half maximal inhibitory concentration of ribavirin, with regard to the growth of the malignant glioma cell lines, was determined from the concentration of ribavirin required for 50% growth inhibition in comparison to the untreated control cells. Furthermore, the current study identified the genes in which the gene expression levels correlated with the ribavirin sensitivity of the malignant glioma cells lines, using a high-density oligonucleotide array. Finally, cell cycle analysis was performed on the U-87MG cell line. It was identified that ribavirin inhibited the growth of all of the malignant glioma cell lines in a dose-dependent manner, although the ribavirin sensitivity varied between each cell line. Of the extracted genes, PDGFRA demonstrated the strongest positive correlation between gene expression level and ribavirin sensitivity. Cell cycle analysis of the U-87MG cell line demonstrated that ribavirin treatment induces G0/G1 arrest and thus may be an effective agent for inhibiting malignant

  9. Mutations at positions 186 and 194 in the HA gene of the 2009 H1N1 pandemic influenza virus improve replication in cell culture and eggs

    Directory of Open Access Journals (Sweden)

    Balabanis Kara

    2010-07-01

    Full Text Available Abstract Obtaining suitable seed viruses for influenza vaccines poses a challenge for public health authorities and manufacturers. We used reverse genetics to generate vaccine seed-compatible viruses from the 2009 pandemic swine-origin influenza virus. Comparison of viruses recovered with variations in residues 186 and 194 (based on the H3 numbering system of the viral hemagglutinin showed that these viruses differed with respect to their ability to grow in eggs and cultured cells. Thus, we have demonstrated that molecular cloning of members of a quasispecies can help in selection of seed viruses for vaccine manufacture.

  10. Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Ritter Peter R

    2010-03-01

    Full Text Available Abstract Background Taurolidine (TRD represents an anti-infective substance with anti-neoplastic activity in many malignant cell lines. So far, the knowledge about the cell death inducing mechanisms and pathways activated by TRD is limited. The aim of this study was therefore, to perform a comparative analysis of cell death induction by TRD simultaneously in different malignant cell lines. Materials and methods Five different malignant cell lines (HT29/Colon, Chang Liver/Liver, HT1080/fibrosarcoma, AsPC-1/pancreas and BxPC-3/pancreas were incubated with increasing concentrations of TRD (100 μM, 250 μM and 1000 μM for 6 h and 24 h. Cell viability, apoptosis and necrosis were analyzed by FACS analysis (Propidiumiodide/AnnexinV staining. Additionally, cells were co-incubated with the caspase Inhibitor z-VAD, the radical scavenger N-Acetylcystein (NAC and the Gluthation depleting agent BSO to examine the contribution of caspase activation and reactive oxygen species in TRD induced cell death. Results All cell lines were susceptible to TRD induced cell death without resistance toward this anti-neoplastic agent. However, the dose response effects were varying largely between different cell lines. The effect of NAC and BSO co-treatment were highly different among cell lines - suggesting a cell line specific involvement of ROS in TRD induced cell death. Furthermore, impact of z-VAD mediated inhibition of caspases was differing strongly among the cell lines. Conclusion This is the first study providing a simultaneous evaluation of the anti-neoplastic action of TRD across several malignant cell lines. The involvement of ROS and caspase activation was highly variable among the five cell lines, although all were susceptible to TRD induced cell death. Our results indicate, that TRD is likely to provide multifaceted cell death mechanisms leading to a cell line specific diversity.

  11. Exometabolom analysis of breast cancer cell lines: Metabolic signature.

    Science.gov (United States)

    Willmann, Lucas; Erbes, Thalia; Halbach, Sebastian; Brummer, Tilman; Jäger, Markus; Hirschfeld, Marc; Fehm, Tanja; Neubauer, Hans; Stickeler, Elmar; Kammerer, Bernd

    2015-08-21

    Cancer cells show characteristic effects on cellular turnover and DNA/RNA modifications leading to elevated levels of excreted modified nucleosides. We investigated the molecular signature of different subtypes of breast cancer cell lines and the breast epithelial cell line MCF-10A. Prepurification of cell culture supernatants was performed by cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer. Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism. We observed major differences of metabolite excretion pattern between the breast cancer cell lines and MCF-10A, just as well as between the different breast cancer cell lines themselves. Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level. Modified nucleosides have great potential as biomarkers in due consideration of the heterogeneity of breast cancer that is reflected by the different molecular subtypes of breast cancer. Our data suggests that the metabolic signature of breast cancer cell lines might be a more subtype-specific tool to predict breast cancer, rather than a universal approach.

  12. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  13. Characterisation of thyroid medullary carcinoma TT cell line.

    Science.gov (United States)

    Zabel, M; Grzeszkowiak, J

    1997-01-01

    TT cell line is the best known stabilized cell line derived from the human medullary thyroid carcinoma. The ultrastructural characteristics of these cells include well developed rough endoplasmic reticulum, a prominent Golgi apparatus and a considerable number of secretory granules. Numerous hormones were immunocytochemically demonstrated in TT cells of which calcitonin and calcitonin gene-related peptide (CGRP) are the products of the same gene but an alternative RNA processing. TT cells were found to produce some other hormones as well, namely ACTH, neurotensin, enkephalin, PTHrP, gastrin-releasing peptide (GRP), serotonin but also functional proteins of the chromogranin group, synaptophysin, NSE, calbindin and tyrosine hydroxylase. Some marker proteins have been detected in the cytosol (CEA) and in the cytoskeleton (alpha-tubulin, cytokeratin). The influence of numerous factors on the secretory activity of these cells has been demonstrated so far, including effects of 1,25-dihydroxycholecalciferol, glucocorticoids, sex steroids, cAMP, gastrin-releasing peptide, sodium butyrate, phorbol esters, ionomycin and forskolin. The investigators performed on the TT cell line demonstrate that this is the most reliable model system for the human parafollicular cells developed so far, in comparison to other cell lines derived from the medullary carcinoma of the thyroid.

  14. Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

    transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

  15. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

    Sampaio, Julio L; Gerl, Mathias J; Klose, Christian

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...... to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more hydroxylated as required...

  16. Heterozygous embryonic stem cell lines derived from nonhuman primate parthenotes.

    Science.gov (United States)

    Dighe, Vikas; Clepper, Lisa; Pedersen, Darlene; Byrne, James; Ferguson, Betsy; Gokhale, Sumita; Penedo, M Cecilia T; Wolf, Don; Mitalipov, Shoukhrat

    2008-03-01

    Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients.

  17. Transportation characteristics of nolatrexed in three tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    LI Yi-lei; ZHAO Ai-guo; WU Shu-guang

    2002-01-01

    Objective:To investigate the association of the transportation characteristics of nolatrexed in tumor cells with its drug sensitivity. Methods: The sensitivity of 3 tumor cell lines, C6, SRS82 and LoVo, to nolatrexed were determined by growth inhibition study. After exposure to 20 μmol/L nolatrexed at different time intervals ranging from 0 to 30 min, or to nolatrexed at different concentrations ranging from 0 to 40μmol/L for 10 min, the intracellular drug concentration was measured using high-performance liquid chromatography. Results: C6 was the most sensitive cell line among the three, with sensitivity 6. 8-fold and 13.8-fold those of SRS-82 and LoVo cells respectively. Transportation of nolatrexed in the 3 cell lines were qualitatively similar, which rapidly achieved steady-state within 5 min, and linear relationship between the intracellular and extracellular drug concentration was observed. The intracellular steady-state level achieved in C6 was significantly higher than those in the other two cell lines, the latter having comparable levels. Conclusion: Nolatrexed enters the cell very quickly and different transport capacities are involved in the generation of varied sensitivity to nolatrexed in tumor cells.

  18. Continuous production of erythropoietin by an established human renal carcinoma cell line: development of the cell line

    Energy Technology Data Exchange (ETDEWEB)

    Sherwood, J.B.; Shouval, D.

    1986-01-01

    Establishment of a stable, transformed human renal carcinoma cell line that produces erythropoietin in vitro and has maintained this function continuously since 1981 and for > 150 passages in monolayer culture was accomplished by transplantation of human renal clear cell carcinoma tissue from a patient with erythrocytosis into an immunosuppressed athymic mouse. In addition to its immunocrossreactivity with native human urinary erythropoietin, the tumor erythropoietin demonstrates biological activity in the in vitro mouse erythroid colony-forming unit assay and in tumor-bearing nude mice. The cloned renal carcinoma cell line has an abnormal human karyotype and has ultrastructural features characteristic of human renal clear cell carcinoma. This cell line provides a reproducible model system for the production of an erythropoietin-like material and for the study of its synthesis and secretion.

  19. Derivation of human embryonic stem cell line Genea019

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea019 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype, female Allele pattern and unaffected Htt CAG repeat length, compared to HD affected sibling Genea020. Pluripotency of Genea019 was demonstrated with 75% of cells expressing Nanog, 89% Oct4, 48% Tra1-60 and 85% SSEA4, a Pluritest Pluripotency score of 22.97, Novelty score of 1.42, tri-lineage teratoma formation and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination.

  20. Guidelines for the use of cell lines in biomedical research.

    Science.gov (United States)

    Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

    2014-09-09

    Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

  1. Intranasal immunization with recombinant HA and mast cell activator C48/80 elicits protective immunity against 2009 pandemic H1N1 influenza in mice.

    Directory of Open Access Journals (Sweden)

    Shu Meng

    Full Text Available BACKGROUND: Pandemic influenza represents a major threat to global health. Vaccination is the most economic and effective strategy to control influenza pandemic. Conventional vaccine approach, despite being effective, has a number of major deficiencies including limited range of protection, total dependence on embryonated eggs for production, and time consuming for vaccine production. There is an urgent need to develop novel vaccine strategies to overcome these deficiencies. METHODOLOGY/PRINCIPAL FINDINGS: The major objective of this work was to develop a novel vaccine strategy combining recombinant haemagglutinin (HA protein and a master cell (MC activator C48/80 for intranasal immunization. We demonstrated in BALB/c mice that MC activator C48/80 had strong adjuvant activity when co-administered with recombinant HA protein intranasally. Vaccination with C48/80 significantly increased the serum IgG and mucosal surface IgA antibody responses against HA protein. Such increases correlated with stronger and durable neutralizing antibody activities, offering protection to vaccinated animals from disease progression after challenge with lethal dose of A/California/04/2009 live virus. Furthermore, protected animals demonstrated significant reduction in lung virus titers, minimal structural alteration in lung tissues as well as higher and balanced production of Th1 and Th2 cytokines in the stimulated splenocytes when compared to those without C48/80. CONCLUSIONS/SIGNIFICANCE: The present study demonstrates that the novel vaccine approach of combining recombinant HA and mucosal adjuvant C48/80 is safe and effective in eliciting protective immunity in mice. Future studies on the mechanism of action of C48/80 and potential combination with other vaccine strategies such as prime and boost approach may help to induce even more potent and broad immune responses against viruses from various clades.

  2. Differential Proteomics in Malignant and Normal Liver Cell Lines

    Institute of Scientific and Technical Information of China (English)

    LIU Zhi-jun; WANG Bin; YAN Zhi-yong; QIAN Dong-meng; SONG Xu-xia; Ding Shou-yi; BAI Zhi-qiang

    2007-01-01

    Objective: To detect differential protein expression in malignant and normal liver cell lines in vitro using the SELDI ProteinChip platform, for investigating the pathogenesis of liver cancer. Methods: Two cell lines, human normal liver cell line L02 and hepatoma cell line SMMC-7721 were cultured routinely, harvested in good condition and lysed. After quantification, the supernatant of the lysate was tested by IMAC3 (Immobilized Mental Affinity Capture) and WCX2 (Weak Cation Exchange) chips on the SELDI-TOF-MS ProteinChip reader. Results: Protein expression differed between the malignant and normal liver cell lines. A total of 20 differentially expressed proteins were found, among which, 7 were captured by the IMAC3 chip and 14 by the WCX2 chip. Peaks at 5,419, 7,979 and 11,265 Da were higher and at 8,103, 8,492, 10,160 and 11,304 Da lower in SMMC-7721 cells by the IMAC3 chip; peaks at 7,517, 7,945 and 7,979 Da were higher and at 5,061, 5,551, 5,818, 7,439, 9,401,10,100, 10,312, 11,621, 11,662, 11,830 and 12,772 Da lower in SMMC-7721 cells by the WCX2 chip. Interestingly, both chips captured the 7,979 Da peak. In addition, the 11,081 Da peak corresponded precisely with the molecular mass of the calcium binding protein S100A10, which may participate in the formation of liver cancer in association with p36. Conclusion: Detecting differential protein expression in malignant and normal liver cell lines using the SELDI ProteinChip platform was simple, sensitive and repeatable. The results we obtained can serve as a basis for investigating the pathogenesis of liver cancer and aid the discovery of new therapeutic targets.

  3. Reconstruction of endometrium from human endometrial side population cell lines.

    Directory of Open Access Journals (Sweden)

    Irene Cervelló

    Full Text Available Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic stem cell (SSC population recently identified by several groups using the side population (SP technique. We previously demonstrated that endometrial SP displays genotypic, phenotypic and the functional capability to develop human endometrium after subcutaneous injection in NOD-SCID mice. We have now established seven human endometrial SP (hESP cell lines (ICE 1-7: four from the epithelial and three from the stromal fraction, respectively. SP cell lines were generated under hypoxic conditions based on their cloning efficiency ability, cultured for 12-15 passages (20 weeks and cryopreserved. Cell lines displayed normal 46XX karyotype, intermediate telomerase activity pattern and expressed mRNAs encoding proteins that are considered characteristic of undifferentiated cells (Oct-4, GDF3, DNMT3B, Nanog, GABR3 and those of mesodermal origin (WT1, Cardiac Actin, Enolase, Globin, REN. Phenotype analysis corroborated their epithelial (CD9+ or stromal (vimentin+ cell origin and mesenchymal (CD90+, CD73+ and CD45⁻ attributes. Markers considered characteristic of ectoderm or endoderm were not detected. Cells did not express either estrogen receptor alpha (ERα or progesterone receptor (PR. The hESP cell lines were able to differentiate in vitro into adipocytes and osteocytes, which confirmed their mesenchymal origin. Finally, we demonstrated their ability to generate human endometrium when transplanted beneath the renal capsule of NOD-SCID mice. These findings confirm that SP cells exhibit key features of human endometrial SSC and open up new possibilities for the understanding of gynecological disorders such as endometriosis or Asherman syndrome. Our cell lines can be a valuable model to investigate new targets for endometrium proliferation in endometriosis.

  4. Establishment, immortalisation and characterisation of pteropid bat cell lines.

    Directory of Open Access Journals (Sweden)

    Gary Crameri

    Full Text Available BACKGROUND: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. METHODOLOGY/FINDINGS: Black flying foxes (Pteropus alecto were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. CONCLUSIONS/SIGNIFICANCE: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

  5. Monoclonal antibodies against the human leukemia cell line K 562.

    Science.gov (United States)

    Böttger, V; Hering, S; Jantscheff, P; Micheel, B

    1985-01-01

    Three monoclonal antibodies raised against K 562, a cell line originally established from a patient with chronic myeloid leukemia (CML) in terminal blast crisis, were selected according to their distinct reaction pattern. Whereas two antibodies (ZIK-C1-A/C5 and ZIK-C1-A/H5 also designated C and H) recognized antigens, present on K 562 cells and other immature and mature hematopoietic cells (cell lines and normal blood and bone marrow cells), antibody ZIK-C1-A/D9 also designated Y showed an exclusive binding to K 562 cells. The results obtained (here and in the following paper) indicate, that antibody ZIK-C1-A/D9 defines an early differentiation antigen of hematopoiesis or a leukemia-associated antigen.

  6. Comparative Metabolic Flux Profiling of Melanoma Cell Lines

    Science.gov (United States)

    Scott, David A.; Richardson, Adam D.; Filipp, Fabian V.; Knutzen, Christine A.; Chiang, Gary G.; Ronai, Ze'ev A.; Osterman, Andrei L.; Smith, Jeffrey W.

    2011-01-01

    Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the “reverse” (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma. PMID:21998308

  7. Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies

    OpenAIRE

    Krampe, Britta; Al-Rubeai, Mohamed

    2010-01-01

    Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members o...

  8. Skin Biopsy and Patient-Specific Stem Cell Lines

    Science.gov (United States)

    Li, Yao; Nguyen, Huy V.; Tsang, Stephen H.

    2016-01-01

    The generation of patient-specific induced pluripotent stem (iPS) cells permits the development of next-generation patient-specific systems biology models reflecting personalized genomics profiles to better understand pathophysiology. In this chapter, we describe how to create a patient-specific iPS cell line. There are three major steps: (1) performing a skin biopsy procedure on the patient; (2) extracting human fibroblast cells from the skin biopsy tissue; and (3) reprogramming patient-specific fibroblast cells into the pluripotent stem cell stage. PMID:26141312

  9. The YUMM lines: a series of congenic mouse melanoma cell lines with defined genetic alterations.

    Science.gov (United States)

    Meeth, Katrina; Wang, Jake Xiao; Micevic, Goran; Damsky, William; Bosenberg, Marcus W

    2016-09-01

    The remarkable success of immune therapies emphasizes the need for immune-competent cancer models. Elegant genetically engineered mouse models of a variety of cancers have been established, but their effective use is limited by cost and difficulties in rapidly generating experimental data. Some mouse cancer cell lines are transplantable to immunocompetent host mice and have been utilized extensively to study cancer immunology. Here, we describe the Yale University Mouse Melanoma (YUMM) lines, a comprehensive system of mouse melanoma cell lines that are syngeneic to C57BL/6, have well-defined human-relevant driver mutations, and are genomically stable. This will be a useful tool for the study of tumor immunology and genotype-specific cancer biology.

  10. Human cell lines: A promising alternative for recombinant FIX production.

    Science.gov (United States)

    de Sousa Bomfim, Aline; Cristina Corrêa de Freitas, Marcela; Picanço-Castro, Virgínia; de Abreu Soares Neto, Mário; Swiech, Kamilla; Tadeu Covas, Dimas; Maria de Sousa Russo, Elisa

    2016-05-01

    Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca(+2) and Mg(+2) ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca(+2) and 1 mM Mg(+2) resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins.

  11. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

    Energy Technology Data Exchange (ETDEWEB)

    Medina, D.; Oborn, C.J. (Baylor College of Medicine, Houston, TX (USA)); Li, M.L.; Bissell, M.J. (Univ. of California, Berkeley (USA))

    1987-09-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.

  12. Analysis of LINE-1 expression in human pluripotent cells.

    Science.gov (United States)

    Muñoz-Lopez, Martin; Garcia-Cañadas, Marta; Macia, Angela; Morell, Santiago; Garcia-Perez, Jose L

    2012-01-01

    Half of the human genome is composed of repeated DNA, and some types are mobile within our genome (transposons and retrotransposons). Despite their abundance, only a small fraction of them are currently active in our genome (Long Interspersed Element-1 (LINE-1), Alu, and SVA elements). LINE-1 or L1 elements are a family of active non-LTR retrotransposons, the ongoing mobilization of which still impacts our genome. As selfish DNA elements, L1 activity is more prominent in early human development, where new insertions would be transmitted to the progeny. Here, we describe the conventional methods aimed to determine the expression level of LINE-1 elements in pluripotent human cells.

  13. Establishment of cell suspension line of Populus tomentosa Carr

    Institute of Scientific and Technical Information of China (English)

    YAO Na; ZHANG Zhi-yi; AN Xin-min; YANG Kai

    2008-01-01

    Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subeultured into a MS liquid medium supplemented with 1.5 mg·L-1 2,4-D. The best subculture medium was MS+ 0.8 mg·L-1 2,4-D + 30 g·L-1 sucrose with a subculture cycle of seven days.

  14. Graphene Oxide Nanoribbons Induce Autophagic Vacuoles in Neuroblastoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Emanuela Mari

    2016-11-01

    Full Text Available Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2 and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS, mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells.

  15. Dipeptidyl peptidase IV in two human glioma cell lines

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    A Sedo

    2009-12-01

    Full Text Available There is growing evidence that dipeptidyl peptidase IV [DPP-IV, EC 3.4.14.5] takes part in the metabolism of biologically active peptides participating in the regulation of growth and transformation of glial cells. However, the knowledge on the DPP-IV expression in human glial and glioma cells is still very limited. In this study, using histochemical and biochemical techniques, the DPP-IV activity was demonstrated in two commercially available human glioma cell lines of different transformation degree, as represented by U373 astrocytoma (Grade III and U87 glioblastoma multiforme (Grade IV lines. Higher total activity of the enzyme, as well as its preferential localisation in the plasma membrane, was observed in U87 cells. Compared to U373 population, U87 cells were morphologically more pleiomorphic, they were cycling at lower rate and expressing less Glial Fibrillary Acidic Protein. The data revealed positive correlation between the degree of transformation of cells and activity of DPP-IV. Great difference in expression of this enzyme, together with the phenotypic differences of cells, makes these lines a suitable standard model for further 57 studies of function of this enzyme in human glioma cells.

  16. Oral bioavailability of glyphosate: studies using two intestinal cell lines.

    Science.gov (United States)

    Vasiluk, Luba; Pinto, Linda J; Moore, Margo M

    2005-01-01

    Glyphosate is a commonly used nonselective herbicide that inhibits plant growth through interference with the production of essential aromatic amino acids. In vivo studies in mammals with radiolabeled glyphosate have shown that 34% of radioactivity was associated with intestinal tissue 2 h after oral administration. The aim of our research was to investigate the transport, binding, and toxicity of glyphosate to the cultured human intestinal epithelial cell line, Caco-2, and the rat small intestinal crypt-derived cell line, ileum epithelial cells-18 (IEC-18). An in vitro analysis of the transport kinetics of [14C]-glyphosate showed that 4 h after exposure, approximately 8% of radiolabeled glyphosate moved through the Caco-2 monolayer in a dose-dependent manner. Binding of glyphosate to cells was saturable and approximately 4 x 10(11) binding sites/cell were estimated from bound [14C]. Exposure of Caco-2 cells to > or =10 mg/ml glyphosate reduced transmembrane electrical resistance (TEER) by 82 to 96% and increased permeability to [3H]-mannitol, indicating that paracellular permeability increased in glyphosate-treated cells. At 10-mg/ml glyphosate, both IEC-18 and Caco-2 cells showed disruption in the actin cytoskeleton. In Caco-2 cells, significant lactate dehydrogenase leakage was observed when cells were exposed to 15 mg/ml of glyphosate. These data indicate that at doses >10 mg/ml, glyphosate significantly disrupts the barrier properties of cultured intestinal cells.

  17. Derivation of a Homozygous Human Androgenetic Embryonic Stem Cell Line.

    Science.gov (United States)

    Ding, Chenhui; Huang, Sunxing; Qi, Quan; Fu, Rui; Zhu, Wanwan; Cai, Bing; Hong, Pingping; Liu, Zhengxin; Gu, Tiantian; Zeng, Yanhong; Wang, Jing; Xu, Yanwen; Zhao, Xiaoyang; Zhou, Qi; Zhou, Canquan

    2015-10-01

    Human embryonic stem cells (hESCs) have long been considered as a promising source for cell replacement therapy. However, one major obstacle for the use of these cells is immune compatibility. Histocompatible human parthenogenetic ESCs have been reported as a new method for generating human leukocyte antigen (HLA)-matched hESCs. To further investigate the possibility of obtaining histocompatible stem cells from uniparental embryos, we tried to produce androgenetic haploid human embryos by injecting a single spermatozoon into enucleated human oocyte, and establish human androgenetic embryonic stem (hAGES) cell lines from androgenetic embryos. In the present study, a diploid hAGES cell line has been established, which exhibits typical features of human ESCs, including the expression of pluripotency markers, having differentiation potential in vitro and in vivo, and stable propagation in an undifferentiated state (>P40). Bisulfite sequencing of the H19, Snrpn, Meg3, and Kv imprinting control regions suggested that hAGES cells maintained to a certain extent a sperm methylation pattern. Genome-wide single nucleotide polymorphism, short tandem repeat, and HLA analyses revealed that the hAGES cell genome was highly homozygous. These results suggest that hAGES cells from spermatozoon could serve as a useful tool for studying the mechanisms underlying genomic imprinting in humans. It might also be used as a potential resource for cell replacement therapy as parthenogenetic stem cells.

  18. Antiproliferative effect of Tualang honey on oral squamous cell carcinoma and osteosarcoma cell lines

    Directory of Open Access Journals (Sweden)

    Ismail Noorliza M

    2010-09-01

    Full Text Available Abstract Background The treatment of oral squamous cell carcinomas (OSCC and human osteosarcoma (HOS includes surgery and/or radiotherapy which often lead to reduced quality of life. This study was aimed to study the antiproliferative activity of local honey (Tualang on OSCC and HOS cell lines. Methods Several concentrations of Tualang honey (1% - 20% were applied on OSCC and HOS cell lines for 3, 6, 12, 24, 48 and 72 hours. Morphological characteristics were observed under light and fluorescent microscope. Cell viability was assessed using MTT assay and the optical density for absorbance values in each experiment was measured at 570 nm by an ELISA reader. Detection of cellular apoptosis was done using the Annexin V-FITC Apoptosis Detection Kit. Results Morphological appearance showed apoptotic cellular changes like becoming rounded, reduction in cell number, blebbed membrane and apoptotic nuclear changes like nuclear shrinkage, chromatin condensation and fragmented nucleus on OSCC and HOS cell lines. Cell viability assay showed a time and dose-dependent inhibitory effect of honey on both cell lines. The 50% inhibitory concentration (IC50 for OSCC and HOS cell lines was found to be 4% and 3.5% respectively. The maximum inhibition of cell growth of ≥80% was obtained at 15% for both cell lines. Early apoptosis was evident by flow cytometry where percentage of early apoptotic cells increased in dose and time dependent manner. Conclusion Tualang honey showed antiproliferative effect on OSCC and HOS cell lines by inducing early apoptosis.

  19. Identification and Characterization of Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Valentina Pozzi

    2015-05-01

    Full Text Available Background/Aims: Head and neck squamous cell carcinoma (HNSCC ranks sixth worldwide for tumor-related mortality. A subpopulation of tumor cells, termed cancer stem cells (CSCs, has the ability to support cancer growth. Therefore, profiling CSC-enriched populations could be a reliable tool to study cancer biology. Methods: We performed phenotypic characterization of 7 HNSCC cell lines and evaluated the presence of CSCs. CSCs from Hep-2 cell line and HNSCC primary cultures were enriched through sphere formation and sphere-forming cells have been characterized both in vitro and in vivo. In addition, we investigated the expression levels of Nicotinamide N-methyltransferase (NNMT, an enzyme overexpressed in several malignancies. Results: CSC markers were markedly expressed in Hep-2 cell line, which was found to be highly tumorigenic. CSC-enriched populations displayed increased expression of CSC markers and a strong capability to form tumors in vivo. We also found an overexpression of CSC markers in tumor formed by CSC-enriched populations. Interestingly, NNMT levels were significantly higher in CSC-enriched populations compared with parental cells. Conclusion: Our study provides an useful procedure for CSC identification and enrichment in HNSCC. Moreover, results obtained seem to suggest that CSCs may represent a promising target for an anticancer therapy.

  20. Boldine: a potential new antiproliferative drug against glioma cell lines.

    Science.gov (United States)

    Gerhardt, Daniéli; Horn, Ana Paula; Gaelzer, Mariana Maier; Frozza, Rudimar Luiz; Delgado-Cañedo, Andrés; Pelegrini, Alessandra Luiza; Henriques, Amélia T; Lenz, Guido; Salbego, Christianne

    2009-12-01

    Malignant gliomas are the most common and devastating primary tumors of the central nervous system. Currently no efficient treatment is available. This study evaluated the effect and underlying mechanisms of boldine, an aporphine alkaloid of Peumus boldus, on glioma proliferation and cell death. Boldine decreased the cell number of U138-MG, U87-MG and C6 glioma lines at concentrations of 80, 250 and 500 muM. We observed that cell death caused by boldine was cell-type specific and dose-dependent. Exposure to boldine for 24 h did not activate key mediators of apoptosis. However, it induced alterations in the cell cycle suggesting a G(2)/M arrest in U138-MG cells. Boldine had no toxic effect on non-tumor cells when used at the same concentrations as those used on tumor cells. Based on these results, we speculate that boldine may be a promising compound for evaluation as an anti-cancer agent.

  1. Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, R.F.; Waide, J.J.; Lechner, J.F.

    1995-12-01

    A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

  2. LINEing germ and embryonic stem cells' silencing of retrotransposons.

    Science.gov (United States)

    Ishiuchi, Takashi; Torres-Padilla, Maria-Elena

    2014-07-01

    Almost half of our genome is occupied by transposable elements. Although most of them are inactive, one type of non-long terminal repeat (LTR) retrotransposon, long interspersed nuclear element 1 (LINE1), is capable of retrotransposition. Two studies in this issue, Pezic and colleagues (pp. 1410-1428) and Castro-Diaz and colleagues (pp. 1397-1409), provide novel insight into the regulation of LINE1s in human embryonic stem cells and mouse germ cells and shed new light on the conservation of complex mechanisms to ensure silencing of transposable elements in mammals.

  3. Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies.

    Science.gov (United States)

    Krampe, Britta; Al-Rubeai, Mohamed

    2010-07-01

    Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.

  4. Influence of Epinastine Hydrochloride, an H1-Receptor Antagonist, on the Function of Mite Allergen-Pulsed Murine Bone Marrow-Derived Dendritic Cells In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Ken-Zaburo Oshima

    2009-01-01

    Full Text Available There is established concept that dendritic cells (DCs play essential roles in the development of allergic immune responses. However, the influence of H1 receptor antagonists on DC functions is not well defined. The aim of the present study was to examine the effect of epinastine hydrochloride (EP, the most notable histamine H1 receptor antagonists in Japan, on Dermatophagoides farinae (Der f-pulsed mouse bone marrow-derived DCs in vitro and in vivo. EP at more than 25 ng/mL could significantly inhibit the production of IL-6, TNF- and IL-10 from Der f-pulsed DCs, which was increased by Der f challenge in vitro. On the other hand, EP increased the ability of Der f-pulsed DCs to produce IL-12. Intranasal instillation of Der f-pulsed DCs resulted in nasal eosinophilia associated with a significant increase in IL-5 levels in nasal lavage fluids. Der f-pulsed and EP-treated DCs significantly inhibited nasal eosinophila and reduced IL-5. These results indicate that EP inhibits the development of Th2 immune responses through the modulation of DC functions and results in favorable modification of clinical status of allergic diseases.

  5. Cellular and Phenotypic Characterization of Canine Osteosarcoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Marie E. Legare, Jamie Bush, Amanda K. Ashley, Taka Kato, William H. Hanneman

    2011-01-01

    Full Text Available Canine and human osteosarcoma (OSA have many similarities, with the majority of reported cases occurring in the appendicular skeleton, gender predominance noted, high rate of metastasis at the time of presentation, and a lack of known etiology for this devastating disease. Due to poor understanding of the molecular mechanisms underlying OSA, we have characterized seven different OSA canine cell lines: Abrams, D17, Grey, Hughes, Ingles, Jarques, and Marisco and compared them to U2, a human OSA cell line, for the following parameters: morphology, growth, contact inhibition, migrational tendencies, alkaline phosphatase staining, heterologous tumor growth, double-strand DNA breaks, and oxidative damage. All results demonstrated the positive characteristics of the Abrams cell line for use in future studies of OSA. Of particular interest, the robust growth of a subcutaneous tumor and rapid pulmonary metastasis of the Abrams cell line in an immunocompromised mouse shows incredible potential for the future use of Abrams as a canine OSA model. Further investigations utilizing a canine cell model of OSA, such as Abrams, will be invaluable to understanding the molecular events underlying OSA, pharmaceutical inhibition of metastasis, and eventual prevention of this devastating disease.

  6. Osmotic stress affects functional properties of human melanoma cell lines

    CERN Document Server

    La Porta, Caterina A M; Pasini, Maria; Laurson, Lasse; Alava, Mikko J; Zapperi, Stefano; Amar, Martine Ben

    2015-01-01

    Understanding the role of microenvironment in cancer growth and metastasis is a key issue for cancer research. Here, we study the effect of osmotic pressure on the functional properties of primary and metastatic melanoma cell lines. In particular, we experimentally quantify individual cell motility and transmigration capability. We then perform a circular scratch assay to study how a cancer cell front invades an empty space. Our results show that primary melanoma cells are sensitive to a low osmotic pressure, while metastatic cells are less. To better understand the experimental results, we introduce and study a continuous model for the dynamics of a cell layer and a stochastic discrete model for cell proliferation and diffusion. The two models capture essential features of the experimental results and allow to make predictions for a wide range of experimentally measurable parameters.

  7. PLC-γ1 is involved in the inflammatory response induced by influenza A virus H1N1 infection.

    Science.gov (United States)

    Zhu, Liqian; Yuan, Chen; Ding, Xiuyan; Xu, Shuai; Yang, Jiayun; Liang, Yuying; Zhu, Qiyun

    2016-09-01

    We have previously reported that phosphoinositide-specific phospholipase γ1 (PLC-γ1) signaling is activated by influenza virus H1N1 infection and mediates efficient viral entry in human epithelial cells. In this study, we show that H1N1 also activates PLCγ-1 signaling in human promonocytic cell line -derived macrophages. Surprisingly, the activated PLCγ-1 signaling is not important for viral replication in macrophages, but is involved in the virus-induced inflammatory responses. PLC-γ1-specific inhibitor U73122 strongly inhibits the H1N1 virus-induced NF-κB signaling, blocking the up-regulation of TNF-α, IL-6, MIP-1α, and reactive oxidative species. In a positive feedback loop, IL-1β and TNF-α activate the PLCγ-1 signaling in both epithelial and macrophage cell lines. In summary, we have shown for the first time that the PLCγ-1 signaling plays an important role in the H1N1-induced inflammatory responses. Our study suggests that targeting the PLCγ-1 signaling is a potential antiviral therapy against H1N1 by inhibiting both viral replication and excessive inflammation.

  8. Optimized protocol for derivation of human embryonic stem cell lines.

    Science.gov (United States)

    Camarasa, María Vicenta; Galvez, Víctor Miguel; Brison, Daniel Roy; Bachiller, Daniel

    2012-09-01

    For the past 12 years, the biology and applications of human embryonic stem cells (hESCs) have received great attention from the scientific community. Derivatives of the first hESC line obtained by J. Thomson's group (Science 282(5391):1145-1147, 1998) have been used in clinical trials in patients with spinal cord injury, and other hESC lines have now been used to generate cells for use in treating blindness (Lancet 379(9817):713-720, 2012). In addition to the classical protocol based on mouse or human feeder layers using open culture methods (In Vitro Cellular & Developmental Biology - Animal 46(3-4):386-394, 2010; Stem Cells 23(9):1221-1227, 2005; Nature Biotechnology 24(2):185-187, 2006; Human Reproduction 21(2):503-511, 2006; Human Reproduction 20(8):2201-2206, 2005; Fertility and Sterility 83(5):1517-1529, 2005), novel hESC lines have been derived xeno-free (without using animal derived reagents) (PLoS One 5 (4):1024-1026, 2010), feeder-free (without supporting cell monolayers) (Lancet 365(9471):1601-1603, 2005), in microdrops under oil (In Vitro Cellular & Developmental Biology - Animal 46(3-4):236-41, 2010) and in suspension with ROCK inhibitor (Nature Biotechnology 28(4):361-4, 2010). Regardless of the culture system, successful hESC derivation usually requires optimization of embryo culture, the careful and timely isolation of its inner cell mass (ICM), and precise culture conditions up to the establishment of pluripotent cell growth during hESC line derivation. Herein we address the crucial steps of the hESC line derivation protocol, and provide tips to apply quality control to each step of the procedure.

  9. Cysteine modified polyaniline films improve biocompatibility for two cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Yslas, Edith I., E-mail: eyslas@exa.unrc.edu.ar [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Cavallo, Pablo; Acevedo, Diego F.; Barbero, César A. [Departamento de Química, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Rivarola, Viviana A. [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina)

    2015-06-01

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using L-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV–visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86° ± 1 to 90° ± 1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering. - Highlights: • A new surface PANI-Cys was produced on films of polyethylene terephthalate. • The relationship between surface characteristics and biocompatibility is analyzed. • The PANI-Cys film presents good biocompatibility for two cell lines.

  10. Anti-proliferative effect of a compound isolated from Cassia auriculata against human colon cancer cell line HCT 15.

    Science.gov (United States)

    Esakkirajan, M; Prabhu, N M; Arulvasu, C; Beulaja, M; Manikandan, R; Thiagarajan, R; Govindaraju, K; Prabhu, D; Dinesh, D; Babu, G; Dhanasekaran, G

    2014-01-01

    The compound was isolated from leaves of Cassia auriculata and its structure was characterized using high-performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LC-MS), UV-vis spectroscopy (UV-vis), fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (NMR). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, cytotoxicity, nuclear morphology and lactate dehydrogenase assay of isolated compound was tested against human colon cancer cell line HCT 15. The isolated compound, 4-(4-chlorobenzyl)-2,3,4,5,6,7-hexahydro-7-(2-ethoxyphenyl)benzo[h][1,4,7]triazecin-8(1H)-one at 25μg/ml concentration and by 48h showed 50% inhibition of human colon cancer cells (HCT 15). The results suggest that isolated compound from C. auriculata has potential to prevent colon cancer cell line.

  11. UOK 268 Cell Line for Hereditary Leiomyomatosis and Renal Cell Carcinoma | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Cancer Institute’s Urologic Oncology Branch seeks parties to co-develop the UOK 262 immortalized cell line as research tool to study aggressive hereditary leiomyomatosis and renal cell carcinoma (HLRCC)-associated recurring kidney cancer.

  12. Third-line chemotherapy for small cell lung cancer

    NARCIS (Netherlands)

    de Jong, WK; ten Hacken, NHT; Groen, HJM

    2006-01-01

    Efficacy of third-line chemotherapy treatment for small cell lung cancer (SCLC) is unknown. We present our experience with third-tine chemotherapy for recurrent SCLC. Between January 1996 and July 2004 all. consecutive patients treated for SCLC were retrospectively studied. We recorded patient chara

  13. UV light blocks EGFR signalling in human cancer cell lines

    DEFF Research Database (Denmark)

    Olsen, BB; Neves-Petersen, M T; Klitgaard, S

    2007-01-01

    UV light excites aromatic residues, causing these to disrupt nearby disulphide bridges. The EGF receptor is rich in aromatic residues near the disulphide bridges. Herein we show that laser-pulsed UV illumination of two different skin-derived cancer cell lines i.e. Cal-39 and A431, which both...

  14. Silicon Carbide Tiles for Sidewall Lining in Aluminium Electrolysis Cells

    Institute of Scientific and Technical Information of China (English)

    RUANBo; ZHAOJunguo; 等

    1999-01-01

    The paper introduces the nitride bonded silicon carbide used for sidewall lining in aluminium eletrolysis cells ,including technical process,main properties and application results.Comparison tests on various physical properties of silicon carbide products made by LIRR and other producers worldwide have also been conducted in an independent laboratory.

  15. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Science.gov (United States)

    2011-03-24

    ...; Identification of Human Cell Lines Project AGENCY: National Institute of Standards and Technology (NIST...) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All... for Biotechnology Information (NCBI) and will be used to differentiate among cell lines, as...

  16. Translocation of histone H1 subtypes between chromatin and cytoplasm during mitosis in normal human fibroblasts.

    Science.gov (United States)

    Gréen, Anna; Lönn, Anita; Peterson, Kajsa Holmgren; Ollinger, Karin; Rundquist, Ingemar

    2010-05-01

    Histone H1 is an important constituent of chromatin, which undergoes major structural rearrangements during mitosis. However, the role of H1, multiple H1 subtypes, and H1 phosphorylation is still unclear. In normal human fibroblasts, phosphorylated H1 was found located in nuclei during prophase and in both cytoplasm and condensed chromosomes during metaphase, anaphase, and telophase as detected by immunocytochemistry. Moreover, we detected remarkable differences in the distribution of the histone H1 subtypes H1.2, H1.3, and H1.5 during mitosis. H1.2 was found in chromatin during prophase and almost solely in the cytoplasm of metaphase and early anaphase cells. In late anaphase, it appeared in both chromatin and cytoplasm and again in chromatin during telophase. H1.5 distribution pattern resembled that of H1.2, but H1.5 was partitioned between chromatin and cytoplasm during metaphase and early anaphase. H1.3 was detected in chromatin in all cell cycle phases. We propose therefore, that H1 subtype translocation during mitosis is controlled by phosphorylation, in combination with H1 subtype inherent affinity. We conclude that H1 subtypes, or theirphosphorylated forms, may leave chromatin in a regulated way to give access for chromatin condensing factors or transcriptional regulators during mitosis.

  17. Crude subcellular fractionation of cultured mammalian cell lines

    Directory of Open Access Journals (Sweden)

    Holden Paul

    2009-12-01

    Full Text Available Abstract Background The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples. Findings We have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step. Conclusion We describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.

  18. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

    Directory of Open Access Journals (Sweden)

    Chen Lei

    2011-06-01

    Full Text Available Abstract Background Cancer stem cells (CSCs are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44. Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

  19. THP-1 cell line: an in vitro cell model for immune-modulation approach : Review

    NARCIS (Netherlands)

    Chanput, W.; Mes, J.J.; Wichers, H.J.

    2014-01-01

    THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review a

  20. Human Embryonic St me Cell Lines fromthe Chinese Population and Differentiation to Liver and Muscle Cell Types

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    We have established 6 hES cell lines from IVF surplus blastocysts. Characterization of these lines have shown that 4 of the 6 lines meet all of the criterion (Science) for hES cell lines and 2 of them display most characteristics of hES cells but do not form teratoma. In order to produce hES cell lines without using mouse feeders, we have produced a hES cell line using feeders derived from hES cells themselves, and showed that hES-derived feeders are capable of supporting the derivation of new hES cell line...

  1. The response of a human bronchial epithelial cell line to histamine: Intracellular calcium changes and extracellular release of inflammatory mediators

    Energy Technology Data Exchange (ETDEWEB)

    Noah, T.L.; Paradiso, A.M.; Madden, M.C.; McKinnon, K.P.; Devlin, R.B. (Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill (United States))

    1991-11-01

    Epithelial cells are likely to modulate inflammation and tissue repair in the airways, but the factors responsible for these processes remain unclear. Because human airway epithelia are infrequently available for in vitro studies, transformed epithelial cell lines are of interest as models. The authors therefore investigated the response of an SV-40/adenovirus-transformed human bronchial epithelial cell line (BEAS-2B) to histamine, a mediator with relevance for airway diseases. The intracellular calcium response to histamine (10(-4) M) was measured, using Fura-2 and microspectrofluorimetry. Histamine induced a transient increase in intracellular calcium that originated from intracellular sources; this effect was inhibited by the H1 receptor antagonist diphenhydramine, suggesting that BEAS cells retain functioning histamine receptors. BEAS cells were grown to confluence on microporous, collagen-coated filters, allowing measurement of vectorial release of soluble mediators. Monolayers exposed to histamine for 30 min released interleukin-6 and fibronectin in the apical direction, in a dose-dependent manner. Little eicosanoid production was induced by histamine, either in the apical or the basolateral direction, although BEAS cells constitutively produced small amounts of prostaglandin E2 and 15-HETE. However, these cells formed large amounts of eicosanoids in response to ozone exposure as a positive control. Comparison of their data with published reports for human airway epithelia in primary culture suggests that the BEAS cell line is, in a number of respects, a relevant model for the study of airway epithelial responses to a variety of stimuli.

  2. Cytotoxicity and genotoxicity of phenazine in two human cell lines.

    Science.gov (United States)

    McGuigan, Claire F; Li, Xing-Fang

    2014-06-01

    Phenazine was recently identified as a drinking water disinfection byproduct (DBP), but little is known of its toxic effects. We examined in vitro cytotoxicity and genotoxicity of phenazine (1.9-123 μM) in HepG2 and T24 cell lines. Cytotoxicity was determined by an impedance-based real-time cell analysis instrument. The BrdU (5-bromo-2'-deoxyuridine) proliferation and MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assays were used to examine mechanisms of cytotoxicity. Genotoxicity was determined using the alkaline comet assay. Concentration-dependent cytotoxicity was observed in HepG2 cells, primarily due to an antiproliferative effect (BrdU 24 h IC50: 11 μM; 48 h IC50: 7.8 μM) observed as low as 1.9 μM. T24 cells experienced a minor antiproliferative effect (BrdU 24 h IC50: 47 μM; 48 h IC50: 17 μM). IC50 values for HepG2 proliferation and viability were 54-77% lower compared to T24 cells. In both cell lines, IC50 values for proliferation were 66-90% lower than those for viability. At phenazine concentrations producing equivalent cytotoxicity, HepG2 cells (1.9-30.8 μM) experienced no significant genotoxic effects, while T24 cells (7.7-123 μM) experienced significant genotoxicity at ⩾61.5 μM. While these effects were seen at phenazine concentrations above those found in disinfected water, the persistence of the antiproliferative effect and the differential toxicity in each cell line deserves further study.

  3. Dissipative electron drift modes in the H1-NF stellarator

    Energy Technology Data Exchange (ETDEWEB)

    Nadeem, M.; Rafiq, T.; Persson, M. [Chalmers Univ. of Technology, Goeteborg (Sweden). Dept. of Electromagnetics

    2001-09-01

    A resistive drift wave model is developed from the two fluids theory, and the associated eigenvalue problem is solved using the ballooning mode formalism for the 3D equilibrium magnetic field configuration of the H-1NF stellarator. The frequency spectrum and the localization of electron drift modes are driven unstable by collisional dissipation of electrons along the magnetic field lines. (orig.)

  4. Effect of cell confluence on production of cloned mice using an inbred embryonic stem cell line.

    Science.gov (United States)

    Gao, Shaorong; McGarry, Michelle; Ferrier, Tricia; Pallante, Benedetta; Priddle, Helen; Gasparrini, Bianca; Fletcher, Judy; Harkness, Linda; De Sousa, Paul; McWhir, Jim; Wilmut, Ian

    2003-02-01

    Mice have been successfully cloned from both somatic cells and hybrid embryonic stem (ES) cells. Heterozygosity of the donor ES cell genome has been suggested as a crucial factor for long-term survival of cloned mice. In the present study, an inbred ES cell line, HM-1 (129/Ola), and a well-tested ES cell line, R1 (129/Sv x 129/Sv-CP), were used as donor cells to evaluate the developmental potential of nuclear transfer embryos. We found that ES cell confluence dramatically affects the developmental potential of reconstructed embryos. With the ES cell line HM-1 and 80-90% confluence, 49% of reconstructed embryos developed to the morula/blastocyst stage, 9% of these embryos developed to live pups when transferred to the surrogate mothers, and 5 of 18 live pups survived to adulthood. By contrast, at 60-70% confluence, only 22% of embryos developed to the morula/blastocyst stage, and after transfer, only a single fetus reached term. Consistent with previous reports, the nuclei of R1 ES cells were also shown to direct development to term, but no live pups were derived from cells at later passages (>20). Our results show that the developmental potential of reconstructed embryos is determined by both cell confluence and cell passage. These results also demonstrate that the inbred ES cell line, HM-1, can be used to produce viable cloned mice, although less efficiently than most heterozygous ES cell lines.

  5. Relation of cell proliferation to expression of peripheral benzodiazepine receptors in human breast cancer cell lines.

    Science.gov (United States)

    Beinlich, A; Strohmeier, R; Kaufmann, M; Kuhl, H

    2000-08-01

    Peripheral benzodiazepine receptor (PBR) agonist [(3)H]Ro5-4864 has been shown to bind with high affinity to the human breast cancer cell line BT-20. Therefore, we investigated different human breast cancer cell lines with regard to binding to [(3)H]Ro5-4864 and staining with the PBR-specific monoclonal antibody 8D7. Results were correlated with cell proliferation characteristics. In flow cytometric analysis, the estrogen receptor (ER)-negative breast cancer cell lines BT-20, MDA-MB-435-S, and SK-BR-3 showed significantly higher PBR expression (relative fluorescence intensity) than the ER-positive cells T47-D, MCF-7 and BT-474 (Pdiazepam-binding inhibitor are possibly involved in the regulation of cell proliferation of human breast cancer cell lines.

  6. Mitochondrial DNA sequence analysis of two mouse hepatocarcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Ji-Gang Dai; Xia Lei; Jia-Xin Min; Guo-Qiang Zhang; Hong Wei

    2005-01-01

    AIM: To study genetic difference of mitochondrial DNA (mtDNA)between two hepatocarcinoma cell lines (Hca-F and Hca-P)with diverse metastatic characteristics and the relationship between mtDNA changes in cancer cells and their oncogenic phenotype.METHODS: Mitochondrial DNA D-loop, tRNAMet+Glu+Ile and ND3gene fragments from the hepatocarcinoma cell lines with 1100, 1126 and 534 bp in length respectively were analysed by PCR amplification and restriction fragment length polymorphism techniques. The D-loop 3' end sequence of the hepatocarcinoma cell lines was determined by sequencing.RESULTS: No amplification fragment length polymorphism and restriction fragment length polymorphism were observed in tRNAMet+Glu+Ile,ND3 and D-loop of mitochondrial DNA of the hepatocarcinoma cells. Sequence differences between Hca-F and Hca-P were found in mtDNA D-loop.CONCLUSION: Deletion mutations of mitochondrial DNA restriction fragment may not play a significant role in carcinogenesis. Genetic difference of mtDNA D-loop between Hca-F and Hca-P, which may reflect the environmental and genetic influences during tumor progression, could be linked to their tumorigenic phenotypes.

  7. Centrosomal dysregulation in human metastatic melanoma cell lines.

    Science.gov (United States)

    Charters, Geoffrey A; Stones, Clare J; Shelling, Andrew N; Baguley, Bruce C; Finlay, Graeme J

    2011-09-01

    Correct partitioning of the replicated genome during mitosis is orchestrated by centrosomes, and chromosomal instability is a commonly reported feature of human cancer. Melanomas are notorious for their genetic instability and rapid clonal evolution that may be manifested as aggressive growth and facile generation of therapy-resistant variants. We characterized the centrosomal status, ploidy, and gene status (TP53, CDKN2A/B, BRAF, and NRAS) of 15 human metastatic melanoma cell lines. Cells were labelled for pericentrin (a centrosomal marker), DNA and α-tubulin, and scored for centrosome morphology, supernumerary centrosomes, and mitotic symmetry. The incidence of supernumerary centrosomes correlated with that of gross centrosomal abnormalities (r = 0.90), mitotic asymmetry (r = 0.90), and, surprisingly, increased content of G/M cells (r = 0.79). Centrosomal numerical dysregulation, observed in all cell lines, was found not to be specifically related to the status of any of the characterized gene mutations that were found in 13/15 cell lines. We conclude that centrosomal dysregulation may arise from multiple mechanisms and may drive the generation of genetic and phenotypic diversity in melanoma.

  8. BRITER: a BMP responsive osteoblast reporter cell line.

    Directory of Open Access Journals (Sweden)

    Prem Swaroop Yadav

    Full Text Available BACKGROUND: BMP signaling pathway is critical for vertebrate development and tissue homeostasis. High-throughput molecular genetic screening may reveal novel players regulating BMP signaling response while chemical genetic screening of BMP signaling modifiers may have clinical significance. It is therefore important to generate a cell-based tool to execute such screens. METHODOLOGY/PRINCIPAL FINDINGS: We have established a BMP responsive reporter cell line by stably integrating a BMP responsive dual luciferase reporter construct in the immortalized calvarial osteoblast cells isolated from tamoxifen inducible Bmp2; Bmp4 double conditional knockout mouse strain. This cell line, named BRITER (BMP Responsive Immortalized Reporter cell line, responds robustly, promptly and specifically to exogenously added BMP2 protein. The sensitivity to added BMP may be further increased by depleting the endogenous BMP2 and BMP4 proteins. CONCLUSION: As the dynamic range of the assay (for BMP responsiveness is very high for BRITER and as it responds specifically and promptly to exogenously added BMP2 protein, BRITER may be used effectively for chemical or molecular genetic screening for BMP signaling modifiers. Identification of novel molecular players capable of influencing BMP signaling pathway may have clinical significance.

  9. Differences in radiosensitivity between three HER2 overexpressing cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Steffen, Ann-Charlott; Tolmachev, Vladimir; Stenerloew, Bo [Uppsala University, Unit of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden); Goestring, Lovisa [Affibody AB, Bromma (Sweden); Palm, Stig [Sahlgrenska Academy at Goeteborg University, Department of Radiation Physics, Goeteborg (Sweden); Carlsson, Joergen [Uppsala University, Unit of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden); Rudbeck Laboratory, Biomedical Radiation Sciences, Uppsala (Sweden)

    2008-06-15

    HER2 is a potential target for radionuclide therapy, especially when HER2 overexpressing breast cancer cells are resistant to Herceptin {sup registered} treatment. Therefore, it is of interest to analyse whether HER2 overexpressing tumour cells have different inherent radiosensitivity. The radiosensitivity of three often used HER2 overexpressing cell lines, SKOV-3, SKBR-3 and BT-474, was analysed. The cells were exposed to conventional photon irradiation, low linear energy transfer (LET), to characterise their inherent radiosensitivity. The analysis was made with clonogenic survival and growth extrapolation assays. The cells were also exposed to alpha particles, high LET, from {sup 211}At decays using the HER2-binding affibody molecule {sup 211}At-(Z{sub HER2:4}){sub 2} as targeting agent. Assays for studies of internalisation of the affibody molecule were applied. SKOV-3 cells were most radioresistant, SKBR-3 cells were intermediate and BT-474 cells were most sensitive as measured with the clonogenic and growth extrapolation assays after photon irradiation. The HER2 dependent cellular uptake of {sup 211}At was qualitatively similar for all three cell lines. However, the sensitivity to the alpha particles from {sup 211}At differed; SKOV-3 was most resistant, SKBR-3 intermediate and BT-474 most sensitive. These differences were unexpected because it is assumed that all types of cells should have similar sensitivity to high-LET radiation. The sensitivity to alpha particle exposure correlated with internalisation of the affibody molecule and with size of the cell nucleus. There can be differences in radiosensitivity, which, if they also exist between patient breast cancer cells, are important to consider for both conventional radiotherapy and for HER2-targeted radionuclide therapy. (orig.)

  10. Effect of 8-Chloroadenosine on Undifferentiatied HL-60 Cell Line

    Institute of Scientific and Technical Information of China (English)

    CUIJing-rong; HUIYu; XIANGYou-qing; ZHANGLi-he

    2003-01-01

    Aim To study the effect of 8-chloroadenosine (8-CA)on undifferentiatied HL-60 cell line. Methods The IC50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 540 nm (SRB).Morphology of HL-60 cells was observed under a scanning electron microscope and a transmission electron microscope. The differentiation of HL-60 cells was examined by nitro blue tetrazolium reduction (NBT) and acid phosphatase assay. The cycle of HL-60 cells was analyzed by flow cytometry. Results 8-CA inhibited proliferation of eight human cancer cell lines.The IC50 ranked in the following order; KB (0.05 μmol·L-1 ) < HL-60 (0.25 μmol·L-1) < Bel-7402 (0.56μmol·L-1 )< MCF-7 (0.65μmol·L-1) < HCT (0.79 μmol·L-1) < HeLa (0.89μmol·L-1) < BGC-823 ( 1.149μmol·L-1) cell surface shortened, and the shape of HL-60 cells nuclei changed to kidney-shaped, horse shoe-shaped and bilob ated after treatment with 8-CA. Meanwhile, 8-CA promoted NBT reduction and increased activity of acid phosphatase in HL-60 ceils in a time and concentration-dependent manner. Flow cytometry analysis indicated that 8-CA induced an appreciable increase of the cell population in G1 phase with a marked reduction in S phase. Conclusion 8-CA can induce differentiation of HL-60 cells and block the cells at G1 phase, thus inhibiting proliferation of HL-60 cells.

  11. Plasmids and packaging cell lines for use in phage display

    Science.gov (United States)

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  12. Influenza virus A/Beijing/501/2009(H1N1 NS1 interacts with β-tubulin and induces disruption of the microtubule network and apoptosis on A549 cells.

    Directory of Open Access Journals (Sweden)

    Xueqing Han

    Full Text Available NS1 of influenza A virus is a key multifunctional protein that plays various roles in regulating viral replication mechanisms, host innate/adaptive immune responses, and cellular signalling pathways. These functions rely on its ability to participate in a multitude of protein-protein and protein-RNA interactions. To gain further insight into the role of NS1, a tandem affinity purification (TAP method was utilized to find unknown interaction partner of NS1. The protein complexes of NS1 and its interacting partner were purified from A549 cell using TAP-tagged NS1 as bait, and co-purified cellular factors were identified by mass spectrometry (MS. We identified cellular β-tubulin as a novel interaction partner of NS1. The RNA-binding domain of NS1 interacts with β-tubulin through its RNA-binding domain, as judged by a glutathione S-transferase (GST pull-down assay with the GST-fused functional domains of NS1. Immunofluorescence analysis further revealed that NS1 with β-tubulin co-localized in the nucleus. In addition, the disruption of the microtubule network and apoptosis were also observed on NS1-transfected A549 cells. Our findings suggest that influenza A virus may utilize its NS1 protein to interact with cellular β-tubulin to further disrupt normal cell division and induce apoptosis. Future work will illustrate whether this interaction is uniquely specific to the 2009 pandemic H1N1 virus.

  13. Atypical Epstein-Barr viral genomic structure in lymphoma tissue and lymphoid cell lines.

    Science.gov (United States)

    Tang, Weihua; Fan, Hongxin; Schroeder, Jane; Dunphy, Cherie H; Bryant, Ronald J; Fedoriw, Yuri; Gulley, Margaret L

    2013-06-01

    Epstein-Barr virus (EBV) DNA is found within the malignant cells of some subtypes of lymphoma, and viral presence is being exploited for improved diagnosis, monitoring, and management of affected patients. Recent work suggests that viral genomic polymorphism, such as partial deletion of the viral genome, could interfere with virus detection in tumor tissues. To test for atypical forms of the EBV genome, 98 lymphomas and 6 infected cell lines were studied using a battery of 6 quantitative polymerase chain reaction assays targeting disparate sections of EBV DNA. Fifty of the lymphomas (51%) had no amplifiable EBV DNA, and 38 lymphomas (39%) had low-level EBV infection that was deemed incidental based on EBV-encoded RNA (EBER) in situ hybridization results. The remaining 10 lymphomas (10%) had high EBV loads and EBER localization to malignant cells by EBER in situ hybridization. All 10 represented lymphoma subtypes were previously associated with EBV (Burkitt, diffuse large B-cell, or T-cell type), whereas no remnants of EBV were detected in other lymphoma subtypes (follicular, small lymphocytic, mantle cell, or marginal zone type). Interestingly, 4 of the 10 infected lymphomas had evidence of atypical viral genomes, including 3 of 4 infected T-cell lymphomas with aberrant loss of LMP2 amplicons, and a single diffuse large B-cell lymphoma lacking the central part of the viral genome spanning BamH1W, BZLF1, and EBNA1 gene segments. A reasonable screening strategy for infected malignancy involves applying EBER1 and LMP1 quantitative polymerase chain reaction assays and confirming that values exceeding 2000 copies of EBV per 100,000 cells have EBER localization to malignant cells.

  14. Survey of Differentially Methylated Promoters in Prostate Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Yipeng Wang

    2005-08-01

    Full Text Available DNA methylation, copy number in the genomes of three immortalized prostate epithelial, five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, PC3MLN4 were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme Hpall, followed by linker ligation, polymerase chain reaction (PCR amplification, labeling, hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5% showed differential hybridization between immortalized prostate epithelial, cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, TSPY previously observed in prostate cancer, 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, WIT-1. The majority of genes that appear to be both differentially methylated, differentially regulated between prostate epithelial, cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.

  15. Highly efficient site-specific transgenesis in cancer cell lines

    Directory of Open Access Journals (Sweden)

    Michael Iacovos P

    2012-12-01

    Full Text Available Abstract Background Transgenes introduced into cancer cell lines serve as powerful tools for identification of genes involved in cancer. However, the random nature of genomic integration site of a transgene highly influences the fidelity, reliability and level of its expression. In order to alleviate this bottleneck, we characterized the potential utility of a novel PhiC31 integrase-mediated site-specific insertion system (PhiC31-IMSI for introduction of transgenes into a pre-inserted docking site in the genome of cancer cells. Methods According to this system, a “docking-site” was first randomly inserted into human cancer cell lines and clones with a single copy were selected. Subsequently, an “incoming” vector containing the gene of interest was specifically inserted in the docking-site using PhiC31. Results Using the Pc-3 and SKOV-3 cancer cell lines, we showed that transgene insertion is reproducible and reliable. Furthermore, the selection system ensured that all surviving stable transgenic lines harbored the correct integration site. We demonstrated that the expression levels of reporter genes, such as green fluorescent protein and luciferase, from the same locus were comparable among sister, isogenic clones. Using in vivo xenograft studies, we showed that the genetically altered cancer cell lines retain the properties of the parental line. To achieve temporal control of transgene expression, we coupled our insertion strategy with the doxycycline inducible system and demonstrated tight regulation of the expression of the antiangiogenic molecule sFlt-1-Fc in Pc-3 cells. Furthermore, we introduced the luciferase gene into the insertion cassette allowing for possible live imaging of cancer cells in transplantation assays. We also generated a series of Gateway cloning-compatible intermediate cassettes ready for high-throughput cloning of transgenes and demonstrated that PhiC31-IMSI can be achieved in a high throughput 96-well plate

  16. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

    Science.gov (United States)

    Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

    2016-02-03

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts.

  17. Correlation between Twist expression and multidrug resistance of breast cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Yue-Xi Wang; Xiao-Mei Chen; Jun Yan; Zhi-Ping Li

    2016-01-01

    Objective:To study the correlation between Twist expression and multidrug resistance of breast cancer cell lines. Methods:Human breast cancer cell lines MCF-7, cisplatin-resistant human breast cancer cell lines MCF-7/DDP, doxorubicin-resistant human breast cancer cell lines MCF-7/Adr and taxol-resistant human breast cancer cell lines MCF/PTX were cultured, Twist in human breast cancer cell lines MCF-7 was overexpressed and treated with doxorubicin, and then cell viability and expression levels of EMT marker molecules and related signaling pathway molecules were detected. Results:mRNA contents and protein contents of Twist in drug-resistant breast cancer cell lines MCF-7/DDP, MCF-7/Adr and MCF/PTX were higher than those in MCF-7 cell lines;after doxorubicin treatment, inhibitory rates of cell viability in MCF-7 cell lines were higher than those in MCF-7/Adr and MCF-7/Twist cell lines;E-cadherin expression levels in MCF-7/Adr cell lines and MCF-7/Twist cell lines were lower than those in MCF-7 cell lines, and mRNA contents and protein contents of N-cadherin, Vimentin, TGF-β, Smad, Wnt,β-catenin, TNF-αand NF-kB were higher than those in MCF-7 cell lines. Conclusion:Increased expression of Twist is associated with the occurrence of drug resistance in breast cancer cells.

  18. "Helicobacter Pylori Attachment To 7 Mamalian Cell Lines "

    Directory of Open Access Journals (Sweden)

    N. Rahimi-Fard

    2006-06-01

    Full Text Available Background and Aim: Helicobacter pylori is the etiologic agent of chronic –active gastritis, gastroduodenal ulcers in humans, and a co-factor in the occurrence of gastric cancer and mucosa-associated lymphoid tumors, Adhesion of H.pylori to the gastric mucosa is a critical and also initial step in the pathogenesis of the disease. Bacterial adhesion inhibitory agents provide a novel pharmacologic approach to the management of infectious diseases. Materials and Methods: 22 H. pylori strains, isolated from the antral biopsies of 49 patients with dyspepsia, gastritis, gastric ulcer, duodenal ulcer,…were assayed by ELISA (UPRto investigate the diversity of attachment to 7 mamalian cell lines. Results: The concentration of H.pylori and cell suspention ,the condition and temperature, can alter the attachment rate.Best bacterial concentration was equal to 1 Mc farland,and for cell suspension was 5*10 cells/ml.90 minutes in 37C incubation period result in maximum attachment. H.pylori can attach to all 7 cell lines, there are no significant differences between 22 H.pylori strains in attachment to cells. The attachment pattern of H.pylori to the cells showed significant reduction respectly from HepII, HeLa, SW742, AGS,HT29/219, HT29 to Caco-2.Maximum attachment were seen to HepII, HeLa and SW742 cells, and among these HepII was the best cells for this purpose. Conclusion: Our studies suggest that Hep II, HeLa and SW742 cells could serve as a suitable in-vitro model for the study of H.pylori adhesions, attachment, inhibition of attachment and detachment assays and among these Hep II cell is prefer recommended.

  19. Multidrug resistance and retroviral transduction potential in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Theilade, M D; Gram, G J; Jensen, P B

    1999-01-01

    for the gibbon ape leukemia virus (GALV-1) receptor or had specificity for the amphotropic murine leukemia virus (MLV-A) receptor were used for transduction of five SCLC cell lines differing by a range of MDR mechanisms. Transduction efficiencies in these cell lines were compared by calculating the percentage...... of blue colonies after X-Gal staining of the cells grown in soft agar. All examined SCLC cell lines were transducible with either vector. Transduction efficiencies varied from 5.7% to 33.5% independent of the presence of MDR. These results indicate that MDR does not severely impair transduction of SCLC...

  20. Luminosity measurement in H1; Mesure de la luminosite pour l'experience H1

    Energy Technology Data Exchange (ETDEWEB)

    Frisson, T

    2006-10-15

    At HERA, luminosity is determined on-line and bunch by bunch by measuring the Bremsstrahlung spectrum from e-p collisions. The Hl collaboration has built a completely new luminosity system in order to sustain the harsh running conditions after the fourfold luminosity increase. Namely, the higher synchrotron radiation doses and the increased event pile-up have governed the design of the two major components, a radiation resistant quartz-fibre electro-magnetic calorimeter, and a fast read-out electronic with on-line energy histogram loading at a rate of 500 kHz. The group was in charge of the electronic and the on-line data analysis of the new luminosity system. In this thesis, I present analysis tools and methods to improve the precision of the luminosity measurement. The energy scale and acceptance calculation methods set out in this thesis permit these values to be determined every four minutes, to an accuracy of 0.5 parts per thousand for the energy scale and 2 parts per thousand for the acceptance. From these results, the degree of accuracy obtained on the luminosity measurement is between 6.5 and 9.5 parts per thousand. These results are currently undergoing validation, with the aim of becoming the standard H1 method. I also studied quasi-elastic Compton events to cross-check the luminosity measurement using the 2003- 2004 and 2005 data. Indeed, this process has a well calculable cross section and a clear experimental signature. The leptonic final state consists of a coplanar e-gamma system, both observable in the central H1 detector. (author)

  1. Feeder-independent continuous culture of the PICM-19 pig liver stem cell line

    Science.gov (United States)

    The PICM-19 pig liver stem cell line is a bipotent cell line, i.e., capable of forming either bile ductules or hepatocyte monolayers in vitro, that was derived from the primary culture of pig embryonic stem cells. The cell line has been strictly feeder-dependent in that cell replication morphology,...

  2. Cytolytic replication of echoviruses in colon cancer cell lines

    Directory of Open Access Journals (Sweden)

    Gullberg Maria

    2011-10-01

    Full Text Available Abstract Background Colorectal cancer is one of the most common cancers in the world, killing nearly 50% of patients afflicted. Though progress is being made within surgery and other complementary treatments, there is still need for new and more effective treatments. Oncolytic virotherapy, meaning that a cancer is cured by viral infection, is a promising field for finding new and improved treatments. We have investigated the oncolytic potential of several low-pathogenic echoviruses with rare clinical occurrence. Echoviruses are members of the enterovirus genus within the family Picornaviridae. Methods Six colon cancer cell lines (CaCo-2, HT29, LoVo, SW480, SW620 and T84 were infected by the human enterovirus B species echovirus 12, 15, 17, 26 and 29, and cytopathic effects as well as viral replication efficacy were investigated. Infectivity was also tested in spheroids grown from HT29 cells. Results Echovirus 12, 17, 26 and 29 replicated efficiently in almost all cell lines and were considered highly cytolytic. The infectivity of these four viruses was further evaluated in artificial tumors (spheroids, where it was found that echovirus 12, 17 and 26 easily infected the spheroids. Conclusions We have found that echovirus 12, 17 and 26 have potential as oncolytic agents against colon cancer, by comparing the cytolytic capacity of five low-pathogenic echoviruses in six colon cancer cell lines and in artificial tumors.

  3. Genetic instability of cell lines derived from a single human small cell carcinoma of the lung

    DEFF Research Database (Denmark)

    Engelholm, S A; Vindeløv, L L; Spang-Thomsen, M

    1985-01-01

    Specimens from a human small cell carcinoma of the lung were established as a cell line in vitro. Flow cytometric DNA analysis demonstrated only one tumor cell population in the parent tumor as well as in the early passages in vitro. After six passages in vitro, two new subpopulations with differ......Specimens from a human small cell carcinoma of the lung were established as a cell line in vitro. Flow cytometric DNA analysis demonstrated only one tumor cell population in the parent tumor as well as in the early passages in vitro. After six passages in vitro, two new subpopulations...

  4. Melatonin and Doxorubicin synergistically induce cell apoptosis in human hepatoma cell lines

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402.Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay.Cell apoptosis was evaluated using TUNEL method and flow cytometry.Apoptosis-r...

  5. Regulation of cholesterol synthesis in four colonic adenocarcinoma cell lines.

    Science.gov (United States)

    Cerda, S R; Wilkinson, J; Broitman, S A

    1995-12-01

    Colon tumor cells, unlike normal human fibroblasts, exhibited an uncoupling of low density lipoprotein (LDL)-derived cholesterol from cellular growth, when endogenous cholesterol synthesis was inhibited by mevinolin, a hydroxymethylglutaryl-CoA reductase (HMG-CoAR) competitive inhibitor [Fabricant, M., and Broitman, S.A. (1990) Cancer Res. 50, 632-636]. Further evaluation of cholesterol metabolism was conducted in two undifferentiated (SW480, SW1417) and two differentiated (HT29, CACO2) colonic adenocarcinoma (adeno-CA) cell lines and an untransformed human fibroblast, AG1519A. Cells grown in monolayer culture to near subconfluency were used to assess endogenous cholesterol synthesis by 14C-acetate incorporation, in response to the following treatments in lipoprotein-deficient serum (LPDS)-supplemented minimum essential medium (MEM): LPDS alone, LDL, mevinolin, mevinolin with LDL, and 25-hydroxy-cholesterol (25-OH-CH). Complete fetal bovine serum (FBS)-supplemented MEM was used as control. All colon tumor lines exhibited similarly high endogenous cholesterol synthesis in both FBS and LPDS relative to the fibroblasts which demonstrated low basal levels in FBS and maximal synthesis in LPDS. LDL treatment did not inhibit cholesterol synthesis in colon tumor cells, but suppressed that in the fibroblast by 70%. Sterol repression of cholesterol synthesis mediated by 25-OH-CH occurred in all cells. Mevinolin caused a reduction in cholesterol synthesis in the colonic cancer cell lines, which was not further decreased by concurrent addition of LDL. In contrast, in mevinolin-treated fibroblasts, LDL further inhibited cholesterol synthesis. When the effect of cell density on cholesterol synthesis regulation was evaluated under conditions of sparse density in SW480 and SW147, results indicated that (i) basal rates of cholesterol synthesis were higher, (ii) LDL inhibited cholesterol synthesis more effectively, and (iii) mevinolin or 25-OH-CH had a more pronounced effect than in

  6. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts

    Institute of Scientific and Technical Information of China (English)

    Qingyun Mai; Yang Yu; Tao Li; Liu Wang; Mei-jue Chen; Shu-zhen Huang; Canquan Zhou; Qi Zhou

    2007-01-01

    Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.

  7. Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

    Institute of Scientific and Technical Information of China (English)

    Jeeranan Manokawinchoke; Thanaphum Osathanon; Prasit Pavasant

    2016-01-01

    Objective: To investigate the influence of Notch signaling on osteoprotegerin (OPG) expression in a human oral squamous cell carcinoma cell line. Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a γ-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG mRNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software. Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 mRNA expres-sion, confirming the intracellular activation of Notch signaling. OPG mRNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a γ-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 mRNA levels, and a marked increase in OPG protein expression was observed. These results implied that Notch signaling regulated OPG expression in HSC-4 cells. However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line (HSC-5) or a human head and neck squamous cell carcinoma cell line (HN22). Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.

  8. Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

    Institute of Scientific and Technical Information of China (English)

    Jeeranan Manokawinchoke; Thanaphum Osathanon; Prasit Pavasant

    2016-01-01

    Objective: To investigate the influence of Notch signaling on osteoprotegerin(OPG)expression in a human oral squamous cell carcinoma cell line.Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a g-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG m RNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software.Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 m RNA expression, confirming the intracellular activation of Notch signaling. OPG m RNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a g-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 m RNA levels, and a marked increase in OPG protein expression was observed.These results implied that Notch signaling regulated OPG expression in HSC-4 cells.However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line(HSC-5) or a human head and neck squamous cell carcinoma cell line(HN22).Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.

  9. Apoptosis in Raji cell line induced by influenza A virus

    Institute of Scientific and Technical Information of China (English)

    李虹; 肖丽英; 李华林; 李婉宜; 蒋中华; 张林; 李明远

    2003-01-01

    Objective To study the apoptotic effects of influenza A virus on the Raji cell line. Methods Cultured Raji cells were infected with influenza A virus at a multiplicity of infection (m.o.i) of 20 and the effects of apoptosis were detected at different time points post infection using the following methods: electron microscope, DNA agarose gel electrophoresis, PI stained flow cytometry (FCM) and Annexin-V FITC/PI stained FCM.Results Raji cells infected with influenza A virus showed changes of morphology apoptotis, DNA agarose electrophoresis also demonstrated a ladder-like pattern of DNA fragments in a time-dependent manner. PI stained FCM showed "apoptosis peak" and FITC/PI stained FCM showed apoptotic cells. Quantitative analysis indicated that the percentage of apoptotic Raji cells increased after infection, and cycloheximide (CHX), an eukaryotic transcription inhibitor, could effectively inhibit the apoptotic effects of influenza A virus in vitro.Conclusions Influenza A virus can induce apoptosis in Raji cell line suggesting that it may lead to a potential method for tumor therapy.

  10. Absence of C-type virus production in human leukemic B cell, T cell and null cell lines.

    Directory of Open Access Journals (Sweden)

    Ogura,Hajime

    1978-06-01

    Full Text Available Electron microscope observation of cultured human leukemic B cell, T cell and null cell lines and reverse transcriptase assay of the culture supernatants were all negative for the presence of C-type virus. Bat cell line, which propagates primate C-type viruses well, was cocultivated with the human leukemic cell lines, in the hope of amplification of virus if present. Three weeks after mixed culture, the culture supernatants were again examined for reverse transcriptase activity and the cells were tested for syncytia formation by cocultivation with rat XC, human KC and RSb cell lines. All these tests, except for the positive control using a simian sarcoma virus, were negative, suggesting that no C-type was produced from these human leukemic cell lines.

  11. Growth dynamics and cyclin expression in cutaneous T-cell lymphoma cell lines

    Directory of Open Access Journals (Sweden)

    Edyta Biskup

    2010-05-01

    Full Text Available We have investigated cell growth dynamics and cyclins B1 and E expression in cell lines derived from mycosis fungoides (MyLa, Sézary syndrome (SeAx, and CD30+ lympho-proliferative diseases (Mac1, Mac2a, JK. Mac1 and Mac2a had the highest growth rate (doubling time 18-28 h, >90% cycling cells whereas SeAx was proliferating slowly (doub-ling time 55 h, approximately 35% cycling cells. Expression of cyclin B1 correlated positively with doubling time whereas expression of cyclin E was unscheduled and constant across the investigated cell lines. All cell lines exhibited high expression of PCNA. Thus, we concluded that cyclin B1 could be used for rapid screening of cell proliferation in malignant lymphocytes derived from cutaneous T-cell lymphoma.

  12. Mistaken identity of widely used esophageal adenocarcinoma cell line TE-7.

    Science.gov (United States)

    Boonstra, Jurjen J; van der Velden, Albertina W; Beerens, Erwin C W; van Marion, Ronald; Morita-Fujimura, Yuiko; Matsui, Yasuhisa; Nishihira, Tetsuro; Tselepis, Chris; Hainaut, Pierre; Lowe, Anson W; Beverloo, Berna H; van Dekken, Herman; Tilanus, Hugo W; Dinjens, Winand N M

    2007-09-01

    Cancer of the esophagus is the seventh leading cause of cancer death worldwide. Esophageal carcinoma cell lines are useful models to study the biological and genetic alterations in these tumors. An important prerequisite of cell line research is the authenticity of the used cell lines because the mistaken identity of a cell line may lead to invalid conclusions. Estimates indicate that up to 36% of the cell lines are of a different origin or species than supposed. The TE series, established in late 1970s and early 1980s by Nishihira et al. in Japan, is one of the first esophageal cancer cell line series that was used throughout the world. Fourteen TE cell lines were derived from human esophageal squamous cell carcinomas and one, TE-7, was derived from a primary esophageal adenocarcinoma. In numerous studies, this TE-7 cell line was used as a model for esophageal adenocarcinoma because it is one of the few esophageal adenocarcinoma cell lines existing. We investigated the authenticity of the esophageal adenocarcinoma cell line TE-7 by xenografting, short tandem repeat profiling, mutation analyses, and array-comparative genomic hybridization and showed that cell line TE-7 shared the same genotype as the esophageal squamous cell carcinoma cell lines TE-2, TE-3, TE-12, and TE-13. In addition, for more than a decade, independent TE-7 cultures from Japan, United States, United Kingdom, France, and the Netherlands had the same genotype. Examination of the TE-7 cell line xenograft revealed the histology of a squamous cell carcinoma. We conclude that the TE-7 cell line, used in several laboratories throughout the world, is not an adenocarcinoma, but a squamous cell carcinoma cell line. Furthermore, the cell lines TE-2, TE-3, TE-7, TE-12, and TE-13 should be regarded as one single squamous cell carcinoma cell line.

  13. Changes in the viral distribution pattern after the appearance of the novel influenza A H1N1 (pH1N1 virus in influenza-like illness patients in Peru.

    Directory of Open Access Journals (Sweden)

    Victor Alberto Laguna-Torres

    Full Text Available BACKGROUND: We describe the temporal variation in viral agents detected in influenza like illness (ILI patients before and after the appearance of the ongoing pandemic influenza A (H1N1 (pH1N1 in Peru between 4-January and 13-July 2009. METHODS: At the health centers, one oropharyngeal swab was obtained for viral isolation. From epidemiological week (EW 1 to 18, at the US Naval Medical Research Center Detachment (NMRCD in Lima, the specimens were inoculated into four cell lines for virus isolation. In addition, from EW 19 to 28, the specimens were also analyzed by real time-polymerase-chain-reaction (rRT-PCR. RESULTS: We enrolled 2,872 patients: 1,422 cases before the appearance of the pH1N1 virus, and 1,450 during the pandemic. Non-pH1N1 influenza A virus was the predominant viral strain circulating in Peru through (EW 18, representing 57.8% of the confirmed cases; however, this predominance shifted to pH1N1 (51.5% from EW 19-28. During this study period, most of pH1N1 cases were diagnosed in the capital city (Lima followed by other cities including Cusco and Trujillo. In contrast, novel influenza cases were essentially absent in the tropical rain forest (jungle cities during our study period. The city of Iquitos (Jungle had the highest number of influenza B cases and only one pH1N1 case. CONCLUSIONS: The viral distribution in Peru changed upon the introduction of the pH1N1 virus compared to previous months. Although influenza A viruses continue to be the predominant viral pathogen, the pH1N1 virus predominated over the other influenza A viruses.

  14. Establishment and characterization of primary lung cancer cell lines from Chinese population

    Institute of Scientific and Technical Information of China (English)

    Chao ZHENG; Yi-hua SUN; Xiao-lei YE; Hai-quan CHEN; Hong-bin JI

    2011-01-01

    Aim: To establish and characterize primary lung cancer cell lines from Chinese population.Methods: Lung cancer specimens or pleural effusions were collected from Chinese lung cancer patients and cultured in vitro with ACL4 medium (for non-small cell lung carcinomas (NSCLC)) or HITES medium (for small cell lung carcinomas (SCLC)) supplemented with 5%FBS. All cell lines were maintained in culture for more than 25 passages. Most of these cell lines were further analyzed for oncogenic mutations, karyotype, cell growth kinetics, and tumorigenicity in nude mice.Results: Eight primary cell lines from Chinese lung cancer patients were established and characterized, including seven NSCLC cell lines and one SCLC cell line. Five NSCLC cell lines were found to harbor epidermal growth factor receptor (EGFR) kinase domain mutations.Conclusion: These well-characterized primary lung cancer cell lines from Chinese population provide a unique platform for future studies of the ethnic differences in lung cancer biology and drug response.

  15. Efficient genetic method for establishing Drosophila cell lines unlocks the potential to create lines of specific genotypes.

    Science.gov (United States)

    Simcox, Amanda; Mitra, Sayan; Truesdell, Sharon; Paul, Litty; Chen, Ting; Butchar, Jonathan P; Justiniano, Steven

    2008-08-01

    Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene Ras(V12) (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of Ras(V12) is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype.

  16. Efficient genetic method for establishing Drosophila cell lines unlocks the potential to create lines of specific genotypes.

    Directory of Open Access Journals (Sweden)

    Amanda Simcox

    2008-08-01

    Full Text Available Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene Ras(V12 (a constitutively activated form of Ras profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of Ras(V12 is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype.

  17. Apoptosis induced by propolis in human hepatocellular carcinoma cell line.

    Science.gov (United States)

    Choi, Y H; Lee, W Y; Nam, S Y; Choi, K C; Park, Y E

    1999-07-01

    Propolis has been reported to exhibit a wide spectrum of activities including antibiotic, antiviral, anti-inflammatory, immunostimulatory and tumor carcinostatic properties. We showed propolis induced apoptosis in a human hepatoma cell line (SNU449) by FITC-Annexin V/PI staining. We also compared the apoptosis inducing effect between Korean and Commercial (Sigma # p-1010) propolis. There was no difference on apoptosis between them.

  18. Effect of histone deacetylase inhibitor on proliferation of biliary tract cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Li-Ning Xu; Xin Wang; Sheng-Quan Zou

    2008-01-01

    AIM: To explore the effect of histone deacetylase inhibitor, trichostatin A (TSA) on the growth of biliary tract cancer cell lines (gallbladder carcinoma cell line and cholangiocarcinoma cell line) in v/vo and in vitro,and to investigate the perspective of histone deacetylase inhibitor in its clinical application.METHODS: The survival rates of gallbladder carcinoma cell line (Mz-ChA-I cell line) and cholangiocarcinoma cell lines (QBC939, KMBC and OZ cell lines) treated with various doses of TSA were detected by methylthiazoy tetrazolium (MTT) assay.A nude mouse model of transplanted gallbladder carcinoma (Mz-ChA-I cell line)was successfully established, and changes in the growth of transplanted tumor after treated with TSAwere measured.RESULTS: TSA could inhibit the proliferation of gallbladder carcinoma cell line (Mz-ChA-I cell line) and cholangiocarcinoma cell lines (QBC939, KMBC and OZ cell lines) in a dose-dependent manner.After the nude mouse model of transplanted gallbladder carcinoma (Mz-ChA-I cell line) was successfully established, the growth of cancer was inhibited in the model, after treated with TSA.CONCLUSION: TSA can inhibit the growth of cholangiocarcinoma and gallbladder carcinoma cell lines in vitro and in vivo.

  19. Impact of host cell line adaptation on quasispecies composition and glycosylation of influenza A virus hemagglutinin.

    Directory of Open Access Journals (Sweden)

    Jana Verena Roedig

    Full Text Available The genome of influenza A viruses is constantly changing (genetic drift resulting in small, gradual changes in viral proteins. Alterations within antibody recognition sites of the viral membrane glycoproteins hemagglutinin (HA and neuraminidase (NA result in an antigenetic drift, which requires the seasonal update of human influenza virus vaccines. Generally, virus adaptation is necessary to obtain sufficiently high virus yields in cell culture-derived vaccine manufacturing. In this study detailed HA N-glycosylation pattern analysis was combined with in-depth pyrosequencing analysis of the virus genomic RNA. Forward and backward adaptation from Madin-Darby Canine Kidney (MDCK cells to African green monkey kidney (Vero cells was investigated for two closely related influenza A virus PR/8/34 (H1N1 strains: from the National Institute for Biological Standards and Control (NIBSC or the Robert Koch Institute (RKI. Furthermore, stability of HA N-glycosylation patterns over ten consecutive passages and different harvest time points is demonstrated. Adaptation to Vero cells finally allowed efficient influenza A virus replication in Vero cells. In contrast, during back-adaptation the virus replicated well from the very beginning. HA N-glycosylation patterns were cell line dependent and stabilized fast within one (NIBSC-derived virus or two (RKI-derived virus successive passages during adaptation processes. However, during adaptation new virus variants were detected. These variants carried "rescue" mutations on the genomic level within the HA stem region, which result in amino acid substitutions. These substitutions finally allowed sufficient virus replication in the new host system. According to adaptation pressure the composition of the virus populations varied. In Vero cells a selection for "rescue" variants was characteristic. After back-adaptation to MDCK cells some variants persisted at indifferent frequencies, others slowly diminished and even

  20. Pseudoislet of hybrid cellular spheroids from commercial cell lines.

    Science.gov (United States)

    Jo, Y H; Nam, B M; Kim, B Y; Nemeno, J G; Lee, S; Yeo, J E; Yang, W; Park, S H; Kim, Y S; Lee, J I

    2013-10-01

    Investigators conducting diabetes-related research have focused on islet transplantation as a radical therapy for type 1 diabetes mellitus. Pancreatic islet isolation, an essential process, is a very demanding work because of the proteolytic enzymes, species, treatment time, and individual difference. Replacement of primary isolated pancreatic islets must be carried out continuously for various in vitro tests, making primary isolated islets a useful tool for cell transplantation research. Hence, we sought to develop pseudoislets from commercial pancreas-derived cell lines. In this study, we used RIN-5F and RIN-m cells, which secrete insulin, somatostatin, or glucagon. To manufacture hybrid cellular spheroids, the cells were cultured under hanging drop plate and nonadhesive plate methods. We observed that hybrid cellular pseudoislets exhibited an oval shape, with sizes ranging from 590 to 1200 μm. Their morphology was similar to naïve islets. Cell line pseudoislets secreted and expressed insulin, glucagon, and somatostatin, as confirmed by reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry analyses. Thus, the current artificially manufactured biomimetic pseudoislets resembled pancreatic islets of the endocrine system, appearing as cellular aggregates that secreted insulin, glucagon, and somatostatin. Enhanced immunoisolation techniques may lead to the development of new islet sources for pancreatic transplantation through this pseudoislet strategy.

  1. Propranolol induced chromosomal aberrations in Chinese hamster ovary cell line

    Directory of Open Access Journals (Sweden)

    Mozhgan Sedigh-Ardekani

    2013-03-01

    Full Text Available Propranolol (PL, a non-selective beta-blocker, is a cardiovascular drug widely used to treat hypertension. The present study was concerned with assessing the cytogenetic effects of this drug on Chinese hamster ovary (CHO cell line. MTT assay was then carried out to determine the cytotoxicity index (IC50 of the drug. The IC50 value of PL was 0.43±0.02 mM. To investigate the clastogenic effects of the drug, chromatid and chromosome breaks and polyploidy in metaphases were analyzed. CHO cells were exposed to different concentrations of the drug (0.1, 0.2, 0.3, 0.4 mM for 24 hours. Considering that PL has liver metabolism, experiments were carried out in the presence and absence of the metabolic activation system (S9 mix. Mitomycin-C and sodium arsenite were used as positive controls. It was observed that in cells treated with different PL concentrations as 0.1, 0.2 and 0.3 mM, the frequency of chromatid and chromosome breaks as well as polyploidy increased when compared with untreated CHO cells. The addition of S9 mix significantly decreased the chromatid breaks, chromosome breaks and polyploidy compared to the treatment of PL alone. It is concluded that, PL causes chromatid and chromosome aberrations in CHO cell line and the metabolic activation system (S9 mix, playing an important role in drug cytotoxicity reduction.

  2. Off-line test of the KISS gas cell

    Energy Technology Data Exchange (ETDEWEB)

    Hirayama, Yoshikazu, E-mail: yoshikazu.hirayama@kek.jp [Institute of Particle and Nuclear Studies (IPNS), High Energy Accelerator Research Organization (KEK), Ibaraki 305-0801 (Japan); Watanabe, Yutaka; Imai, Nobuaki; Ishiyama, Hironobu; Jeong, Sun-Chan; Miyatake, Hiroari; Oyaizu, Michihiro [Institute of Particle and Nuclear Studies (IPNS), High Energy Accelerator Research Organization (KEK), Ibaraki 305-0801 (Japan); Kim, Yung Hee [Seoul National University, Seoul 151 742 (Korea, Republic of); Mukai, Momo [Tsukuba University, Ibaraki 305 0006 (Japan); Matsuo, Yukari; Sonoda, Tetsu; Wada, Michiharu [Nishina Center for Accelerator-Based Science, RIKEN, Wako, Saitama 351 0198 (Japan); Huyse, Mark; Kudryavtsev, Yuri; Van Duppen, Piet [Instituut voor Kern-en Stralingsfysica, KU Leuven, B-3001 Leuven (Belgium)

    2013-12-15

    Highlights: • Construction of the KEK Isotope Separation System (KISS) at RIKEN. • Ionization scheme of an iron. • Measurement of transport time profile in a gas cell. -- Abstract: The KEK Isotope Separation System (KISS) has been constructed at RIKEN to study the β-decay properties of neutron-rich isotopes with neutron numbers around N = 126 for application to astrophysics. A key component of KISS is a gas cell filled with argon gas at a pressure of 50 kPa to stop and collect the unstable nuclei, where the isotopes of interest will be selectively ionized using laser resonance ionization. We have performed off-line tests to study the basic properties of the gas cell and of KISS using nickel and iron filaments placed in the gas cell.

  3. In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor

    DEFF Research Database (Denmark)

    Damstrup, L; Rude Voldborg, B; Spang-Thomsen, M;

    1998-01-01

    analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16......-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition...... of this antibody resulted in a significant reduction of the in vitro invasion in three selected EGFR-positive cell lines. Our results show that only EGFR-positive SCLC cell lines had the in vitro invasive phenotype, and it is therefore suggested that the EGFR might play an important role for the invasion potential...

  4. Generation, isolation, and maintenance of rodent mast cells and mast cell lines

    DEFF Research Database (Denmark)

    Jensen, Bettina M; Swindle, Emily J; Iwaki, Shoko;

    2006-01-01

    Antigen-mediated mast cell activation, with subsequent mediator release, is a major initiator of the inflammatory allergic response associated with such conditions as asthma. A comprehensive understanding of the principles involved in this process therefore is key to the development of novel...... therapies for the treatment of these disease states. In vitro models of mast cell function have allowed significant progress to be made in the recognition of the fundamental principles of mast cell activation via the high-affinity IgE receptor (FcvarepsilonRI) and, more recently, other receptors expressed...... on mast cells. In addition to human mast cells, the major cell culture systems employed to investigate these responses are rat and mouse peritoneal mast cells, mouse bone-marrow-derived mast cells, the rat basophilic leukemia cell line RBL-2H3, and the mouse MC/9 mast cell line. In this unit, we describe...

  5. Impairment of cell cycle progression by aflatoxin B1 in human cell lines.

    Science.gov (United States)

    Ricordy, R; Gensabella, G; Cacci, E; Augusti-Tocco, G

    2002-05-01

    Aflatoxin B1 is a mycotoxin produced by Aspergillus flavus and Aspergillus parasiticum, which may be present as a food contaminant. It is known to cause acute toxic effects and act as a carcinogenic agent. The carcinogenic action has been related to its ability to form unstable adducts with DNA, which represent possible mutagenic sites. On the other hand, the primary cellular target responsible for its toxic action has not yet been clearly identified. Previous data suggested a possible correlation between cell proliferation and responsiveness to aflatoxin toxicity. These observations led us to investigate the effect of the toxin on cell cycle progression of three human cell lines (HepG2, SK-N-MC and SK-N-SH derived from liver and nervous tissue tumours); they were shown to display different responses to toxin exposure and have different growth kinetics. We performed analysis of the cell cycle, DNA synthesis and expression of p21 and p53 in the presence and absence of the toxin in all cell lines exposed. The results of cell cycle cytofluorometric analysis show significant alterations of cell cycle progression as a result of toxin treatment. In all cell lines exposure to a 24 h toxin treatment causes a dose-dependent accumulation in S phase, however, the ability to recover from impairment to traverse S phase varies in the cell lines under study. SK-N-MC cells appear more prone to resume DNA synthesis when the toxin is removed, while the other two cell lines maintain a significant inhibition of DNA synthesis, as indicated by cytofluorimetry and [(3)H]dTR incorporation. The level of p53 and p21 expression in the three cell lines was examined by western blot analysis and significant differences were detected. The ready resumption of DNA synthesis displayed by SK-N-MC cells could possibly be related to the absence of p53 control of cell cycle progression.

  6. Preparation of cell lines for single-cell analysis of transcriptional activation dynamics.

    Science.gov (United States)

    Rafalska-Metcalf, Ilona U; Janicki, Susan M

    2013-01-01

    Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, which can be used for real-time high-resolution imaging of transcriptional activation dynamics in single cells.

  7. Establishment and characterization of feeder-cell-dependent bovine fetal liver cell lines

    Science.gov (United States)

    The establishment and initial characterization of bovine fetal liver cell lines is described. Bovine fetal hepatocytes were cultured from the liver of a 34-day bovine fetus by physical disruption of the liver tissue. Released liver cells and clumps of cells were plated on STO feeder layers and wer...

  8. Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research.

    Science.gov (United States)

    Kniss, Douglas A; Summerfield, Taryn L

    2014-08-01

    Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research.

  9. H1-antihistamines induce vacuolation in astrocytes through macroautophagy

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Wei-Wei; Yang, Ying; Wang, Zhe; Shen, Zhe; Zhang, Xiang-Nan [Department of Pharmacology, Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Zhejiang Province Key Laboratory of Neurobiology, School of Basic Medical Sciences, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058 (China); Wang, Guang-Hui [College of Pharmaceutical Sciences, Soochow University, Suzhou, 215123 (China); Chen, Zhong, E-mail: chenzhong@zju.edu.cn [Department of Pharmacology, Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Zhejiang Province Key Laboratory of Neurobiology, School of Basic Medical Sciences, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058 (China)

    2012-04-15

    H1-antihistamines induce vacuolation in vascular smooth muscle cells, which may contribute to their cardiovascular toxicity. The CNS toxicity of H1-antihistamines may also be related to their non-receptor-mediated activity. The aim of this study was to investigate whether H1-antihistamines induce vacuolation in astrocytes and the mechanism involved. The H1-antihistamines induced large numbers of giant vacuoles in astrocytes. Such vacuoles were marked with both the lysosome marker Lysotracker Red and the alkalescent fluorescence dye monodansylcadaverine, which indicated that these vacuoles were lysosome-like acidic vesicles. Quantitative analysis of monodansylcadaverine fluorescence showed that the effect of H1-antihistamines on vacuolation in astrocytes was dose-dependent, and was alleviated by extracellular acidification, but aggravated by extracellular alkalization. The order of potency to induce vacuolation at high concentrations of H1-antihistamines (diphenhydramine > pyrilamine > astemizole > triprolidine) corresponded to their pKa ranking. Co-treatment with histamine and the histamine receptor-1 agonist trifluoromethyl toluidide did not inhibit the vacuolation. Bafilomycin A1, a vacuolar (V)-ATPase inhibitor, which inhibits intracellular vacuole or vesicle acidification, clearly reversed the vacuolation and intracellular accumulation of diphenhydramine. The macroautophagy inhibitor 3-methyladenine largely reversed the percentage of LC3-positive astrocytes induced by diphenhydramine, while only partly reversing the number of monodansylcadaverine-labeled vesicles. In Atg5{sup −/−} mouse embryonic fibroblasts, which cannot form autophagosomes, the number of vacuoles induced by diphenhydramine was less than that in wild-type cells. These results indicated that H1-antihistamines induce V-ATPase-dependent acidic vacuole formation in astrocytes, and this is partly mediated by macroautophagy. The pKa and alkalescent characteristic of H1-antihistamines may be the

  10. Effects of Genistein on Cell Cycle and Apoptosis of Two Murine Melanoma Cell Lines

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The effects of genistein on several tumor cell lines were investigated to study the effects of genistein on cell growth, cell cycle, and apoptosis of two murine melanoma cell lines, B16 and K1735M2. These two closely related murine melanoma cell lines, however, have different responses to the genistein treatment. Genistein inhibits the growth of both the B16 and K1735M2 cell lines and arrests the growth at the G2/M phase. After treatment with 60 μmol/L genistein for 72 h, apoptosis and caspase activities were detected in B16 cells, while such effects were not found in K1735M2. Further tests showed that after genistein treatment the protein content and mRNA levels of p53 increased in B16, but remained the same in K1735M2. The protein content and mRNA levels of p21WAF1/CIP1 increased in both cell lines after treatment.The results show that genistein might induce apoptosis in B16 cells by damaging the DNA, inhibiting topoisomerase Ⅱ, increasing p53 expression, releasing cytochrome c from the mitochondria, and activating the caspases which will lead to apoptosis.

  11. Detection of tumor stem cell markers in pancreatic carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Monika Olempska; Patricia Alice Eisenach; Ole Ammerpohl; Hendrik Ungefroren; Fred Fandrich; Holger Kalthoff

    2007-01-01

    BACKGROUND: Cancer of the pancreas is the fourth leading cause of cancer death in industrialized countries. In malignancy, actively proliferating cells may be effectively targeted and killed by anti-cancer therapies, but stem cells may survive and support re-growth of the tumor. Thus, new strategies for the treatment of cancer clearly will also have to target cancer stem cells. The goal of the present study was to determine whether pancreatic carcinoma cell growth may be driven by a subpopulation of cancer stem cells. Because previous data implicated ABCG2 and CD133 as stem cell markers in hematopoietic and neural stem/progenitor cells, we analyzed the expression of these two proteins in pancreatic carcinoma cell lines. METHODS:Five established pancreatic adenocarcinoma cell lines were analyzed. Total RNA was isolated and real-time RT-PCR was performed to determine the expression of ABCG2 and CD133. Surface expression of ABCG2 and CD133 was analyzed by lfow cytometric analysis. RESULTS:All pancreatic carcinoma cell lines tested expressed signiifcantly higher levels of ABCG2 than non-malignant ifbroblasts or two other malignant non-pancreatic cell lines, i.e., SaOS2 osteosarcoma and SKOV3 ovarian cancer. Elevated CD133 expression was found in two out of ifve pancreatic carcinoma cell lines tested. Using lfow cytometric analysis we conifrmed surface expression of ABCG2 in all ifve lines. Yet, CD133 surface expression was detectable in the two cell lines, A818-6 and PancTu1, which exhibited higher mRNA levels. CONCLUSIONS: Two stem cell markers, ABCG2 and CD133 are expressed in pancreatic carcinoma cell lines. ABCG2 and/or CD133 positive cells may represent subpopulation of putative cancer stem cells also in this malignancy. Because cancer stem cells are thought to be responsible for tumor initiation and its recurrence after an initial response to chemotherapy, they may be a very promising target for new drug developments.

  12. Chromosome abnormalities in Japanese Burkitt lymphoma cell lines.

    Directory of Open Access Journals (Sweden)

    Hamasaki,Kazuhide

    1982-02-01

    Full Text Available Six established Japanese Burkitt lymphoma (BL cell lines including one case with null cell type were studied by chromosomal banding techniques. The modal chromosome number was diploid or nearly diploid in five cases and hyperdiploid in one case. The marker chromosome 14q+ was observed in four of the six cases; the origin of the extra band was a chromosome 8 in three including the null cell case but could not be identified in the other. The two cases lacking the 14q+ marker had variant translocations involving the long arm of chromosome 8, one of which carried a translocation, t(8;22 (q24;q13 and the other a translocation, t(2;8 (p12;q24. Although structural and/or numerical aberrations were found in all six cell lines, chromosome 8 was the one most consistently involved. This frequent involvement of chromosome 8 in aberrations; therefore, may be an important event in the development of BL rather than the presence of a 14q+ marker chromosome.

  13. Characterization of cell lines stably transfected with rubella virus replicons

    Energy Technology Data Exchange (ETDEWEB)

    Tzeng, Wen-Pin; Xu, Jie [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States); Frey, Teryl K., E-mail: tfrey@gsu.edu [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States)

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  14. Cloned goats (Gapra hircus) from foetal fibroblast cell lines

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Mammalian cloning has been one of the most active research topics in the world.Cloning with in vitro culured foetal fibroblast cells,in comparison with embryonic cells,can be used not only to theoretically study the embryonic or cellular development and differentiation in mammals,but also to utilize the unlimited fibroblast cells to produce large numbers of clonings.The preliminary results are as follows:(i) The division and development of the cloned embryos with embryonic donor cells and goat foetal fibroblast donor cells were 55%,77% and 35%,31%,respectively.There is no significant statistical difference between them.(ii) These studies result in the birth of two cloned goats derived from two 30-day foetal fibroblast cell lines,which are the first cloned mammals from somatic cells in China.This project has established a technological data base for the furture research on adult mammalian somatic cloning and nucleocytoplasmic interactions in animal development,and a novel technique for the cloning of animals with a high-level expression of transgene(s).

  15. LINE-1 induces hTERT and ensures telomere maintenance in tumour cell lines.

    Science.gov (United States)

    Aschacher, T; Wolf, B; Enzmann, F; Kienzl, P; Messner, B; Sampl, S; Svoboda, M; Mechtcheriakova, D; Holzmann, K; Bergmann, M

    2016-01-01

    A hallmark of cancer cells is an activated telomere maintenance mechanism, which allows prolonged survival of the malignant cells. In more than 80% of tumours, telomeres are elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Cancer cells are also characterized by expression of active LINE-1 elements (L1s, long interspersed nuclear elements-1). L1 elements are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Using L1-knockdown (KD), we show for the first time that L1 is critical for telomere maintenance in telomerase-positive tumour cells. The reduced length of telomeres in the L1-KD-treated cells correlated with an increased rate of telomere dysfunction foci, a reduced expression of shelterin proteins and an increased rate of anaphase bridges. The decreased telomere length was associated with a decreased telomerase activity and decreased telomerase mRNA level; the latter was increased upon L1 overexpression. L1-KD also led to a decrease in mRNA and protein expression of cMyc and KLF-4, two main transcription factors of telomerase and altered mRNA levels of other stem-cell-associated proteins such as CD44 and hMyb, as well as a corresponding reduced growth of spheroids. The KD of KLF-4 or cMyc decreased the level of L1-ORF1 mRNA, suggesting a specific reciprocal regulation with L1. Thus, our findings contribute to the understanding of L1 as a pathogenicity factor in cancer cells. As L1 is only expressed in pathophysiological conditions, L1 now appears to be target in the rational treatment of telomerase-positive cancer.

  16. Molecular signatures in response to Isoliquiritigenin in lymphoblastoid cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jae-Eun; Hong, Eun-Jung; Nam, Hye-Young [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of); Hwang, Meeyul [Research Center for Biomedical Resource of Oriental Medicine, Daegu Haany University (Korea, Republic of); Kim, Ji-Hyun [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of); Han, Bok-Ghee, E-mail: bokghee@nih.go.kr [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of); Jeon, Jae-Pil, E-mail: jpjeon@cdc.go.kr [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer We identified the inhibitory effect of ISL on cell proliferation of LCLs. Black-Right-Pointing-Pointer We found ISL-induced genes and miRNAs through microarray approach. Black-Right-Pointing-Pointer ISL-treated LCLs represented gene expression changes in cell cycle and p53 pathway. Black-Right-Pointing-Pointer We revealed 12 putative mRNA-miRNA functional pairs associated with ISL effect. -- Abstract: Isoliquiritigenin (ISL) has been known to induce cell cycle arrest and apoptosis of various cancer cells. However, genetic factors regulating ISL effects remain unclear. The aim of this study was to identify the molecular signatures involved in ISL-induced cell death of EBV-transformed lymphoblastoid cell lines (LCLs) using microarray analyses. For gene expression and microRNA (miRNA) microarray experiments, each of 12 LCL strains was independently treated with ISL or DMSO as a vehicle control for a day prior to total RNA extraction. ISL treatment inhibited cell proliferation of LCLs in a dose-dependent manner. Microarray analysis showed that ISL-treated LCLs represented gene expression changes in cell cycle and p53 signaling pathway, having a potential as regulators in LCL survival and sensitivity to ISL-induced cytotoxicity. In addition, 36 miRNAs including five miRNAs with unknown functions were differentially expressed in ISL-treated LCLs. The integrative analysis of miRNA and gene expression profiles revealed 12 putative mRNA-miRNA functional pairs. Among them, miR-1207-5p and miR-575 were negatively correlated with p53 pathway- and cell cycle-associated genes, respectively. In conclusion, our study suggests that miRNAs play an important role in ISL-induced cytotoxicity in LCLs by targeting signaling pathways including p53 pathway and cell cycle.

  17. Internalization of cystatin C in human cell lines.

    Science.gov (United States)

    Ekström, Ulf; Wallin, Hanna; Lorenzo, Julia; Holmqvist, Bo; Abrahamson, Magnus; Avilés, Francesc X

    2008-09-01

    Altered protease activity is considered important for tumour invasion and metastasis, processes in which the cysteine proteases cathepsin B and L are involved. Their natural inhibitor cystatin C is a secreted protein, suggesting that it functions to control extracellular protease activity. Because cystatins added to cell cultures can inhibit polio, herpes simplex and coronavirus replication, which are intracellular processes, the internalization and intracellular regulation of cysteine proteases by cystatin C should be considered. The extension, mechanism and biological importance of this hypothetical process are unknown. We investigated whether internalization of cystatin C occurs in a set of human cell lines. Demonstrated by flow cytometry and confocal microscopy, A-431, MCF-7, MDA-MB-453, MDA-MB-468 and Capan-1 cells internalized fluorophore-conjugated cystatin C when exposed to physiological concentrations (1 microm). During cystatin C incubation, intracellular cystatin C increased after 5 min and accumulated for at least 6 h, reaching four to six times the baseline level. Western blotting showed that the internalized inhibitor was not degraded. It was functionally intact and extracts of cells exposed to cystatin C showed a higher capacity to inhibit papain and cathepsin B than control cells (decrease in enzyme activity of 34% and 37%, respectively). The uptake of labelled cystatin C was inhibited by unlabelled inhibitor, suggesting a specific pathway for the internalization. We conclude that the cysteine protease inhibitor cystatin C is internalized in significant quantities in various cancer cell lines. This is a potentially important physiological phenomenon not previously described for this group of inhibitors.

  18. Expression of cadherin and NCAM in human small cell lung cancer cell lines and xenografts

    DEFF Research Database (Denmark)

    Rygaard, K; Møller, C; Bock, E

    1992-01-01

    characterised, the cadherin family and the Ig superfamily member, neural cell adhesion molecule (NCAM). We investigated expression of these two adhesion molecule families in small cell lung cancer (SCLC) cell lines and xenografts by immunoblotting. Nineteen tumours established from 15 patients with SCLC were...... embryonic development, which may play a role in connection with tumour invasion and metastasis, was found in 14/18 NCAM expressing SCLC tumours. Individual tumours grown as cell lines and as nude mouse xenografts showed no qualitative differences in cadherin or NCAM expression....

  19. Creation and characterization of a cell-death reporter cell line for hepatitis C virus infection

    Science.gov (United States)

    Chen, Zhilei; Simeon, Rudo; Chockalingam, Karuppiah; Rice, Charles M.

    2010-01-01

    The present study describes the creation and characterization of a hepatoma cell line, n4mBid, that supports all stages of the hepatitis C virus (HCV) life cycle and strongly reports HCV infection by a cell-death phenotype. The n4mBid cell line is derived from the highly HCV-permissive Huh-7.5 hepatoma cell line and contains a modified Bid protein (mBid) that is cleaved and activated by the HCV serine protease NS3-4A. N4mBid exhibited a 10–20 fold difference in cell viability between the HCV-infected and mock-infected states, while the parental Huh-7.5 cells showed <2 fold difference under the same conditions. The pronounced difference in n4mBid cell viability between the HCV- and mock-infected states in a 96-well plate format points to its usefulness in cell survival-based high-throughput screens for anti-HCV molecules. The degree of cell death was found to be proportional to the intracellular load of HCV. HCV-low n4mBid cells, expressing an anti-HCV short hairpin RNA, showed a significant growth advantage over naïve cells and could be rapidly enriched after HCV infection, suggesting the possibility of using n4mBid cells for the cell survival-based selection of genetic anti-HCV factors. PMID:20188762

  20. Characterization of hybrids between bovine (MDBK) and mouse (L-cell) cell lines.

    Science.gov (United States)

    Chinchar, V G; Floyd, A D; Chinchar, G D; Taylor, M W

    1979-02-01

    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient mutants of a bovine kidney cell line (MDBK) were selected following mutagenesis with ethylmethane sulfonate or ICR-170G. MDBK mutants were hybridized to thymidine kinase-deficient L cells and selected in HAT medium. Parental and hybrid cells were characterized for isozyme patterns of lactic dehydrogenase malate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate oxalate transaminase. Chromosomes of MDBK can be distinguished from mouse L cells by configuration and by fluorescent staining with Hoechst 33-258 stain. Hybrid cells contained both MDBK and L-cell chromosomes and had elevated DNA content. MDBK cells are normally restrictive for mengovirus replication. Both permissive and restrictive hybrids were found. Our data indicate that there was preferential loss of MDBK chromosomes in the hybrid cell lines.

  1. New model for gastroenteropancreatic large-cell neuroendocrine carcinoma: establishment of two clinically relevant cell lines.

    Directory of Open Access Journals (Sweden)

    Andreas Krieg

    Full Text Available Recently, a novel WHO-classification has been introduced that divided gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN according to their proliferation index into G1- or G2-neuroendocrine tumors (NET and poorly differentiated small-cell or large-cell G3-neuroendocrine carcinomas (NEC. Our knowledge on primary NECs of the GEP-system is limited due to the rarity of these tumors and chemotherapeutic concepts of highly aggressive NEC do not provide convincing results. The aim of this study was to establish a reliable cell line model for NEC that could be helpful in identifying novel druggable molecular targets. Cell lines were established from liver (NEC-DUE1 or lymph node metastases (NEC-DUE2 from large cell NECs of the gastroesophageal junction and the large intestine, respectively. Morphological characteristics and expression of neuroendocrine markers were extensively analyzed. Chromosomal aberrations were mapped by array comparative genomic hybridization and DNA profiling was analyzed by DNA fingerprinting. In vitro and in vivo tumorigenicity was evaluated and the sensitivity against chemotherapeutic agents assessed. Both cell lines exhibited typical morphological and molecular features of large cell NEC. In vitro and in vivo experiments demonstrated that both cell lines retained their malignant properties. Whereas NEC-DUE1 and -DUE2 were resistant to chemotherapeutic drugs such as cisplatin, etoposide and oxaliplatin, a high sensitivity to 5-fluorouracil was observed for the NEC-DUE1 cell line. Taken together, we established and characterized the first GEP large-cell NEC cell lines that might serve as a helpful tool not only to understand the biology of these tumors, but also to establish novel targeted therapies in a preclinical setup.

  2. Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines.

    Science.gov (United States)

    Bock, Christoph; Kiskinis, Evangelos; Verstappen, Griet; Gu, Hongcang; Boulting, Gabriella; Smith, Zachary D; Ziller, Michael; Croft, Gist F; Amoroso, Mackenzie W; Oakley, Derek H; Gnirke, Andreas; Eggan, Kevin; Meissner, Alexander

    2011-02-04

    The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines.

  3. Fluorouracil selectively enriches stem-like cells in the lung adenocarcinoma cell line SPC.

    Science.gov (United States)

    Shi, Mu-mu; Xiong, Yan-lei; Jia, Xin-shan; Li, Xin; Zhang, Li; Li, Xiao-lei; Wang, En-Hua

    2013-06-01

    Most adult stem cells are in the G0 or quiescent phase of the cell cycle and account for only a small percentage of the cells in the tissue. Thus, isolation of stem cells from tissues for further study represents a major challenge. This study sought to enrich cancer stem cells and explore cancer stem-like cell clones using 5-fluorouracil (5-FU) in the lung adenocarcinoma cell line, SPC. Proliferation inhibition was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, according to which half maximal inhibitory concentration values were calculated. Expression levels of stem cell markers after treatment with 5-FU were examined using immunofluorescence and Western blotting. Additionally, side population (SP) cells were sorted using FACS. Properties of SP cells were evaluated by using Transwell, colony-forming assays, and tumor formation experiments. 5-FU greatly inhibits proliferation, especially of cells in S phase. SP cells possess greater invasive potential, higher clone-forming potential, and greater tumor-forming ability than non-SP cells. Treatment with 5-FU enriches the SP cells with stem cell properties in human lung adenocarcinoma cell lines.

  4. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    Directory of Open Access Journals (Sweden)

    Rajvi H Mehta

    2014-01-01

    Full Text Available The ability to successfully derive human embryonic stem cells (hESC lines from human embryos following in vitro fertilization (IVF opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ′discarded′ or ′spare′ fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. In case a couple does not desire to ′cryopreserve′ their embryos then all the embryos remaining following embryo transfer can be considered ′spare′ or if a couple is no longer in need of the ′cryopreserved′ embryos then these also can be considered as ′spare′. But, the question raised by the ethicists is, "what about ′slightly′ over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to ′discarded′ embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of ′discarding′ embryos. What would be the criteria for discarding embryos and the potential ′use′ of ESC derived from the ′abnormal appearing′ embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.

  5. Isolation and characterization of cancer stem-like cells from MHCC97H Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Shanyong Yi; Kejun Nan; Aihua Yuan; Chuangxin Lu

    2009-01-01

    Objective:To identify and isolate CD133 positive cancer stem-like cells (CD133+ cells) from the highly invasive human hepatocellular carcinoma cell line(MHCC97H), and examine their potential for clonogenicity and tumorigenicity. Methods: CD133+ and CD133- cells were isolated from MHCC97H cell line by magnetic bead cell sorting(MACS), and the potentials of CD133+ cells for colony formation and tumorigenicity were evaluated by soft agar cloning and tumor formation following nude mice inoculation. Results:CD133+ cells represent a minority(0.5-2.0%) of the tumor cell population with a greater colony-forming efficiency and greater tumor production ability. The colony-forming efficiency of CD133+ cells in soft agar was significantly higher than CD133- cells(36.8±1.4 vs 12.9±0.8, P<0.05).After 6 weeks, 3/5 mice inoculated with 1 × 103 CD133+ cells, 4/5 with 1 × 104 CD133+ cells and 5/5 with 1 × 105 CD133+ cells developed detectable tumors at the injection site, while only one tumor was found in mice treated with same numbers of CD133- cells. Conclusion: CD133 may be a hallmark of liver cancer stem cells (CSC) in human hepatocellular carcinoma(HCC), because the CD133+ cells identified and isolated with anti-CD133 labeled magnetic beads from MHCC97H cell line exhibit high potentials for clonogenicity and tumorigenicity. These CD133+ cells might contribute to hepatocarcinogenesis, as well as the growth and recurrence of human HCC, and therefore may be a useful target for anti-cancer therapy.

  6. Effects of small interfering RNAs targeting fascin on human esophageal squamous cell carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Garcia Jose

    2010-06-01

    Full Text Available Abstract Background Fascin induces membrane protrusions and cell motility. Fascin overexpression was associated with poor prognosis, and its downregulation reduces cell motility and invasiveness in esophageal squamous cell carcinoma (ESCC. Using a stable knockdown cell line, we revealed the effect of fascin on cell growth, cell adhesion and tumor formation. Methods We examined whether fascin is a potential target in ESCC using in vitro and in vivo studies utilizing a specific siRNA. We established a stable transfectant with downregulated fascin from KYSE170 cell line. Results The fascin downregulated cell lines showed a slower growth pattern by 40.3% (p In vivo, the tumor size was significantly smaller in the tumor with fascin knockdown cells than in mock cells by 95% at 30 days after inoculation. Conclusions These findings suggest that fascin overexpression plays a role in tumor growth and progression in ESCC and that cell death caused by its downregulation might be induced by cell adhesion loss. This indicates that targeting fascin pathway could be a novel therapeutic strategy for the human ESCC.

  7. Multiple H1-antihistamine-induced urticaria.

    Science.gov (United States)

    Inomata, Naoko; Tatewaki, Satoko; Ikezawa, Zenro

    2009-04-01

    H(1)-antihistamines are widely used in the treatment of various allergic diseases. Particularly, a cornerstone of the management of chronic idiopathic urticaria is treatment with H(1)-antihistamines. However, a few cases of H(1)-antihistamine-induced urticaria have been reported. A 34-year-old woman presented with a 4-month history of recurrent urticaria, which was prominently exacerbated by the administration of H(1)-antihistamines. The patient consented to a provocation test of fexofenadine among drugs including cetirizine and hydroxyzine, which were suspected of inducing severe symptoms in episodes. One hour after challenge with 12 mg fexofenadine (one-fifth of the therapeutic dose), a urticarial reaction rapidly developed on nearly the entire body with remarkably increased levels of plasma histamine (190 nmol/L) and plasma leukotriene B4 (150 pg/mL). In challenge tests with other antihistamines, generalized urticaria occurred 5 and 1 h after intake of 10 mg loratadine and 10 mg bepotastine, respectively, whereas challenges with chlorpheniramine, mequitazine and azelastine were all negative. Skin prick tests with H(1)-antihistamines used in the challenges were all negative, indicating that the urticarial reactions after challenges with the causative drugs might not be immunoglobulin E-mediated. Among the causative drugs in our case, cetirizine and hydroxyzine are the piperazine derivatives, whereas fexofenadine, bepotastine, ebastine and loratadine are the piperidine derivatives. The chemical structures of both derivatives are very similar. Therefore, in this case, H(1)-antihistamine-induced urticaria may have been due to cross-reactivity between metabolites of these drugs, but not to drugs before metabolization. Hypersensitivity to H(1)-antihistamines should be considered when urticarial lesions worsen after H(1)-antihistamine treatment.

  8. Differential carbohydrate binding and cell surface glycosylation of human cancer cell lines.

    Science.gov (United States)

    Arndt, Nadia X; Tiralongo, Joe; Madge, Paul D; von Itzstein, Mark; Day, Christopher J

    2011-09-01

    Currently there is only a modest level knowledge of the glycosylation status of immortalised cell lines that are commonly used in cancer biology as well as their binding affinities to different glycan structures. Through use of glycan and lectin microarray technology, this study has endeavoured to define the different bindings of cell surface carbohydrate structures to glycan-binding lectins. The screening of breast cancer MDA-MB435 cells, cervical cancer HeLa cells and colon cancer Caco-2, HCT116 and HCT116-FM6 cells was conducted to determine their differential bindings to a variety of glycan and lectin structures printed on the array slides. An inverse relationship between the number of glycan structures recognised and the variety of cell surface glycosylation was observed. Of the cell lines tested, it was found that four bound to sialylated structures in initial screening. Secondary screening in the presence of a neuraminidase inhibitor (4-deoxy-4-guanidino-Neu5Ac2en) significantly reduced sialic acid binding. The array technology has proven to be useful in determining the glycosylation signatures of various cell-lines as well as their glycan binding preferences. The findings of this study provide the groundwork for further investigation into the numerous glycan-lectin interactions that are exhibited by immortalised cell lines.

  9. Specific binding of benzodiazepines to human breast cancer cell lines.

    Science.gov (United States)

    Beinlich, A; Strohmeier, R; Kaufmann, M; Kuhl, H

    1999-01-01

    Binding of [3H]Ro5-4864, a peripheral benzodiazepine receptor (PBR) agonist, to BT-20 human, estrogen- (ER) and progesterone- (PR) receptor negative breast cancer cells was characterized. It was found to be specific, dose-dependent and saturable with a single population of binding sites. Dissociation constant (K(D)) was 8.5 nM, maximal binding capacity (Bmax) 339 fM/10(6) cells. Ro5-4864 (IC50 17.3 nM) and PK 11195 (IC50 12.3 nM) were able to compete with [3H]Ro5-4864 for binding, indicating specificity of interaction with PBR. Diazepam was able to displace [3H]Ro5-4864 from binding only at high concentrations (>1 microM), while ODN did not compete for PBR binding. Thymidine-uptake assay showed a biphasic response of cell proliferation. While low concentrations (100 nM) of Ro5-4864, PK 11195 and diazepam increased cell growth by 10 to 20%, higher concentrations (10-100 microM) significantly inhibited cell proliferation. PK 11195, a potent PBR ligand, was able to attenuate growth of BT-20 cells stimulated by 100 nM Ro5-4864 and to reverse growth reduction caused by 1 and 10 microM Ro5-4864, but not by 50 microM and 100 microM. This indicates that the antimitotic activity of higher concentrations of Ro5-4864 is independent of PBR binding. It is suggested, that PBR are involved in growth regulation of certain human breast cancer cell lines, possibly by supplying proliferating cells with energy, as their endogenous ligand is a polypeptide transporting Acyl-CoA.

  10. Development of buffalo (Bubalus bubalis embryonic stem cell lines from somatic cell nuclear transferred blastocysts

    Directory of Open Access Journals (Sweden)

    Syed Mohmad Shah

    2015-11-01

    Full Text Available We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

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  18. File list: Unc.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: Pol.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: Unc.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: His.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: Unc.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: His.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: DNS.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: Pol.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 RNA polymerase Blood Lymphoblastoid cell line... SRX306575,SRX306580,SRX306576,SRX306578 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  6. File list: DNS.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: Unc.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: Pol.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: DNS.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: ALL.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: ALL.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: Oth.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: ALL.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: Oth.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 TFs and others Blood Lymphoblastoid cell line...ciencedbc.jp/kyushu-u/hg19/assembled/Oth.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

  15. Oxidative stress induces hypomethylation of LINE-1 and hypermethylation of the RUNX3 promoter in a bladder cancer cell line.

    Science.gov (United States)

    Wongpaiboonwattana, Wikrom; Tosukhowong, Piyaratana; Dissayabutra, Thasinas; Mutirangura, Apiwat; Boonla, Chanchai

    2013-01-01

    Increased oxidative stress and changes in DNA methylation are frequently detected in bladder cancer patients. We previously demonstrated a relationship between increased oxidative stress and hypomethylation of the transposable long-interspersed nuclear element-1 (LINE-1). Promoter hypermethylation of a tumor suppressor gene, runt-related transcription factor 3 (RUNX3), may also be associated with bladder cancer genesis. In this study, we investigated changes of DNA methylation in LINE-1 and RUNX3 promoter in a bladder cancer cell (UM-UC-3) under oxidative stress conditions, stimulated by challenge with H2O2 for 72 h. Cells were pretreated with an antioxidant, tocopheryl acetate for 1 h to attenuate oxidative stress. Methylation levels of LINE-1 and RUNX3 promoter were measured by combined bisulfite restriction analysis PCR and methylation-specific PCR, respectively. Levels of LINE-1 methylation were significantly decreased in H2O2-treated cells, and reestablished after pretreated with tocopheryl acetate. Methylation of RUNX3 promoter was significantly increased in cells exposed to H2O2. In tocopheryl acetate pretreated cells, it was markedly decreased. In conclusion, hypomethylation of LINE-1 and hypermethylation of RUNX3 promoter in bladder cancer cell line was experimentally induced by reactive oxygen species (ROS). The present findings support the hypothesis that oxidative stress promotes urothelial cell carcinogenesis through modulation of DNA methylation. Our data also imply that mechanistic pathways of ROS-induced alteration of DNA methylation in a repetitive DNA element and a gene promoter might differ.

  16. Analysis of G-banding in tumor cell lines derived from human neural stem cells

    Institute of Scientific and Technical Information of China (English)

    Junhua Zou; Yanhui Li

    2006-01-01

    BACKGROUND: The application of neural stem cell (NSC) is restricted because of its tumorigenesis, and the possible pathogenesis needs investigation.OBJECTIVE: To compare the differences of chromosomal G-banding between human NSCs (hNSCs) derived tumor cell line and hNSCs derived normal cell lines.DESIGN: A randomized controlled observation.SETTING: Building of Anatomy, Peking University Health Science Center.MATERIALS: The hNSC lines and hNSC-derived tumor cell lines were provided by the Research Center of Stem Cells, Peking University; DMEM/F12 (1:1) medium, N2 additive, B27 additive epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were produced by GIBCO BRL Company (USA); fetal bovine serum by HYCLONE Company (USA).METHODS: The experiments were carried out in the Department of Genetics, Peking University Health Science Center from February 2003 to July 2004. Human fetal striatal NSCs were inoculated hypodermically on the right scapular of nude mice; Normal human fetal striatal NSCs were cultured to 5-8 passages as controls. Karyotyping was performed on the 5th passage of hNSC-derived tumor cells at 6 weeks after hN-SC transplantation into nude mice (T1) and tumor cells at 15 weeks after transplantation (T2). Metaphase chromosomes were examined with microscope, G-banding cytogenetic analysis and karyotyping were performed according to the Cytoscan Karyotyping FISH and CGH software system (United biotechnology USA Corporation).MAIN OUTCOME MEASURES: G-banded analytical results of human fetal striatal nerve stem cells derived tumor cell lines (T1 and T2) of metaphase chromosomes were observed.RESULTS: ① Chromosome analysis of hNSC-derived tumor cell lines 1 (T1): Twenty-five well-spread metaphases were randomly selected for analysis. The karyotypes were 64, XX (8, 32%); 65, XX (1, 4%); 67,XX (5, 20%); 68, XX (11, 44%). The modal number of chromosomes in this cell lines was 68, which were all hypotriploid. The analysis of 8 G

  17. The PB1 segment of an influenza A virus H1N1 2009pdm isolate enhances the replication efficiency of specific influenza vaccine strains in cell culture and embryonated eggs.

    Science.gov (United States)

    Mostafa, Ahmed; Kanrai, Pumaree; Ziebuhr, John; Pleschka, Stephan

    2016-03-01

    Influenza vaccine strains (IVSs) contain the haemagglutinin (HA) and neuraminidase (NA) genome segments of relevant circulating strains in the genetic background of influenza A/PR/8/1934 virus (PR8). Previous work has shown that the nature of the PB1 segment may be a limiting factor for the efficient production of IVSs. Here, we showed that the PB1 segment (PB1Gi) from the 2009 pandemic influenza A virus (IAV) A/Giessen/06/2009 (Gi wt, H1N1pdm) may help to resolve (some of) these limitations. We produced a set of recombinant PR8-derived viruses that contained (i) the HA and NA segments from representative IAV strains (H3N2, H5N1, H7N9, H9N2); (ii) the PB1 segment from PR8 or Gi wt, respectively; and (iii) the remaining five genome segments from PR8. Viruses containing the PB1Gi segment, together with the heterologous HA/NA segments and five PR8 segments (5+2+1), replicated to higher titres compared with their 6+2 counterparts containing six PR8 segments and the equivalent heterologous HA/NA segments. Compared with PB1PR8-containing IVSs, viruses with the PB1Gi segment replicated to higher or similar titres in both cell culture and embryonated eggs, most profoundly IVSs of the H5N1 and H7N9 subtype, which are known to grow poorly in these systems. IVSs containing either the PB1Gi or the cognate PB1 segment of the respective specific HA/NA donor strain showed enhanced or similar virus replication levels. This study suggests that substitution of PB1PR8 with the PB1Gi segment may greatly improve the large-scale production of PR8-derived IVSs, especially of those known to replicate poorly in vitro.

  18. Identification of genes associated with cisplatin resistance in human oral squamous cell carcinoma cell line

    Directory of Open Access Journals (Sweden)

    Zhang Ping

    2006-09-01

    Full Text Available Abstract Background Cisplatin is widely used for chemotherapy of head and neck squamous cell carcinoma. However, details of the molecular mechanism responsible for cisplatin resistance are still unclear. The aim of this study was to identify the expression of genes related to cisplatin resistance in oral squamous cell carcinoma cells. Methods A cisplatin-resistant cell line, Tca/cisplatin, was established from a cisplatin-sensitive cell line, Tca8113, which was derived from moderately-differentiated tongue squamous cell carcinoma. Global gene expression in this resistant cell line and its sensitive parent cell line was analyzed using Affymetrix HG-U95Av2 microarrays. Candidate genes involved in DNA repair, the MAP pathway and cell cycle regulation were chosen to validate the microarray analysis results. Cell cycle distribution and apoptosis following cisplatin exposure were also investigated. Results Cisplatin resistance in Tca/cisplatin cells was stable for two years in cisplatin-free culture medium. The IC50 for cisplatin in Tca/cisplatin was 6.5-fold higher than that in Tca8113. Microarray analysis identified 38 genes that were up-regulated and 25 that were down-regulated in this cell line. Some were novel candidates, while others are involved in well-characterized mechanisms that could be relevant to cisplatin resistance, such as RECQL for DNA repair and MAP2K6 in the MAP pathway; all the genes were further validated by Real-time PCR. The cell cycle-regulated genes CCND1 and CCND3 were involved in cisplatin resistance; 24-hour exposure to 10 μM cisplatin induced a marked S phase block in Tca/cisplatin cells but not in Tca8113 cells. Conclusion The Tca8113 cell line and its stable drug-resistant variant Tca/cisplatin provided a useful model for identifying candidate genes responsible for the mechanism of cisplatin resistance in oral squamous cell carcinoma. Our data provide a useful basis for screening candidate targets for early diagnosis

  19. A vertically integrated dynamic RAM-cell: Buried bit line memory cell with floating transfer layer

    NARCIS (Netherlands)

    Mouthaan, Ton; Vertregt, Maarten

    1986-01-01

    A charge injection device has been realized in which charge can be injected on to an MOS-capacitor from a buried layer via an isolated transfer layer. The cell is positioned vertically between word and bit line. LOCOS (local oxidation) is used to isolate the cells and (deep) ion implantation to real

  20. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines.

    Science.gov (United States)

    Qin, J-Z; Xin, H; Nickoloff, B J

    2010-05-28

    Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.

  1. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Qin, J.-Z.; Xin, H. [Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University of Chicago Medical Center (United States); Nickoloff, B.J., E-mail: bnickol@lumc.edu [Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University of Chicago Medical Center (United States)

    2010-05-28

    Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.

  2. Characterisation and Manipulation of Docetaxel Resistant Prostate Cancer Cell Lines

    LENUS (Irish Health Repository)

    O'Neill, Amanda J

    2011-10-07

    Abstract Background There is no effective treatment strategy for advanced castration-resistant prostate cancer. Although Docetaxel (Taxotere®) represents the most active chemotherapeutic agent it only gives a modest survival advantage with most patients eventually progressing because of inherent or acquired drug resistance. The aims of this study were to further investigate the mechanisms of resistance to Docetaxel. Three Docetaxel resistant sub-lines were generated and confirmed to be resistant to the apoptotic and anti-proliferative effects of increasing concentrations of Docetaxel. Results The resistant DU-145 R and 22RV1 R had expression of P-glycoprotein and its inhibition with Elacridar partially and totally reversed the resistant phenotype in the two cell lines respectively, which was not seen in the PC-3 resistant sublines. Resistance was also not mediated in the PC-3 cells by cellular senescence or autophagy but multiple changes in pro- and anti-apoptotic genes and proteins were demonstrated. Even though there were lower basal levels of NF-κB activity in the PC-3 D12 cells compared to the Parental PC-3, docetaxel induced higher NF-κB activity and IκB phosphorylation at 3 and 6 hours with only minor changes in the DU-145 cells. Inhibition of NF-κB with the BAY 11-7082 inhibitor reversed the resistance to Docetaxel. Conclusion This study confirms that multiple mechanisms contribute to Docetaxel resistance and the central transcription factor NF-κB plays an immensely important role in determining docetaxel-resistance which may represent an appropriate therapeutic target.

  3. Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV.

    Science.gov (United States)

    Ustün-Aytekin, Ozlem; Gürhan, Ismet Deliloğlu; Ohura, Kayoko; Imai, Teruko; Ongen, Gaye

    2014-01-01

    Human dipeptidylpeptidase IV (hDPPIV) is an enzyme that is in hydrolase class and has various roles in different parts of human body. Its deficiency may cause some disorders in the gastrointestinal, neurologic, endocrinological and immunological systems of humans. In the present study, hDPPIV enzyme was expressed on Spodoptera frugiperda (Sf9) cell lines as a host cell, and the expression of hDPPIV was obtained by a baculoviral expression system. The enzyme production, optimum multiplicity of infection, optimum transfection time, infected and uninfected cell size and cell behavior during transfection were also determined. For maximum hDPPIV (269 mU mL(-1)) enzyme, optimum multiplicity of infection (MOI) and time were 0.1 and 72 h, respectively. The size of infected cells increased significantly (P Sf9 cell line was applicable to the large scale for hDPPIV expression by using optimized parameters (infection time and MOI) because of its high productivity (4.03 mU m L(-1) h(-1)).

  4. Isolation and Enrichment of Mouse Female Germ Line Stem Cells

    Directory of Open Access Journals (Sweden)

    Somayeh Khosravi-Farsani

    2015-01-01

    Full Text Available Objective: The existence of female germ-line stem cells (FGSCs has been the subject of a wide range of recent studies. Successful isolation and culture of FGSCs could facilitate studies on regenerative medicine and infertility treatments in the near future. Our aim in the present study was evaluation of the most commonly used techniques in enrichment of FGSCs and in establishment of the best procedure. Materials and Methods: In this experimental study, after digesting neonate ovary from C57Bl/6 mice, we performed 2 different isolation experiments: magnetic activated cell sorting (MACS and pre-plating. MACS was applied using two different antibodies against mouse vasa homolog (MVH and stage-specific embryonic antigen-1 (SSEA1 markers. After the cells were passaged and proliferated in vitro, colony-forming cells were characterized using reverse transcription-polymerase chain reaction (RT-PCR (for analysis of expression of Oct4, Nanog, C-kit, Fragilis, Mvh, Dazl, Scp3 and Zp3, alkaline phosphatase (AP activity test and immunocytochemistry. Results: Data showed that colonies can be seen more frequently in pre-plating technique than that in MACS. Using the SSEA1 antibody with MACS, 1.98 ± 0.49% (Mean ± SDV positive cells were yield as compared to the total cells sorted. The colonies formed after pre-plating expressed pluripotency and germ stem cell markers (Oct4, Nanog, C-kit, Fragilis, Mvh and Dazl whereas did not express Zp3 and Scp3 at the mRNA level. Immunocytochemistry in these colonies further confirmed the presence of OCT4 and MVH proteins, and AP activity measured by AP-kit showed positive reaction. Conclusion: We established a simple and an efficient pre-plating technique to culture and to enrich FGSCs from neonatal mouse ovaries.

  5. In vitro reassortment between endemic H1N2 and 2009 H1N1 pandemic swine influenza viruses generates attenuated viruses.

    Directory of Open Access Journals (Sweden)

    Ben M Hause

    Full Text Available The pandemic H1N1 (pH1N1 influenza virus was first reported in humans in the spring of 2009 and soon thereafter was identified in numerous species, including swine. Reassortant viruses, presumably arising from the co-infection of pH1N1 and endemic swine influenza virus (SIV, were subsequently identified from diagnostic samples collected from swine. In this study, co-infection of swine testicle (ST cells with swine-derived endemic H1N2 (MN745 and pH1N1 (MN432 yielded two reassortant H1N2 viruses (R1 and R2, both possessing a matrix gene derived from pH1N1. In ST cells, the reassortant viruses had growth kinetics similar to the parental H1N2 virus and reached titers approximately 2 log(10 TCID(50/mL higher than the pH1N1 virus, while in A549 cells these viruses had similar growth kinetics. Intranasal challenge of pigs with H1N2, pH1N1, R1 or R2 found that all viruses were capable of infecting and transmitting between direct contact pigs as measured by real time reverse transcription PCR of nasal swabs. Lung samples were also PCR-positive for all challenge groups and influenza-associated microscopic lesions were detected by histology. Interestingly, infectious virus was detected in lung samples for pigs challenged with the parental H1N2 and pH1N1 at levels significantly higher than either reassortant virus despite similar levels of viral RNA. Results of our experiment suggested that the reassortant viruses generated through in vitro cell culture system were attenuated without gaining any selective growth advantage in pigs over the parental lineages. Thus, reassortant influenza viruses described in this study may provide a good system to study genetic basis of the attenuation and its mechanism.

  6. Characterization of cancer stem-like cells in the side population cells of human gastric cancer cell line MKN-45

    Institute of Scientific and Technical Information of China (English)

    Hai-hong ZHANG; Ai-zhen CAI; Xue-ming WEI; Li DING; Feng-zhi LI; Ai-ming ZHENG; Da-jiang DAI

    2013-01-01

    Objective:Side population (SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer.Many kinds of cell lines and tissues have demonstrated the presence of SP cells,including several gastric cancer cell lines.This study is aimed to identify the cancer stem-like cells in the SP of gastric cancer cell line MKN-45.Methods:We used fluorescence activated cell sorting (FACS) to sort SP cells in the human gastric carcinoma cell line MKN-45 (cells labeled with Hoechst 33342) and then characterized the cancer stem-like properties of SP cells.Results:This study found that the SP cells had higher clone formation efficiency than major population (MP) cells.Five stemness-related gene expression profiles,including OCT-4,SOX-2,NANOG,CD44,and adenosine triphosphate (ATP)-binding cassette transporters gene ABCG2,were tested in SP and MP cells using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR).Western blot was used to show the difference of protein expression between SP and MP cells.Both results show that there was significantly higher protein expression in SP cells than in MP cells.When inoculated into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice,SP cells show higher tumorigenesis tendency than MP cells.Conclusions:These results indicate that SP cells possess cancer stem cell properties and prove that SP cells from MKN-45 are gastric cancer stem-like cells.

  7. Inhibitory effects of xanthohumol from hops (Humulus lupulus L.) on human hepatocellular carcinoma cell lines.

    Science.gov (United States)

    Ho, Yi-Chien; Liu, Chi-Hsien; Chen, Chien-Nan; Duan, Kow-Jen; Lin, Ming-Tse

    2008-11-01

    Xanthohumol is one of the main flavonoids in hop extracts and in beer. Very few investigations of xanthohumol have studied hepatocellular carcinoma. In this study, the inhibitory effects of xanthohumol on human hepatocellular carcinoma cell lines were investigated. The IC(50) values of xanthohumol for two hepatocellular carcinoma cell lines and one normal hepatocyte cell line were 108, 166 and 211 microm, respectively. Normal murine hepatocyte cell line had more resistance to xanthohumol than hepatocellular carcinoma cell lines. Besides, the inhibitory effects of xanthohumol on human hepatocellular carcinoma cell lines were attributed to apoptosis as indicated in the results of flow cytometry, fluorescent nuclear staining and electrophoresis of oligonucleosomal DNA fragments. Hop xanthohumol was more efficient in the growth inhibition of hepatocellular carcinoma cell lines than the flavonoids silibinin and naringin from thistle and citrus. It was shown for the first time that xanthohumol from hops effectively inhibits proliferation of human hepatocellular carcinoma cells in vitro.

  8. Origin of the U87MG glioma cell line: Good news and bad news.

    Science.gov (United States)

    Allen, Marie; Bjerke, Mia; Edlund, Hanna; Nelander, Sven; Westermark, Bengt

    2016-08-31

    Human tumor-derived cell lines are indispensable tools for basic and translational oncology. They have an infinite life span and are easy to handle and scalable, and results can be obtained with high reproducibility. However, a tumor-derived cell line may not be authentic to the tumor of origin. Two major questions emerge: Have the identity of the donor and the actual tumor origin of the cell line been accurately determined? To what extent does the cell line reflect the phenotype of the tumor type of origin? The importance of these questions is greatest in translational research. We have examined these questions using genetic profiling and transcriptome analysis in human glioma cell lines. We find that the DNA profile of the widely used glioma cell line U87MG is different from that of the original cells and that it is likely to be a bona fide human glioblastoma cell line of unknown origin.

  9. Growth inhibitory activity of Ankaferd hemostat on primary melanoma cells and cell lines

    Science.gov (United States)

    Turk, Seyhan; Malkan, Umit Yavuz; Ghasemi, Mehdi; Hocaoglu, Helin; Mutlu, Duygu; Gunes, Gursel; Aksu, Salih; Haznedaroglu, Ibrahim Celalettin

    2017-01-01

    Objective: Ankaferd hemostat is the first topical hemostatic agent about the red blood cell–fibrinogen relations tested in the clinical trials. Ankaferd hemostat consists of standardized plant extracts including Alpinia officinarum, Glycyrrhiza glabra, Thymus vulgaris, Urtica dioica, and Vitis vinifera. The aim of this study was to determine the effect of Ankaferd hemostat on viability of melanoma cell lines. Methods: Dissimilar melanoma cell lines and primary cells were used in this study. These cells were treated with different concentrations of Ankaferd hemostat to assess the impact of different dosages of the drug. All cells treated with different concentrations were incubated for different time intervals. After the data had been obtained, one-tailed T-test was used to determine whether the Ankaferd hemostat would have any significant inhibitory impact on cell growth. Results: We demonstrated in this study that cells treated with Ankaferd hemostat showed a significant decrease in cell viability compared to control groups. The cells showed different resistances against Ankaferd hemostat which depended on the dosage applied and the time treated cells had been incubated. We also demonstrated an inverse relationship between the concentration of the drug and the incubation time on one hand and the viability of the cells on the other hand, that is, increasing the concentration of the drug and the incubation time had a negative impact on cell viability. Conclusion: The findings in our study contribute to our knowledge about the anticancer impact of Ankaferd hemostat on different melanoma cells. PMID:28293423

  10. Neuroblastoma cell lines contain pluripotent tumor initiating cells that are susceptible to a targeted oncolytic virus.

    Directory of Open Access Journals (Sweden)

    Yonatan Y Mahller

    Full Text Available BACKGROUND: Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers. METHODOLOGY/PRINCIPAL FINDINGS: Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice. CONCLUSIONS/SIGNIFICANCE: These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.

  11. Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M;

    1992-01-01

    Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data...... demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression...... of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell...

  12. Cell Motility and Invasiveness of Neurofibromin-Deficient Neural Crest Cells and Malignant Triton Tumor Lines

    Science.gov (United States)

    2005-06-01

    immunoblotting techniques to characterize signaling pathways activated by TGF-beta and PDGF-BB in MPNST -like sarcoma cell lines isolated from cisNfl+/-;p53...mouse model to include characterizations of genomic instability in the context of malignant transformation, and to test possible modifiers of MPNST ...growth and invasiveness. 15. SUBJECT TERMS neurofibromatosis type 1; neural crest cells; cell motility and Migration; PDGF; TGF-beta; MPNST

  13. Cuba vs H1N1 Influenza

    Directory of Open Access Journals (Sweden)

    Gail Reed

    2011-04-01

    Full Text Available El Comité Editorial de MediSur agradece a Gail Reed, editora de Medicc Review la autorización expresa, para reproducir el artículo titulado “Cuba vs H1N1 Influenza”. Este trabajo resume el esfuerzo realizado por todos los organismos en Cuba y en especial el Ministerio de Salud Pública en la lucha para disminuir los efectos de la influenza H1N1 en la población. El artículo original se puede encontrar en: Reed G. Faceoff: Cuba vs H1N1 Influenza. MEDICC Review. 2010; 12(1:6-12. Disponible en: http://www.medicc.org/mediccreview/index.php?issue=11

  14. Gene expression analysis of cell death induction by Taurolidine in different malignant cell lines

    Directory of Open Access Journals (Sweden)

    Weyhe Dirk

    2010-10-01

    Full Text Available Abstract Background The anti-infective agent Taurolidine (TRD has been shown to have cell death inducing properties, but the mechanism of its action is largely unknown. The aim of this study was to identify potential common target genes modulated at the transcriptional level following TRD treatment in tumour cell lines originating from different cancer types. Methods Five different malignant cell lines (HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3 were incubated with TRD (100 μM, 250 μM and 1000 μM. Proliferation after 8 h and cell viability after 24 h were analyzed by BrdU assay and FACS analysis, respectively. Gene expression analyses were carried out using the Agilent -microarray platform to indentify genes which displayed conjoint regulation following the addition of TRD in all cell lines. Candidate genes were subjected to Ingenuity Pathways Analysis and selected genes were validated by qRT-PCR and Western Blot. Results TRD 250 μM caused a significant inhibition of proliferation as well as apoptotic cell death in all cell lines. Among cell death associated genes with the strongest regulation in gene expression, we identified pro-apoptotic transcription factors (EGR1, ATF3 as well as genes involved in the ER stress response (PPP1R15A, in ubiquitination (TRAF6 and mitochondrial apoptotic pathways (PMAIP1. Conclusions This is the first conjoint analysis of potential target genes of TRD which was performed simultaneously in different malignant cell lines. The results indicate that TRD might be involved in different signal transduction pathways leading to apoptosis.

  15. In vitro Acute Cytotoxicity of Abamectin to the Gill Cell Line of Flounder Paralichthy olivaceus

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The cytotoxicity of abamectin to the Gill Cell Line of Flounder (FG cell line) was examined in this study. It was found that the exposure of FG cells to abamectin caused the decreases of both cell growth rate and antioxidant enzyme activities, and the increase of intracellular O2- content. It was proposed that the reduction of antioxidant enzyme activities in FG cells caused the accumulation of O2- content in FG cells, leading to the change of cell morphology and even the death of cells. The results showed that FG cell line is suitable for the evaluation of the acute toxicity of abamectin.

  16. Tooth regeneration from newly established cell lines from a molar tooth germ epithelium.

    Science.gov (United States)

    Komine, Akihiko; Suenaga, Momoko; Nakao, Kazuhisa; Tsuji, Takashi; Tomooka, Yasuhiro

    2007-04-13

    In order to investigate tooth development, several cell lines of the dental epithelium and ectomesenchyme have been established. However, no attempt has been reported to regenerate teeth with cell lines. Here, we have established several clonal cell lines of the dental epithelium from a p53-deficient fetal mouse. They expressed specific markers of the dental epithelium such as ameloblastin and amelogenin. A new method has been developed to bioengineer tooth germs with dental epithelial and mesenchymal cells. Reconstructed tooth germs with cell lines and fetal mesenchymal cells were implanted under kidney capsule. The germs regenerated teeth with well-calcified structures as seen in natural tooth. Germs without the cell lines developed bone. This is the first success to regenerate teeth with dental epithelial cell lines. They are useful models in vitro for investigation of mechanisms in morphogenesis and of cell lineage in differentiation, and for clinical application for tooth regeneration.

  17. Electrophysiological Characteristics of Embryonic Stem Cell-Derived Cardiomyocytes are Cell Line-Dependent

    Directory of Open Access Journals (Sweden)

    Tobias Hannes

    2015-01-01

    Full Text Available Background: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs of different murine ESC lines. Methods: Two wild-type (D3 and R1 and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7 were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC promoter. Action potentials (APs were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Results: Spontaneous AP frequency and AP duration (APD as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Conclusion: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.

  18. Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line

    Directory of Open Access Journals (Sweden)

    Behra Martine

    2012-01-01

    Full Text Available Abstract Background Because of the structural and molecular similarities between the two systems, the lateral line, a fish and amphibian specific sensory organ, has been widely used in zebrafish as a model to study the development/biology of neuroepithelia of the inner ear. Both organs have hair cells, which are the mechanoreceptor cells, and supporting cells providing other functions to the epithelium. In most vertebrates (excluding mammals, supporting cells comprise a pool of progenitors that replace damaged or dead hair cells. However, the lack of regenerative capacity in mammals is the single leading cause for acquired hearing disorders in humans. Results In an effort to understand the regenerative process of hair cells in fish, we characterized and cloned an egfp transgenic stable fish line that trapped tnks1bp1, a highly conserved gene that has been implicated in the maintenance of telomeres' length. We then used this Tg(tnks1bp1:EGFP line in a FACsorting strategy combined with microarrays to identify new molecular markers for supporting cells. Conclusions We present a Tg(tnks1bp1:EGFP stable transgenic line, which we used to establish a transcriptional profile of supporting cells in the zebrafish lateral line. Therefore we are providing a new set of markers specific for supporting cells as well as candidates for functional analysis of this important cell type. This will prove to be a valuable tool for the study of regeneration in the lateral line of zebrafish in particular and for regeneration of neuroepithelia in general.

  19. Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation

    Institute of Scientific and Technical Information of China (English)

    Jie Du; Xiaoqing Gao; Li Deng; Nengbin Chang; Huailin Xiong; Yu Zheng

    2014-01-01

    Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro-tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su-pernatant were signiifcantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes-enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen-chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.

  20. In vitro platinum drug chemosensitivity of human cervical squamous cell carcinoma cell lines with intrinsic and acquired resistance to cisplatin.

    OpenAIRE

    Mellish, K. J.; Kelland, L R; Harrap, K. R.

    1993-01-01

    The platinum drug chemosensitivity of five human cervical squamous cell carcinoma cell lines (HX/151, HX/155, HX/156, HX/160 and HX/171) derived from previously untreated patients has been determined. Compared to our data obtained previously using human ovarian carcinoma cell lines, all five lines were relatively resistant to cisplatin, carboplatin, iproplatin and tetraplatin. One of the lines (HX/156) was exceptionally sensitive to the novel platinum (IV) ammine/amine dicarboxylates JM216 [b...

  1. Expression of tumor antigens on primary ovarian cancer cells compared to established ovarian cancer cell lines

    Science.gov (United States)

    Kloudová, Kamila; Hromádková, Hana; Partlová, Simona; Brtnický, Tomáš; Rob, Lukáš; Bartůňková, Jiřina; Hensler, Michal; Halaška, Michael J.; Špíšek, Radek; Fialová, Anna

    2016-01-01

    In order to select a suitable combination of cancer cell lines as an appropriate source of antigens for dendritic cell-based immunotherapy of ovarian cancer, we analyzed the expression level of 21 tumor associated antigens (BIRC5, CA125, CEA, DDX43, EPCAM, FOLR1, Her-2/neu, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MUC-1, NY-ESO-1, PRAME, p53, TPBG, TRT, WT1) in 4 established ovarian cancer cell lines and in primary tumor cells isolated from the high-grade serous epithelial ovarian cancer tissue. More than 90% of tumor samples expressed very high levels of CA125, FOLR1, EPCAM and MUC-1 and elevated levels of Her-2/neu, similarly to OVCAR-3 cell line. The combination of OV-90 and OVCAR-3 cell lines showed the highest overlap with patients' samples in the TAA expression profile. PMID:27323861

  2. Development of improved vaccine cell lines against rotavirus

    Science.gov (United States)

    Wu, Weilin; Orr-Burks, Nichole; Karpilow, Jon; Tripp, Ralph A.

    2017-01-01

    Rotavirus is a major cause of severe gastroenteritis among very young children. In developing countries, rotavirus is the major cause of mortality in children under five years old, causing up to 20% of all childhood deaths in countries with high diarrheal disease burden, with more than 90% of these deaths occurring in Africa and Asia. Rotavirus vaccination mimics the first infection without causing illness, thus inducing strong and broad heterotypic immunity against prospective rotavirus infections. Two live vaccines are available, Rotarix and RotaTeq, but vaccination efforts are hampered by high production costs. Here, we present a dataset containing a genome-wide RNA interference (RNAi) screen that identified silencing events that enhanced rotavirus replication. Evaluated against several rotavirus vaccine strains, hits were validated in a Vero vaccine cell line as well as CRISPR/Cas9 generated cells permanently and stably lacking the genes that affect RV replication. Knockout cells were dramatically more permissive to RV replication and permitted an increase in rotavirus replication. These data show a means to improve manufacturing of rotavirus vaccine. PMID:28248921

  3. Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Williams, K.; Chubb, C.; Huberman, E.; Giometti, C.S.

    1997-07-01

    High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.

  4. Comprehensive characterization of genomic instability in pluripotent stem cells and their derived neuroprogenitor cell lines

    Directory of Open Access Journals (Sweden)

    Nestor Luis Lopez Corrales

    2012-12-01

    Full Text Available The genomic integrity of two human pluripotent stem cells and their derived neuroprogenitor cell lines was studied, applying a combination of high-resolution genetic methodologies. The usefulness of combining array-comparative genomic hybridization (aCGH and multiplex fluorescence in situ hybridization (M-FISH techniques should be delineated to exclude/detect a maximum of possible genomic structural aberrations. Interestingly, in parts different genomic imbalances at chromosomal and subchromosomal levels were detected in pluripotent stem cells and their derivatives. Some of the copy number variations were inherited from the original cell line, whereas other modifications were presumably acquired during the differentiation and manipulation procedures. These results underline the necessity to study both pluripotent stem cells and their differentiated progeny by as many approaches as possible in order to assess their genomic stability before using them in clinical therapies.

  5. Lipid analysis of eight human breast cancer cell lines with ToF-SIMS.

    Science.gov (United States)

    Robinson, Michael A; Graham, Daniel J; Morrish, Fionnuala; Hockenbery, David; Gamble, Lara J

    2015-06-28

    In this work, four triple negative (TN) cell lines, three ER+ and PR+ receptor positive (RP) cell lines, and one ER+, PR+, and HER2+ cell line were chemically distinguished from one another using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and principal component analysis (PCA). PCA scores separation was observed between the individual cell lines within a given classification (TN and RP) and there were distinctly different trends found in the fatty acid and lipid compositions of the two different classifications. These trends indicated that the RP cell lines separated out based on the carbon chain length of the lipids while the TN cell lines showed separation based on cholesterol-related peaks (in the positive ion data). Both cell types separated out by trends in fatty acid chain length and saturation in the negative ions. These chemical differences may be manifestations of unique metabolic processes within each of the different cell lines. Additionally, the HER2+ cell line was distinguished from three other RP cell types as having a unique distribution of fatty acids including anticorrelation to 18-carbon chain fatty acids. As these cell lines could not be grown in the same growth media, a combination of chemical fixation, rinsing, C60 (+) presputtering, and selection of cellular regions-of-interest is also presented as a successful method to acquire ToF-SIMS data from cell lines grown in different media.

  6. Cell cycle analysis and cytotoxic potential of Ruta graveolens against human tumor cell lines.

    Science.gov (United States)

    Varamini, P; Soltani, M; Ghaderi, A

    2009-01-01

    There are reports on the presence of various compounds exerting different biological activities in Ruta graveolens, a plant of Rutaceae family. The aim of the present study was to evaluate in vitro cytotoxicity of the total extract of R. graveolens against tumor cell lines of different origin. Aerial parts of the plant was extracted with 70% ethanol by sonication method and cytotoxic activity was examined on RAJI, RAMOS, RPMI8866, U937, Jurkat, MDA-MB-453, MCF-7, LNCap-FGC-10, 5637, HeLa, SK-OV-3, A549, Mehr-80 and also peripheral blood mononuclear cells (PBMC) by the use of WST-1 assay. Results were expressed as IC(50) values. R. graveolens extract showed high cytotoxic activity against RAJI and RAMOS, two Burkitt's lymphoma cell lines, with an IC(50) equal to 24.3 microg/ml and 35.2 microg/ml respectively and LNCap-FGC-10, a prostate adenocarcinoma cell line with an IC(50) equal to 27.6 microg/ml as well as Mehr-80, a newly established Large Cell Lung Carcinoma (IC(50)=46.2 microg/ml). No significant anti-proliferative activity was observed on other cell lines including MCF-7, MDA-MB-453, SK-OV-3, HeLa, 5637, JURKAT and RPMI8866. Adverse cytotoxic effect of R. graveolens was investigated against PBMCs and a significantly lower effect of this extract (IC(50)=104 microg/ml) was seen on normal cells compared with RAJI and RAMOS, two haematopoietic cell lines.

  7. Single-cell printing to form three-dimensional lines of olfactory ensheathing cells

    Energy Technology Data Exchange (ETDEWEB)

    Othon, Christina M; Ringeisen, Bradley R [Naval Research Laboratory/Code 6113, 4555 Overlook Ave. SW, Washington, DC 20375 (United States); Wu Xingjia; Anders, Juanita J [Department of Anatomy, Physiology and Genetics, Uniformed Services University of Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814 (United States)], E-mail: ringeisen@nrl.navy.mil

    2008-09-01

    Biological laser printing (BioLP(TM)) is a unique tool capable of printing high resolution two- and three-dimensional patterns of living mammalian cells, with greater than 95% viability. These results have been extended to primary cultured olfactory ensheathing cells (OECs), harvested from adult Sprague-Dawley rats. OECs have been found to provide stimulating environments for neurite outgrowth in spinal cord injury models. BioLP is unique in that small load volumes ({approx}{mu}Ls) are required to achieve printing, enabling low numbers of OECs to be harvested, concentrated and printed. BioLP was used to form several 8 mm lines of OECs throughout a multilayer hydrogel scaffold. The line width was as low as 20 {mu}m, with most lines comprising aligned single cells. Fluorescent confocal microscopy was used to determine the functionality of the printed OECs, to monitor interactions between printed OECs, and to determine the extent of cell migration throughout the 3D scaffold. High-resolution printing of low cell count, harvested OECs is an important advancement for in vitro study of cell interactions and functionality. In addition, these cell-printed scaffolds may provide an alternative for spinal cord repair studies, as the single-cell patterns formed here are on relevant size scales for neurite outgrowth.

  8. Derivation and characterization of matched cell lines from primary and recurrent serous ovarian cancer

    Directory of Open Access Journals (Sweden)

    Létourneau Isabelle J

    2012-08-01

    Full Text Available Abstract Background Cell line models have proven to be effective tools to investigate a variety of ovarian cancer features. Due to the limited number of cell lines, particularly of the serous subtype, the heterogeneity of the disease, and the lack of cell lines that model disease progression, there is a need to further develop cell line resources available for research. This study describes nine cell lines derived from three ovarian cancer cases that were established at initial diagnosis and at subsequent relapse after chemotherapy. Methods The cell lines from three women diagnosed with high-grade serous ovarian cancer (1369, 2295 and 3133 were derived from solid tumor (TOV and ascites (OV, at specific time points at diagnosis and relapse (R. Primary treatment was a combination of paclitaxel/carboplatin (1369, 3133, or cisplatin/topotecan (2295. Second line treatment included doxorubicin, gemcitabine and topotecan. In addition to molecular characterization (p53, HER2, the cell lines were characterized based on cell growth characteristics including spheroid growth, migration potential, and anchorage independence. The in vivo tumorigenicity potential of the cell lines was measured. Response to paclitaxel and carboplatin was assessed using a clonogenic assay. Results All cell lines had either a nonsense or missense TP53 mutations. The ability to form compact spheroids or aggregates was observed in six of nine cell lines. Limited ability for migration and anchorage independence was observed. The OV3133(R cell line, formed tumors at subcutaneous sites in SCID mice. Based on IC50 values and dose response curves, there was clear evidence of acquired resistance to carboplatin for TOV2295(R and OV2295(R2 cell lines. Conclusion The study identified nine new high-grade serous ovarian cancer cell lines, derived before and after chemotherapy that provides a unique resource for investigating the evolution of this common histopathological subtype of ovarian

  9. Effect of Docosahexaenoic Acid on Cell Cycle Pathways in Breast Cell Lines With Different Transformation Degree.

    Science.gov (United States)

    Rescigno, Tania; Capasso, Anna; Tecce, Mario Felice

    2016-06-01

    n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), abundant in fish, have been shown to affect development and progression of some types of cancer, including breast cancer. The aim of our study was to further analyze and clarify the effects of these nutrients on the molecular mechanisms underlying breast cancer. Following treatments with DHA we examined cell viability, death, cell cycle, and some molecular effects in breast cell lines with different transformation, phenotypic, and biochemical characteristics (MCF-10A, MCF-7, SK-BR-3, ZR-75-1). These investigations showed that DHA is able to affect cell viability, proliferation, and cell cycle progression in a different way in each assayed breast cell line. The activation of ERK1/2 and STAT3 pathways and the expression and/or activation of molecules involved in cell cycle regulation such as p21(Waf1/Cip1) and p53, are very differently regulated by DHA treatments in each cell model. DHA selectively: (i) arrests non tumoral MCF-10A breast cells in G0 /G1 cycle phase, activating p21(Waf1/Cip1) , and p53, (ii) induces to death highly transformed breast cells SK-BR-3, reducing ERK1/2 and STAT3 phosphorylation and (iii) only slightly affects each analyzed process in MCF-7 breast cell line with transformation degree lower than SK-BR-3 cells. These findings suggest a more relevant inhibitory role of DHA within early development and late progression of breast cancer cell transformation and a variable effect in the other phases, depending on individual molecular properties and degree of malignancy of each clinical case.

  10. A cell-permeable fluorescent polymeric thermometer for intracellular temperature mapping in mammalian cell lines.

    Directory of Open Access Journals (Sweden)

    Teruyuki Hayashi

    Full Text Available Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT in combination with fluorescence lifetime imaging microscopy (FLIM. Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.

  11. A cell-permeable fluorescent polymeric thermometer for intracellular temperature mapping in mammalian cell lines.

    Science.gov (United States)

    Hayashi, Teruyuki; Fukuda, Nanaho; Uchiyama, Seiichi; Inada, Noriko

    2015-01-01

    Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT) in combination with fluorescence lifetime imaging microscopy (FLIM). Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.

  12. [Neuronal differentiation of human small cell lung cancer cell line PC-6 by Solcoseryl].

    Science.gov (United States)

    Shimizu, T

    1997-11-01

    Solcoseryl is composed of extracts from calf blood, and is a drug known to activate tissue respiration. In the present study, I demonstrated the cell biological effects of Solcoseryl on a human small cell lung cancer cell line, PC-6, by analyzing cell morphology, cell growth, expression of neuronal differentiation markers, and the ras proto-oncogene product(ras p21). Exposure of PC-6 cells to Solcoseryl at the concentration of 200 microliters/ml induced (1) cell morphological changes, including neurodendrite-like projections from the cell surface, and (2) complete inhibition of cell growth, that was shown by the loss of Ki-67 expression. Solcoseryl also induced the expression of neurofilament protein and acetylcholinesterase, both of which are markers of neuronal differentiation. Moreover, it upregulated the expression of the ras proto-oncogene product, ras p21. Taken together, these data suggest that Solcoseryl is composed of component(s) which can induce neuronal differentiation of the human small cell lung cancer cell line, PC-6.

  13. Heterotransplantation of human leukemic B-cell, T-cell and null-cell lines in hamsters.

    Directory of Open Access Journals (Sweden)

    Hiraki,Shunkichi

    1979-02-01

    Full Text Available Human leukemic B-cell (BALL-1, T-cell (TALL-1 and null-cell (NALL-1 lines have been established from three patients with acute lymphoblastic leukemia (ALL. To study the heterotransplantability and in vivo growth characteristics, attempts were made to transplant these ALL cell lines into newborn Syrian hamsters treated with rabbit anti-hamster thymocyte serum. Intraperitoneal implantation of 1.8-3.5 x 10(7 cells gave rise to invasive tumors in all recipients after 15 to 41 days. In addition to a common in vivo feature of mesenteric and retroperitoneal tumors, BALL-1 line was characterized by infiltration of the skin, massive ascites and bone marrow invasion. TALL-1 cells infiltrated various organs including the lymph nodes, liver, gallbladder, spleen, bone marrow, central nervous system and eyes. NALL-1 line grew slowly, producing the least tumors, although there were distant metastases in the lungs. Tumor cells were detected in the blood of 2 of 3 BALL-1-bearing hamsters and in the blood of 4 of 5 TALL-1-bearing hamsters. Thus, these three ALL cell lines were found to exhibit a characteristic biological behavior in hamsters, which might be related to the different cell lineage.

  14. Mixed antibody and T cell responses to peanut and the peanut allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6 in an oral sensitization model

    NARCIS (Netherlands)

    Wijk, F. van; Hartgring, S.; Koppelman, S.J.; Pieters, R.; Knippels, L.M.J.

    2004-01-01

    Background: Peanut allergy is known for its severity and persistence through life. Several peanut proteins have been identified as allergenic and are indicated as Ara h 1-7. Very little is known about the mechanisms that underlie sensitization to peanut proteins. Objective: The purpose of the presen

  15. Antiviral Prophylaxis and H1N1

    Centers for Disease Control (CDC) Podcasts

    2011-07-14

    Dr. Richard Pebody, a consultant epidemiologist at the Health Protection Agency in London, UK, discusses the use of antiviral post-exposure prophylaxis and pandemic H1N1.  Created: 7/14/2011 by National Center for Emerging Zoonotic and Infectious Diseases (NCEZID).   Date Released: 7/18/2011.

  16. Drug treatment of cancer cell lines: a way to select for cancer stem cells?

    Science.gov (United States)

    Chiodi, Ilaria; Belgiovine, Cristina; Donà, Francesca; Scovassi, A Ivana; Mondello, Chiara

    2011-03-04

    Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs.

  17. Drug Treatment of Cancer Cell Lines: A Way to Select for Cancer Stem Cells?

    Directory of Open Access Journals (Sweden)

    Ilaria Chiodi

    2011-03-01

    Full Text Available Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs.

  18. Drug Treatment of Cancer Cell Lines: A Way to Select for Cancer Stem Cells?

    Energy Technology Data Exchange (ETDEWEB)

    Chiodi, Ilaria; Belgiovine, Cristina; Donà, Francesca; Scovassi, A. Ivana; Mondello, Chiara, E-mail: mondello@igm.cnr.it [Institute of Molecular Genetics, CNR, via Abbiategrasso 207, 27100 Pavia (Italy)

    2011-03-04

    Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs.

  19. Photodynamic therapy-induced programmed cell death in carcinoma cell lines

    Science.gov (United States)

    He, Xiao-Yan; Sikes, Robert A.; Thomsen, Sharon L.; Chung, L.; Jacques, Steven L.

    1993-06-01

    The mode of cell death following photodynamic therapy (PDT) was investigated from the perspective of programmed cell death (apoptosis). Human prostate carcinoma cells (PC3), human non-small cell lung carcinoma (H322a), and rat mammary carcinoma (MTF7) were treated by PDT following sensitization with dihematoporphyrin ether (DHE). The response of these carcinoma cell lines to PDT was variable. An examination of extracted cellular DNA by gel electrophoresis showed the characteristic DNA ladder pattern indicative of internucleosomal cleavage of DNA during apoptosis. MTF7 and PC3 responded to PDT by inducing apoptosis while H322a had no apoptotic response. The magnitude of the response and the PDT dosage required to induce the effect were different in PC3 and MTF7. MTF7 cells responded with rapid apoptosis at the dose of light and drug that yielded 50% cell death (LD50). In contrast, PC3 showed only marginal apoptosis at the LD50 but had a marked response at the LD85. Furthermore, the onset of apoptosis followed slower kinetics in PC3 (2 hr - 4 hr) than in MTF7 (cells were killed by PDT but failed to exhibit any apoptotic response. This study indicates that apoptosis may occur during PDT induced cell death, but this pathway is not universal for all cancer cell lines.

  20. Interaction between x-irradiated plateau-phase bone marrow stromal cell lines and co-cultivated factor-dependent cell lines leading to leukemogenesis in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Naparstek, E.; Anklesaria, P.; FitzGerald, T.J.; Sakakeeny, M.A.; Greenberger, J.S.

    1987-03-01

    Plateau-phase mouse clonal bone marrow stromal cell lines D2XRII and C3H cl 11 produce decreasing levels of M-CSF (CSF-1), a specific macrophage progenitor cell humoral regulator, following X-irradiation in vitro. The decrease did not go below 40% of control levels, even after irradiation doses of 50,000 rad (500 Gy). In contrast, a distinct humoral regulator stimulating growth of GM-CSF/IL-3 factor-dependent (FD) hematopoietic progenitor cell lines was detected following radiation to doses above 2000 rad. This humoral factor was not detectable in conditioned medium from irradiated cells, weakly detected using factor-dependent target cell populations in agar overlay, and was prominently detected by liquid co-cultivation of factor-dependent cells with irradiated stromal cell cultures. Subclonal lines of FD cells, derived after co-cultivation revealed karyotypic abnormalities and induced myeloblastic tumors in syngeneic mice. Five-eight weeks co-cultivation was required for induction of factor independence and malignancy and was associated with dense cell to cell contact between FD cells and stromal cells demonstrated by light and electron microscopy. Increases in hematopoietic to stromal cell surface area, total number of adherent cells per flask, total non-adherent cell colonies per flask, and cumulative non-adherent cell production were observed after irradiation. The present data may prove very relevant to an understanding of the cell to cell interactions during X-irradiation-induced leukemia.

  1. Histamine as a Radiosensitizer of Malignant Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Rivera, E. S.; Medina, V.; Cricco, G.; Mohamed, N.; Croci, M.; Martin, G.; Nunez, M.; Bergoc, R. M.

    2004-07-01

    It has been established that the treatment with Histamine (Hi) produces a significant growth inhibition of different cell lines derived from human neoplasia. In a model of Knockout mice completely depleted of endogenous Hi, it was observed a significant delay in bone marroe repopulation after whole body irradiation. These results are in agreement with the hypothesis that histamine has a role in the regulation of haematopoiesis as well as an inhibitory effect on apoptosis. The objective of this paper was to study the possible effect of Hi as protector of normal cells and radiosensitizer of malignant ones. To study the effect of Hi on small-intestine and bone marrow, thirty made mice were randomly separeted into two groups: Control irradiated (C), and irradiated receiving Histamine (HI-group). All animals received a single dose of 10 Gy on whole-body employing a ''137Cs source of 189 TB{sub q} (Dose rate: 7.7 Gy/min) calibrated with TLD 700 dosimeter. Hi-group recieved a daily se injection (0.1 mg/kg) starting 20 hs before irradiation. Mice were sacrificed 5 days after irradiation. Histopathological analysis indicated that intestinal mucosae of C group showed important injury, whist mucosae of Hi-treated mice showed mild mucosal atrophy with conservation of villous projections and absence of vascular congestive changes. In order to investigate the effect of Hi on radiosensitivity of transformed cells, MDA-MB-231 (human breast carcinoma cells) were irradiated in vitro with doses ranging from 0 to 10 Gy. Results of radiobiological parameters indicate a significant increase on radiosensitivity of malignant cells. Employing specific fluorescent dyes and flow cytometric analysis we determined that the intracellular levels of hydrogen peroxide (H{sub 2}O{sub 2}) are significant increased by Hi 10 {mu}M in control and also in irradiated MDA-MB-231 cells, while the levels of superoxide (SO{sub 2}) were not significantly modified by Hi-treatment. (Author) 9 refs.

  2. Evaluation of Stem Cell Markers, CD44/CD24 in Breast Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Masoud Hashemi Arabi

    2014-05-01

    Four breast cancer cell lines, MCF-7 ، T47D ، MDA-MB231 and MDA-MB468 were purchased from National cell Bank of Iran based in Iran Pasture Institute and were cultured in high glucose DMEM supplemented with 10% FCS. Cells were stained with antiCD44-PE and antiCD24-FITC antibodies and Status of CD44 and CD24 as markers of breast cancer stem cells were evaluated using flow cytometer and fluorescent microscopy.Evaluation of CD44 and CD24 as markers of breast cancer stem cells showed that MDA-MB231 with 97±1.2% CD44+/CD24-/low cells is significantly different from the others that they were mainly CD44 and CD24 positive cells(p

  3. Notch reporter activity in breast cancer cell lines identifies a subset of cells with stem cell activity

    OpenAIRE

    D’Angelo, Rosemarie C.; Ouzounova, Maria; Davis, April; Choi, Daejin; Tchuenkam, Stevie M.; Kim, Gwangil; Luther, Tahra; Quraishi, Ahmed A.; Senbabaoglu, Yasin; Conley, Sarah J; Shawn G Clouthier; Hassan, Khaled A.; Wicha, Max S; Korkaya, Hasan

    2015-01-01

    Developmental pathways such as Notch play a pivotal role in tissue specific stem cell self-renewal as well as in tumor development. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notch+) or reduced activity (Notch-) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplantation assays we investigated the role of Notch ...

  4. In vitro evaluation of a new nitrosourea, TCNU, against human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Roed, H; Vindeløv, L L; Spang-Thomsen, M;

    1987-01-01

    The cytotoxic activity of a new nitrosourea, TCNU, was compared with that of BCNU in five human small cell lung cancer cell lines in vitro. TCNU was found to be equivalent or inferior to BCNU when compared on a microgram to microgram basis. If the potential of in vitro phase II trials for selecti...... of new drugs can be validated, it can be concluded that TCNU is not superior to other nitrosoureas for the treatment of SCCL....

  5. Comparative In Vitro Immune Stimulation Analysis of Primary Human B Cells and B Cell Lines

    Science.gov (United States)

    Van Belle, Kristien; Herman, Jean; Boon, Louis; Waer, Mark

    2016-01-01

    B cell specific immunomodulatory drugs still remain an unmet medical need. Utilisation of validated simplified in vitro models would allow readily obtaining new insights in the complexity of B cell regulation. For this purpose we investigated which human B lymphocyte stimulation assays may be ideally suited to investigate new B lymphocyte immunosuppressants. Primary polyclonal human B cells underwent in vitro stimulation and their proliferation, production of immunoglobulins (Igs) and of cytokines, and expression of cell surface molecules were analysed using various stimuli. ODN2006, a toll-like receptor 9 (TLR9) agonist, was the most potent general B cell stimulus. Subsequently, we investigated on which human B cell lines ODN2006 evoked the broadest immunostimulatory effects. The Namalwa cell line proved to be the most responsive upon TLR9 stimulation and hence may serve as a relevant, homogeneous, and stable B cell model in an in vitro phenotypic assay for the discovery of new targets and inhibitors of the B cell activation processes. As for the read-out for such screening assay, it is proposed that the expression of activation and costimulatory surface markers reliably reflects B lymphocyte activation. PMID:28116319

  6. Oncolytic adenovirus SG600-IL24 selectively kills hepatocellular carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To investigate the effect of oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24 on hepatocellular carcinoma (HCC) cell lines and normal liver cell line. METHODS: HCC cell lines (HepG2, Hep3B and MHCC97L) and normal liver cell line (L02) with a different p53 status were infected with SG600-IL24 and Ad.IL-24, respectively. Melanoma differentiation-associated (MDA)-7/interleukin (IL)-24 mRNA and protein expressions in infected cells were detected by reverse transcription-polym...

  7. Histone H1 phosphorylation occurs site-specifically during interphase and mitosis: identification of a novel phosphorylation site on histone H1.

    Science.gov (United States)

    Sarg, Bettina; Helliger, Wilfried; Talasz, Heribert; Förg, Barbara; Lindner, Herbert H

    2006-03-10

    H1 histones, isolated from logarithmically growing and mitotically enriched human lymphoblastic T-cells (CCRF-CEM), were fractionated by reversed phase and hydrophilic interaction liquid chromatography, subjected to enzymatic digestion, and analyzed by amino acid sequencing and mass spectrometry. During interphase the four H1 subtypes present in these cells differ in their maximum phosphorylation levels: histone H1.5 is tri-, H1.4 di-, and H1.3 and H1.2, only monophosphorylated. The phosphorylation is site-specific and occurs exclusively on serine residues of SP(K/A)K motifs. The phosphorylation sites of histone H1.5 from mitotically enriched cells were also examined. In contrast to the situation in interphase, at mitosis there were additional phosphorylations, exclusively at threonine residues. Whereas the tetraphosphorylated H1.5 arises from the triphosphosphorylated form by phosphorylation of one of two TPKK motifs in the C-terminal domain, namely Thr137 and Thr154, the pentaphosphorylated H1.5 was the result of phosphorylation of one of the tetraphosphorylated forms at a novel nonconsensus motif at Thr10 in the N-terminal tail. Despite the fact that histone H1.5 has five (S/T)P(K/A)K motifs, all of these motifs were never found to be phosphorylated simultaneously. Our data suggest that phosphorylation of human H1 variants occurs nonrandomly during both interphase and mitosis and that distinct serine- or threonine-specific kinases are involved in different cell cycle phases. The order of increased phosphorylation and the position of modification might be necessary for regulated chromatin decondensation, thus facilitating processes of replication and transcription as well as of mitotic chromosome condensation.

  8. Establishment, Growth kinetics, and Susceptibility to AcMNPV of Heat Tolerant Lepidop teran Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Yan-lei Wu; Lei Jiang; Yoshifumi Hashimoto; Robert R.Granados; Guo-xun Li

    2011-01-01

    Lepidopteran heat-tolerant(ht)cell lines have been obtained with sf-9,sf-21 and several Bombyx cells.They have a distinct karyotype,membrane lipid composition,morphology and growth kinetics from the parental cell lines.In this paper,we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility.Adaptation of cell lines sf-9,BTI-TN-5131-4(High5)and BTI-TN-MG1(MG 1)to 33℃ and 35℃ was carried out by shifting the culture temperature between 28℃ and higher temperatures by a gradual stepwise increase in temperature.The process of adaption to a higher culture temperature was accomplished over a period of 2 months.The cell lines with the temperature adaption were designated as sf9-ht33,sf9-ht35,High5-ht33,High5-ht35,MG1-ht33,MG1-ht35.These cell lines have been subcultured over 70 passages.Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines.The population doubling time of heat adapted cell lines were 1-4 h less than these of parental cell lines.Cell shapes did not show obvious change,however,the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption.When the cell lines were infected with Autographa californica nuclear polyhedrosis virus(AcMNPV)at 28℃,33℃,35℃ and 37℃,production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.

  9. Selective migration of neuralized embryonic stem cells to stem cell factor and media conditioned by glioma cell lines

    Directory of Open Access Journals (Sweden)

    Maria Bernard L

    2006-01-01

    Full Text Available Abstract Background Pluripotent mouse embryonic stem (ES cells can be induced in vitro to become neural progenitors. Upon transplantation, neural progenitors migrate toward areas of damage and inflammation in the CNS. We tested whether undifferentiated and neuralized mouse ES cells migrate toward media conditioned by glioma cell lines (C6, U87 & N1321 or Stem Cell Factor (SCF. Results Cell migration assays revealed selective migration by neuralized ES cells to conditioned media as well as to synthetic SCF. Migration of undifferentiated ES cells was extensive, but not significantly different from that of controls (Unconditioned Medium. RT-PCR analysis revealed that all the three tumor cell lines tested synthesized SCF and that both undifferentiated and neuralized ES cells expressed c-kit, the receptor for SCF. Conclusion Our results demonstrate that undifferentiated ES cells are highly mobile and that neural progenitors derived from ES cells are selectively attracted toward factors produced by gliomas. Given that the glioma cell lines synthesize SCF, SCF may be one of several factors that contribute to the selective migration observed.

  10. Selective migration of neuralized embryonic stem cells to stem cell factor and media conditioned by glioma cell lines

    Science.gov (United States)

    Serfozo, Peter; Schlarman, Maggie S; Pierret, Chris; Maria, Bernard L; Kirk, Mark D

    2006-01-01

    Background Pluripotent mouse embryonic stem (ES) cells can be induced in vitro to become neural progenitors. Upon transplantation, neural progenitors migrate toward areas of damage and inflammation in the CNS. We tested whether undifferentiated and neuralized mouse ES cells migrate toward media conditioned by glioma cell lines (C6, U87 & N1321) or Stem Cell Factor (SCF). Results Cell migration assays revealed selective migration by neuralized ES cells to conditioned media as well as to synthetic SCF. Migration of undifferentiated ES cells was extensive, but not significantly different from that of controls (Unconditioned Medium). RT-PCR analysis revealed that all the three tumor cell lines tested synthesized SCF and that both undifferentiated and neuralized ES cells expressed c-kit, the receptor for SCF. Conclusion Our results demonstrate that undifferentiated ES cells are highly mobile and that neural progenitors derived from ES cells are selectively attracted toward factors produced by gliomas. Given that the glioma cell lines synthesize SCF, SCF may be one of several factors that contribute to the selective migration observed. PMID:16436212

  11. Establishment and characterization of a cell line (OMC-9) originating from a human endometrial stromal sarcoma.

    Science.gov (United States)

    Kakuno, Yoshiteru; Yamada, Takashi; Mori, Hiroshi; Narabayashi, Isamu

    2008-05-01

    Cell lines are very useful for clinical and basic research. The establishment of uterine malignant tumor cell lines with unusual histology is especially important. We describe the establishment and characterization of a new human endometrial stromal sarcoma cell line of the uterus. The cell line OMC-9 was established from a tumor mass in the uterine body of a 55-year-old woman. Characteristics of the cell line studied include morphology, chromosome analysis, heterotransplantation, tumor markers and chemosensitivity. This cell line has grown well for 196 months and has been subcultured more than 50 times. Monolayer cultured cells are polygonal in shape, appear to be spindle-shaped or multipolar and have a tendency to pile up without contact inhibition. The cells exhibit a human karyotype with a modal chromosomal number in the diploid range. The cells were able to be transplanted into the subcutis of nude mice and produced tumors resembling the original tumor. OMC-9 cells produced tissue polypeptide antigen. Both CD10, a sensitive and diagnostically useful marker of endometrial stromal neoplasms, and vimentin were identified immunohistochemically in the original tumor and the heterotransplanted tumor. The cells were sensitive to actinomycin D, doxorubicin, carboplatin, cisplatin and etoposide, drugs used commonly in the treatment of gynecologic cancer. Only three reports of uterine endometrial stromal sarcoma cell lines have thus far been reported in the literature. OMC-9 is the first endometrial stromal sarcoma cell line in which CD10 expression and chemosensitivity have been identified.

  12. Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines.

    Science.gov (United States)

    Gardell, Alison M; Qin, Qin; Rice, Robert H; Li, Johnathan; Kültz, Dietmar

    2014-01-01

    Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.

  13. Gene probes to detect cross-culture contamination in hormone producing cell lines

    DEFF Research Database (Denmark)

    Matsuba, I; Lernmark, A; Madsen, Ole Dragsbæk;

    1988-01-01

    Cross-culture contamination of cell lines propagated in continuous culture is a frequent event and particularly difficult to resolve in cells expressing similar phenotypes. We demonstrate that DNA-DNA hybridization to blotted endonuclease-digested cell DNA effectively detects cross-culture...... the effective use of gene probes to control the origin of cell cultures....... contamination to monitor inter-species as well as intra-species cross contamination. An insulin-producing cell-line, Clone-16, originally cloned from a human fetal endocrine pancreatic cell line did not produce human c-peptide as anticipated. DNA from these cells showed no hybridization to the human ALU...

  14. Effect of arginase II on L-arginine depletion and cell growth in murine cell lines of renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Patterson John R

    2008-09-01

    Full Text Available Abstract Background L-arginine is the common substrate for the two isoforms of arginase. Arginase I, highly expressed in the liver and arginase II mainly expressed in the kidney. Arginase I-producing myeloid derived suppressor cells have been shown to inhibit T-cell function by the depletion of L-arginine. On the other hand, arginase II has been detected in patients with cancer and is thought to metabolize L-arginine to L-ornithine needed to sustain rapid tumor growth; however its role in L-arginine depletion is unclear. Thus, in tumor biology, L-arginine metabolism may play a dual role in tumor growth and in the induction of T cell dysfunction. Therefore, we studied in murine renal cell carcinoma (RCC cell lines, the effect of arginase II on tumor cell proliferation and L-arginine depletion. The effect of arginase inhibitors on cell proliferation was also tested. Methods Three murine renal cell carcinoma (mRCC cell lines were tested for the presence of arginase. nor-NOHA, an arginase inhibitor was used to substantiate the effect of arginase on cell growth and L-arginine depletion. Amino acid levels were tested by HPLC. Results Our results show that mRCC cell lines express only arginase II and were able to deplete L-arginine from the medium. Cell growth was independent of the amount of arginase activity expressed by the cells. nor-NOHA significantly (P = 0.01 reduced arginase II activity and suppressed cell growth in cells exhibiting high arginase activity. The depletion of L-arginine by mRCC induced the decrease expression of CD3ζ a key element for T-cell function. Conclusion The results of this study show for the first time that arginase II produced by RCC cell lines depletes L-arginine resulting in decreased expression of CD3ζ. These results indicate that RCC cell lines expressing arginase II can modulate the L-arginine metabolic pathway to regulate both cell growth and T-cell function. Blocking arginase may lead to a decrease in RCC cell

  15. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.

    Science.gov (United States)

    Martin, G R

    1981-12-01

    This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures, derived from isolated single cells, can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells, or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo, including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development.

  16. Network signatures of cellular immortalization in human lymphoblastoid cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Shim, Sung-Mi; Jung, So-Young; Nam, Hye-Young; Kim, Hye-Ryun; Lee, Mee-Hee; Kim, Jun-Woo; Han, Bok-Ghee [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Osong 363-951 (Korea, Republic of); Jeon, Jae-Pil, E-mail: jaepiljeon@hanmail.net [Division of Brain Diseases, Center for Biomedical Science, Korea National Institute of Health, Osong 363-951 (Korea, Republic of)

    2013-11-15

    Highlights: •We identified network signatures of LCL immortalization from transcriptomic profiles. •More than 41% of DEGs are possibly regulated by miRNAs in LCLs. •MicroRNA target genes in LCLs are involved in apoptosis and immune-related functions. •This approach is useful to find functional miRNA targets in specific cell conditions. -- Abstract: Human lymphoblastoid cell line (LCL) has been used as an in vitro cell model in genetic and pharmacogenomic studies, as well as a good model for studying gene expression regulatory machinery using integrated genomic analyses. In this study, we aimed to identify biological networks of LCL immortalization from transcriptomic profiles of microRNAs and their target genes in LCLs. We first selected differentially expressed genes (DEGs) and microRNAs (DEmiRs) between early passage LCLs (eLCLs) and terminally differentiated late passage LCLs (tLCLs). The in silico and correlation analysis of these DEGs and DEmiRs revealed that 1098 DEG–DEmiR pairs were found to be positively (n = 591 pairs) or negatively (n = 507 pairs) correlated with each other. More than 41% of DEGs are possibly regulated by miRNAs in LCL immortalizations. The target DEGs of DEmiRs were enriched for cellular functions associated with apoptosis, immune response, cell death, JAK–STAT cascade and lymphocyte activation while non-miRNA target DEGs were over-represented for basic cell metabolisms. The target DEGs correlated negatively with miR-548a-3p and miR-219-5p were significantly associated with protein kinase cascade, and the lymphocyte proliferation and apoptosis, respectively. In addition, the miR-106a and miR-424 clusters located in the X chromosome were enriched in DEmiR–mRNA pairs for LCL immortalization. In this study, the integrated transcriptomic analysis of LCLs could identify functional networks of biologically active microRNAs and their target genes involved in LCL immortalization.

  17. Induction of enamel matrix protein expression in an ameloblast cell line co-cultured with a mesenchymal cell line in vitro.

    Science.gov (United States)

    Matsumoto, Asako; Harada, Hidemitsu; Saito, Masahiro; Taniguchi, Akiyoshi

    2011-01-01

    Interactions between epithelium and mesenchyme are important for organ and tissue development. In this study, in order to mimic interactions between epithelium and mesenchyme during native tooth development, we constructed three-dimensional culture systems in vitro using a collagen membrane. Two types of collagen membrane-based in vitro culture systems were constructed in which dental epithelial and dental follicle cell lines were cultured. One co-culture method involved inoculation of one cell line into one side of the collagen membrane, and the other cell line into the opposite side of the membrane (sandwich co-culture). As a control, the second method involved culture of one of the cell lines on a culture dish and the second cell line on a collagen membrane, facing away from the first cell line (separate co-culture). The HAT-7 cells were also grown as a monolayer culture on collagen. Ameloblast differentiation in these cultures was investigated by analysis of the mRNA and/or protein expression of ameloblastin and amelogenin. Our results suggest that interaction of epithelial and mesenchymal cells via the extracellular matrix is important for tooth differentiation in vitro. Our culture system should be a useful method for investigation of epithelial-mesenchymal interactions.

  18. The telomerase inhibitor imetelstat depletes cancer stem cells in breast and pancreatic cancer cell lines.

    Science.gov (United States)

    Joseph, Immanual; Tressler, Robert; Bassett, Ekaterina; Harley, Calvin; Buseman, Christen M; Pattamatta, Preeti; Wright, Woodring E; Shay, Jerry W; Go, Ning F

    2010-11-15

    Cancer stem cells (CSC) are rare drug-resistant cancer cell subsets proposed to be responsible for the maintenance and recurrence of cancer and metastasis. Telomerase is constitutively active in both bulk tumor cell and CSC populations but has only limited expression in normal tissues. Thus, inhibition of telomerase has been shown to be a viable approach in controlling cancer growth in nonclinical studies and is currently in phase II clinical trials. In this study, we investigated the effects of imetelstat (GRN163L), a potent telomerase inhibitor, on both the bulk cancer cells and putative CSCs. When breast and pancreatic cancer cell lines were treated with imetelstat in vitro, telomerase activity in the bulk tumor cells and CSC subpopulations were inhibited. Additionally, imetelstat treatment reduced the CSC fractions present in the breast and pancreatic cell lines. In vitro treatment with imetelstat, but not control oligonucleotides, also reduced the proliferation and self-renewal potential of MCF7 mammospheres and resulted in cell death after imetelstat, suggesting a mechanism of action independent of telomere shortening for the effects of imetelstat on the CSC subpopulations. Our results suggest that imetelstat-mediated depletion of CSCs may offer an alternative mechanism by which telomerase inhibition may be exploited for cancer therapy.

  19. Electroporation enhances mitomycin C cytotoxicity on T24 bladder cancer cell line

    DEFF Research Database (Denmark)

    Vasquez, Juan Luis; Gehl, Julie; Hermann, Gregers G

    2012-01-01

    improves the cytotoxicity of mitomycin. In two cell lines, T24 (bladder cancer cell line) and DC3F (Chinese hamster fibroblast), exposure to different concentrations of mitomycin (0.01-2000μM) was tested with and without electroporation (6 pulses of 1kV/cm, duration: 99μs, frequency: 1Hz). Cell viability...

  20. Molecular characterization of neoplastic and normal "sister" lymphoblastoid B-cell lines from chronic lymphocytic leukemia

    DEFF Research Database (Denmark)

    Lanemo Myhrinder, Anna; Hellqvist, Eva; Bergh, Ann-Charlotte;

    2013-01-01

    /short tandem repeat (STR) fingerprinting. Innate B-cell features, i.e. natural Ab production and CD5 receptors, were present in most CLL cell lines, but in none of the normal LCLs. This panel of immortalized CLL-derived cell lines is a valuable reference representing a renewable source of authentic Abs and DNA....

  1. A suspended cell line from Trichoplusia ni (Lepidoptera):Characterization and expression of recombinant proteins

    Institute of Scientific and Technical Information of China (English)

    Min-Juan Meng; Tian-Long Li; Chang-You Li; Guo-Xun Li

    2008-01-01

    A suspended cell line from Trichoplusia ni embryos was established, and its susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV)infection was investigated. This cell line had characteristics distinct from the BTI-Tn5B 14 cell line (Tn5B 1-4) from T. ni in growth, and showed approximately the same responses to AcMNPV infection, production of occlusion bodies, and levels of recombinant protein expression. No clumps were observed at maximum cell density at late-log phase in shakeflask or T-flask cultures, and thus the cells represent a useful new contribution for baculovirus research. The cells consist of two major morphological types: approximately 70% spindle-shaped cells and 30% round cells. The cell line was highly susceptible to virus infection and produced around 107 AcMNPV occlusion bodies per cell, on average.Production of β-galactosidase and secreted alkaline phosphatase was high with 3.97 + 0.13×104 IU/mL and 3.48±0.40 IU/mL, respectively. This cell line may be applicable for studies of scale-up production of viruses or baculovirus-insect cell expression. We also believe the new line can be a source for cell clones with higher production of virus and recombinant proteins compared to the parent or other existing cell lines such as Tn5B 1-4.

  2. Prediction of epigenetically regulated genes in breast cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Loss, Leandro A; Sadanandam, Anguraj; Durinck, Steffen; Nautiyal, Shivani; Flaucher, Diane; Carlton, Victoria EH; Moorhead, Martin; Lu, Yontao; Gray, Joe W; Faham, Malek; Spellman, Paul; Parvin, Bahram

    2010-05-04

    Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP). The pipeline (i) reduces the dimensionality of the methylation data, (ii) associates the reduced methylation data with gene expression data, and (iii) ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i) methylation sites are grouped across the genome to identify regions of interest, and (ii) methylation profles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fxed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically signifcant negative correlation between methylation profles and gene expression in the

  3. Prediction of epigenetically regulated genes in breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Lu Yontao

    2010-06-01

    Full Text Available Abstract Background Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP. The pipeline (i reduces the dimensionality of the methylation data, (ii associates the reduced methylation data with gene expression data, and (iii ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i methylation sites are grouped across the genome to identify regions of interest, and (ii methylation profles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fxed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. Results Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically signifcant negative correlation between

  4. Synergistic combination of valproic acid and oncolytic parvovirus H-1PV as a potential therapy against cervical and pancreatic carcinomas.

    Science.gov (United States)

    Li, Junwei; Bonifati, Serena; Hristov, Georgi; Marttila, Tiina; Valmary-Degano, Séverine; Stanzel, Sven; Schnölzer, Martina; Mougin, Christiane; Aprahamian, Marc; Grekova, Svitlana P; Raykov, Zahari; Rommelaere, Jean; Marchini, Antonio

    2013-10-01

    The rat parvovirus H-1PV has oncolytic and tumour-suppressive properties potentially exploitable in cancer therapy. This possibility is being explored and results are encouraging, but it is necessary to improve the oncotoxicity of the virus. Here we show that this can be achieved by co-treating cancer cells with H-1PV and histone deacetylase inhibitors (HDACIs) such as valproic acid (VPA). We demonstrate that these agents act synergistically to kill a range of human cervical carcinoma and pancreatic carcinoma cell lines by inducing oxidative stress, DNA damage and apoptosis. Strikingly, in rat and mouse xenograft models, H-1PV/VPA co-treatment strongly inhibits tumour growth promoting complete tumour remission in all co-treated animals. At the molecular level, we found acetylation of the parvovirus nonstructural protein NS1 at residues K85 and K257 to modulate NS1-mediated transcription and cytotoxicity, both of which are enhanced by VPA treatment. These results warrant clinical evaluation of H-1PV/VPA co-treatment against cervical and pancreatic ductal carcinomas.

  5. Identifying anti-growth factors for human cancer cell lines through genome-scale metabolic modeling

    DEFF Research Database (Denmark)

    Ghaffari, Pouyan; Mardinoglu, Adil; Asplund, Anna

    2015-01-01

    Human cancer cell lines are used as important model systems to study molecular mechanisms associated with tumor growth, hereunder how genomic and biological heterogeneity found in primary tumors affect cellular phenotypes. We reconstructed Genome scale metabolic models (GEMs) for eleven cell lines...... based on RNA-Seq data and validated the functionality of these models with data from metabolite profiling. We used cell line-specific GEMs to analyze the differences in the metabolism of cancer cell lines, and to explore the heterogeneous expression of the metabolic subsystems. Furthermore, we predicted...... antimetabolites using two cell lines with different phenotypic origins, and found that it is effective in inhibiting the growth of these cell lines. Using immunohistochemistry, we also showed high or moderate expression levels of proteins targeted by the validated antimetabolite. Identified anti-growth factors...

  6. EXPRESSION OF THE O6-METHYLGUANINE-DNA METHYLTRANSFERASE GENE IN EIGHT HUMAN TUMOR CELL LINES

    Institute of Scientific and Technical Information of China (English)

    陈建敏; 章扬培; 吴英

    1994-01-01

    O6-methylguanine-DNA methltransferase(MGMT) gene expression in 6 Mer+(HeLa S3,SMMC-7721,SGC-7901,B-239,AGZY83-a,and Cc801)and 2Mer-(SHG-44,AND HeLa MR) haman tumor cell lines was examined.Southern blot analysis revealed no deletion,amplification,or rearrangement of the MGMT gene in these cell lines.However,-1.0kb transcripts were detected in the 6 Mer+ cell lines but not in the 2 Mer- cell lines by Northern blot analysis.Furthermore,a rough correlation between MGMT activity and mRNA level in these cell lines was observed.These results suggest that transcriptional regulation of the MGMT gene is the molecular basis of the absence of MGMT activity in Mer- cell lines.

  7. Safety and immunogenicity of H1/IC31®, an adjuvanted TB subunit vaccine, in HIV-infected adults with CD4+ lymphocyte counts greater than 350 cells/mm3: a phase II, multi-centre, double-blind, randomized, placebo-controlled trial.

    Directory of Open Access Journals (Sweden)

    Klaus Reither

    Full Text Available Novel tuberculosis vaccines should be safe, immunogenic, and effective in various population groups, including HIV-infected individuals. In this phase II multi-centre, double-blind, placebo-controlled trial, the safety and immunogenicity of the novel H1/IC31 vaccine, a fusion protein of Ag85B-ESAT-6 (H1 formulated with the adjuvant IC31, was evaluated in HIV-infected adults.HIV-infected adults with CD4+ T cell counts >350/mm3 and without evidence of active tuberculosis were enrolled and followed until day 182. H1/IC31 vaccine or placebo was randomly allocated in a 5:1 ratio. The vaccine was administered intramuscularly at day 0 and 56. Safety assessment was based on medical history, clinical examinations, and blood and urine testing. Immunogenicity was determined by a short-term whole blood intracellular cytokine staining assay.47 of the 48 randomised participants completed both vaccinations. In total, 459 mild or moderate and 2 severe adverse events were reported. There were three serious adverse events in two vaccinees classified as not related to the investigational product. Local injection site reactions were more common in H1/IC31 versus placebo recipients (65.0% vs. 12.5%, p = 0.015. Solicited systemic and unsolicited adverse events were similar by study arm. The baseline CD4+ T cell count and HIV viral load were similar by study arm and remained constant over time. The H1/IC31 vaccine induced a persistent Th1-immune response with predominately TNF-α and IL-2 co-expressing CD4+ T cells, as well as polyfunctional IFN-γ, TNF-α and IL-2 expressing CD4+ T cells.H1/IC31 was well tolerated and safe in HIV-infected adults with a CD4+ Lymphocyte count greater than 350 cells/mm3. The vaccine did not have an effect on CD4+ T cell count or HIV-1 viral load. H1/IC31 induced a specific and durable Th1 immune response.Pan African Clinical Trials Registry (PACTR PACTR201105000289276.

  8. The Importance of Physiologically Relevant Cell Lines for Studying Virus–Host Interactions

    Directory of Open Access Journals (Sweden)

    David Hare

    2016-11-01

    Full Text Available Viruses interact intimately with the host cell at nearly every stage of replication, and the cell model that is chosen to study virus infection is critically important. Although primary cells reflect the phenotype of healthy cells in vivo better than cell lines, their limited lifespan makes experimental manipulation challenging. However, many tumor-derived and artificially immortalized cell lines have defects in induction of interferon-stimulated genes and other antiviral defenses. These defects can affect virus replication, especially when cells are infected at lower, more physiologically relevant, multiplicities of infection. Understanding the selective pressures and mechanisms underlying the loss of innate signaling pathways is helpful to choose immortalized cell lines without impaired antiviral defense. We describe the trials and tribulations we encountered while searching for an immortalized cell line with intact innate signaling, and how directed immortalization of primary cells avoids many of the pitfalls of spontaneous immortalization.

  9. CELLULAR BASIS FOR DIFFERENTIAL SENSITIVITY TO CISPLATIN IN HUMAN GERM-CELL TUMOR AND COLON-CARCINOMA CELL-LINES

    NARCIS (Netherlands)

    SARK, MWJ; TIMMERBOSSCHA, H; MEIJER, C; UGES, DRA; SLUITER, WJ; PETERS, WHM; MULDER, NH; DEVRIES, EGE

    1995-01-01

    Cisplatin (CDDP) resistance mechanisms were studied in a model of three germ cell tumour and three colon carcinoma cell lines representing intrinsically CDDP-sensitive and -resistant tumours respectively. The CDDP sensitivity of the cell lines mimicked the clinical situation. The glutathione levels

  10. A comparative study of the FcepsilonRI molecule on human mast cell and basophil cell lines

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Dissing, S; Skov, P S;

    2005-01-01

    Mast cells and basophils express the high-affinity IgE receptor FcepsilonRI. We have analysed the human mast cell line LAD2 and four subclones of the basophil cell line KU812 in order to reveal possible differences concerning the FcepsilonRI surface regulation, anti-IgE-triggered activation...

  11. Restriction of human adenovirus replication in Chinese hamster cell lines and their hybrids with human cells.

    Science.gov (United States)

    Radna, R L; Foellmer, B; Feldman, L A; Francke, U; Ozer, H L

    1987-11-01

    We have found that the replication of human adenovirus (Ad2) is restricted in multiple Chinese hamster cell lines including CHO and V79. The major site of restriction involves differential accumulation of late viral proteins as demonstrated by immunofluorescence assay and polyacrylamide gel electrophoresis with and without prior immunoprecipitation. Synthesis of fiber and penton base are markedly reduced, whereas others, such as the 100K polypeptide, are synthesized efficiently. This pattern of restriction is similar to that previously reported for Ad2 infection of several monkey cell lines; however, the restriction is more marked in the Chinese hamster cell lines. The restriction is most likely due to a deficient cellular function since stable cell hybrids between V79 or CHO and human cells are permissive for virus replication. By analysis of a series of hybrids with reduced numbers of human chromosomes, fiber synthesis was correlated with the presence of the short arm of human chromosome 3. More hybrids showed restoration of fiber synthesis than production of progeny virus, suggesting that more than one unlinked function is required for the latter.

  12. Experimental impact of aspirin exposure on rat intestinal bacteria, epithelial cells and cell line.

    Science.gov (United States)

    Upreti, Raj K; Kannan, A; Pant, A B

    2010-10-01

    Aspirin, a commonly used therapeutic non-steroidal anti-inflammatory drug (NSAID) is known to cause gastric mucosal damage. Intestinal bacteria having a regulatory effect on intestinal homeostasis play significant role in NSAID-induced intestinal injury. Bacteria and specific cell lines are considered to be suitable for toxicity screening and testing of chemicals. Therefore, to evaluate and compare in vitro toxicity, cultures of rat intestinal epithelial cells (IEC), isolated bacteria and IEC-6 cell line were assessed for viability, morphometric analysis, membrane transport enzymes and structural constituents for membrane damage, dehydrogenase activity test for respiratory and energy producing processes and esterase activity test for intra- and extra-cellular degradation, following the post exposure to aspirin (0-50 µg mL(- 1)). Similar pattern of dose-dependent changes in these parameters were observed in three types of cells. Similar in situ effects on IEC validated the in vitro findings. These findings indicate that higher aspirin concentrations may alter cellular functions of IEC and gut bacteria. Furthermore, results suggest that gut bacteria and IEC-6 cell line can be used for the initial screening of gastrointestinal cellular toxicity caused by NSAIDs.

  13. A Human Corneal Epithelial Cell Line Model for Limbal Stem Cell Biology and Limbal Immunobiology.

    Science.gov (United States)

    Shaharuddin, Bakiah; Ahmad, Sajjad; Md Latar, Nani; Ali, Simi; Meeson, Annette

    2016-10-14

    : Limbal stem cell (LSC) deficiency is a visually debilitating condition caused by abnormal maintenance of LSCs. It is treated by transplantation of donor-derived limbal epithelial cells (LECs), the success of which depends on the presence and quality of LSCs within the transplant. Understanding the immunobiological responses of these cells within the transplants could improve cell engraftment and survival. However, human corneal rings used as a source of LSCs are not always readily available for research purposes. As an alternative, we hypothesized that a human telomerase-immortalized corneal epithelial cell (HTCEC) line could be used as a model for studying LSC immunobiology. HTCEC constitutively expressed human leukocyte antigen (HLA) class I but not class II molecules. However, when stimulated by interferon-γ, HTCECs then expressed HLA class II antigens. Some HTCECs were also migratory in response to CXCL12 and expressed stem cell markers, Nanog, Oct4, and Sox2. In addition because both HTCECs and LECs contain side population (SP) cells, which are an enriched LSC population, we used these SP cells to show that some HTCEC SP cells coexpressed ABCG2 and ABCB5. HTCEC SP and non-side population (NSP) cells also expressed CXCR4, but the SP cells expressed higher levels. Both were capable of colony formation, but the NSP colonies were smaller and contained fewer cells. In addition, HTCECs expressed ΔNp63α. These results suggest the HTCEC line is a useful model for further understanding LSC biology by using an in vitro approach without reliance on a supply of human tissue.

  14. Rapid selection and proliferation of CD133+ cells from cancer cell lines: chemotherapeutic implications.

    Directory of Open Access Journals (Sweden)

    Sarah E Kelly

    Full Text Available Cancer stem cells (CSCs are considered a subset of the bulk tumor responsible for initiating and maintaining the disease. Several surface cellular markers have been recently used to identify CSCs. Among those is CD133, which is expressed by hematopoietic progenitor cells as well as embryonic stem cells and various cancers. We have recently isolated and cultured CD133 positive [CD133+] cells from various cancer cell lines using a NASA developed Hydrodynamic Focusing Bioreactor (HFB (Celdyne, Houston, TX. For comparison, another bioreactor, the rotary cell culture system (RCCS manufactured by Synthecon (Houston, TX was used. Both the HFB and the RCCS bioreactors simulate aspects of hypogravity. In our study, the HFB increased CD133+ cell growth from various cell lines compared to the RCCS vessel and to normal gravity control. We observed a +15-fold proliferation of the CD133+ cellular fraction with cancer cells that were cultured for 7-days at optimized conditions. The RCCS vessel instead yielded a (-4.8-fold decrease in the CD133+cellular fraction respect to the HFB after 7-days of culture. Interestingly, we also found that the hypogravity environment of the HFB greatly sensitized the CD133+ cancer cells, which are normally resistant to chemo treatment, to become susceptible to various chemotherapeutic agents, paving the way to less toxic and more effective chemotherapeutic treatment in patients. To be able to test the efficacy of cytotoxic agents in vitro prior to their use in clinical setting on cancer cells as well as on cancer stem cells may pave the way to more effective chemotherapeutic strategies in patients. This could be an important advancement in the therapeutic options of oncologic patients, allowing for more targeted and personalized chemotherapy regimens as well as for higher response rates.

  15. Nuclear motility in glioma cells reveals a cell-line dependent role of various cytoskeletal components.

    Directory of Open Access Journals (Sweden)

    Alexa Kiss

    Full Text Available Nuclear migration is a general term for the movement of the nucleus towards a specific site in the cell. These movements are involved in a number of fundamental biological processes, such as fertilization, cell division, and embryonic development. Despite of its importance, the mechanism of nuclear migration is still poorly understood in mammalian cells. In order to shed light on the mechanical processes underlying nuclear movements, we adapted a micro-patterning based assay. C6 rat and U87 human glioma cells seeded on fibronectin patterns--thereby forced into a bipolar morphology--displayed oscillatory movements of the nucleus or the whole cell, respectively. We found that both the actomyosin system and microtubules are involved in the nuclear/cellular movements of both cell lines, but their contributions are cell-/migration-type specific. Dynein activity was necessary for nuclear migration of C6 cells but active myosin-II was dispensable. On the other hand, coupled nuclear and cellular movements of U87 cells were driven by actomyosin contraction. We explain these cell-line dependent effects by the intrinsic differences in the overall mechanical tension due to the various cytoskeletal elements inside the cell. Our observations showed that the movements of the nucleus and the centrosome are strongly correlated and display large variation, indicating a tight but flexible coupling between them. The data also indicate that the forces responsible for nuclear movements are not acting directly via the centrosome. Based on our observations, we propose a new model for nuclear oscillations in C6 cells in which dynein and microtubule dynamics are the main drivers of nuclear movements. This mechanism is similar to the meiotic nuclear oscillations of Schizosaccharomyces pombe and may be evolutionary conserved.

  16. Micro-RNA expression in cisplatin resistant germ cell tumor cell lines

    Directory of Open Access Journals (Sweden)

    Meineke Viktor

    2011-05-01

    Full Text Available Abstract Background We compared microRNA expression patterns in three cisplatin resistant sublines derived from paternal cisplatin sensitive germ cell tumor cell lines in order to improve our understanding of the mechanisms of cisplatin resistance. Methods Three cisplatin resistant sublines (NTERA-2-R, NCCIT-R, 2102EP-R showing 2.7-11.3-fold increase in drug resistance after intermittent exposure to increasing doses of cisplatin were compared to their parental counterparts, three well established relatively cisplatin sensitive germ cell tumor cell lines (NTERA-2, NCCIT, 2102EP. Cells were cultured and total RNA was isolated from all 6 cell lines in three independent experiments. RNA was converted into cDNA and quantitative RT-PCR was run using 384 well low density arrays covering almost all (738 known microRNA species of human origin. Results Altogether 72 of 738 (9.8% microRNAs appeared differentially expressed between sensitive and resistant cell line pairs (NTERA-2R/NTERA-2 = 43, NCCIT-R/NCCIT = 53, 2102EP-R/2102EP = 15 of which 46.7-95.3% were up-regulated (NTERA-2R/NTERA-2 = 95.3%, NCCIT-R/NCCIT = 62.3%, 2102EP-R/2102EP = 46.7%. The number of genes showing differential expression in more than one of the cell line pairs was 34 between NTERA-2R/NTERA-2 (79% and NCCIT-R/NCCIT (64%, and 3 and 4, respectively, between these two cell lines and 2102EP-R/2102EP (about 27%. Only the has-miR-10b involved in breast cancer invasion and metastasis and has-miR-512-3p appeared to be up-regulated (2-3-fold in all three cell lines. The hsa-miR-371-373 cluster (counteracting cellular senescence and linked with differentiation potency, as well as hsa-miR-520c/-520h (inhibiting the tumor suppressor p21 were 3.9-16.3 fold up-regulated in two of the three cisplatin resistant cell lines. Several new micro-RNA species missing an annotation towards cisplatin resistance could be identified. These were hsa-miR-512-3p/-515/-517/-518/-525 (up to 8.1-fold up

  17. Molecular characterization of permanent cell lines from primary, metastatic and recurrent malignant peripheral nerve sheath tumors (MPNST) with underlying neurofibromatosis-1.

    Science.gov (United States)

    Fang, Yuqiang; Elahi, Abul; Denley, Ryan C; Rao, Pulivarthi H; Brennan, Murray F; Jhanwar, Suresh C

    2009-04-01

    lines was observed, as reported by others. The role of biallelic inactivation of p53 gene in MPNST with underlying NF1 mutations, however, needs further study. Overexpression of Rb1-protein observed in metastatic and recurrent cell lines is indicative of its role in the progression of the disease. One of the most important observations of this study is that Nm23-H1 expression is closely associated with advanced or metastatic disease. In summary, MPNST cell lines derived from a patient with metastatic and recurrent disease with NF1 disorder were characterized and a gene associated with metastatic potential which is amenable to therapeutic and chemo-preventative approaches was identified. These cell lines with extensive characterization of genetic abnormalities are likely to provide important reagents for biochemical, molecular and pharmacological studies related to MPNST.

  18. Technicon H*1 Hematology System: Optical Design Considerations

    Science.gov (United States)

    Colella, G. M.; Tycko, D. H.; Groner, W.

    1988-06-01

    The Technicon H*1 systemTM is a clinical laboratory flow cytometer which performs a complete hematology profile, providing quantitative information on the various types of cells in a blood sample. A light-scattering method, using a HeNe laser, determines in a single flow channel the red cell count, platelet count, and the distributions of red cell volume, red cell hemoglobin concentration, and platelet volume. To accomplish this the scattered light from each red cell in the sample is measured in real time at two angular intervals. The cell volume and the hemoglobin concentration within the cell are derived from these two measurements. Severe accuracy and precision specifications are placed on the medically important red cell count (RBC) and the mean red cell volume (MCV). From the point of view of optical system design, the dominant factor is the requirement that RBC and MCV have precision and accuracy of the order of 2%. Signal-to-noise and scattering-angle definition requirements dictated the choice of a HeNe laser light source. The optics includes an illumination system for producing a sharply defined, uniformly illuminated scattering region and a detection system which must accurately define the accepted scattering angles. In previous cytometric methods for determining MCV only a single quantity was measured for each cell. Such methods cannot disentangle the independent effects of cell size and hemoglobin concentration on the measurement, thus compromising MCV accuracy. The present double-angle scattering method overcomes this accuracy problem. The H*1 red cell method, the supporting optical design and data demonstrating that the use of this technique eliminates interference between the observed red cell indices are presented.

  19. Effects of Dioscin Extracted from Polygonatum Zanlanscianense Pamp on Several Human Tumor Cell Lines

    Institute of Scientific and Technical Information of China (English)

    王钊; 周江兵; 巨勇; 姚沈勤; 张洪钧

    2001-01-01

    Dioscin was extracted and isolated from Polygonatum Zanlanscianense Pamp. The effects of dioscin on HL60, HeLa, H14, and MDA-MB-435 cell lines were studied with the results showing that dioscin dramatically inhibited the growth of the MDA-MB-435, H14, HL60, and HeLa cell lines. The IC50 of dioscin on these cell lines were 2.6, 0.8, 7. 5, and 4.5 μ mol/L respectively.

  20. H-ras transfection of the rat kidney cell line NRK-52E results in increased induction of c-fos, c-jun and hsp70 following sulofenur treatment.

    Science.gov (United States)

    Gu, H; Smith, M W; Phelps, P C; Berezesky, I K; Merriman, R L; Boder, G B; Trump, B F

    1996-09-10

    The effect of the antineoplastic drug sulofenur on the induction of the immediate-early genes (IEG) c-fos and c-jun and the stress gene hsp70 was compared in the rat kidney epithelial-like cell line NRK-52E and a derivative H-ras-transfected (H/1.2NRK-52E) cell line. Fold induction for each gene after sulofenur (500 microM) treatment was greater in H/1.2NRK-52E. The maximum increases for NRK-2E and H/1.2NRK-52E were as follows: c-fos, approximately 10-fold and approximately 18-fold; c-jun, approximately 2.5-fold and approximately 3.6-fold; hsp70, approximately 13-fold and approximately 30-fold. In cells loaded with EGTA/AM or treated in low or no Ca2+ HBSS, c-fos induction was reduced similarly in both cell types. However, inhibition of protein kinases with staurosporin and calphostin C reduced c-fos by 80% in NRK-52E but by only 10-20% in H/1.2NRK.52E. These results indicate that sulofenur-induced IEG elevation is Ca(2+)-dependent and that the requirement for protein kinase C activation is bypassed in H-ras-transfected cells.

  1. A bovine cell line that can be infected by natural sheep scrapie prions.

    Science.gov (United States)

    Oelschlegel, Anja M; Geissen, Markus; Lenk, Matthias; Riebe, Roland; Angermann, Marlies; Schatzl, Herman; Schaetzl, Hermann; Groschup, Martin H

    2015-01-01

    Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice). We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases.

  2. A bovine cell line that can be infected by natural sheep scrapie prions.

    Directory of Open Access Journals (Sweden)

    Anja M Oelschlegel

    Full Text Available Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice. We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases.

  3. Comparison of the effect of interferon on two human hepatoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Crespi, M.; Schoub, B.D.; Lyons, S.F.; Chiu, M.N. (University of the Witwatersrand, Johannesburg (South Africa). Dept. of Virology)

    1985-06-01

    Two human hepatoma cell lines, the PLC/PRF/5 and the Mahlavu cells, which differ in their production of the hepatitis B surface antigen (HBsAg), responded differently to interferon (IFN). After IFN treatment both cell lines were able to inhibit Sindbis virus replication. Oligo A synthetase (E enzyme) could be activated in the PLC/PRF/5 cells although they were not sensitive to exogenous 2 - 5 oligoadenylic acid (2 - 5 A). In contrast, the Mahlavu cells were sensitive to exogenous 2 - 5 A, but unable to activate the E enzyme. Both cell lines were unable to stimulate phosphorylation of the exogenous initiator factor eIF-2.

  4. Inhibition of geranylgeranylation mediates sensitivity to CHOP-induced cell death of DLBCL cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Ageberg, Malin, E-mail: Malin.Ageberg@med.lu.se [Division of Hematology and Transfusion Medicine, Lund University, BMC C14, 221 84 Lund (Sweden); Rydstroem, Karin, E-mail: Karin.Rydstom@skane.se [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Linden, Ola, E-mail: Ola.Linden@skane.se [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Linderoth, Johan, E-mail: Johan.Linderoth@skane.se [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Jerkeman, Mats, E-mail: Mats.Jerkeman@skane.se [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Drott, Kristina, E-mail: Kristina.Drott@med.lu.se [Division of Hematology and Transfusion Medicine, Lund University, BMC C14, 221 84 Lund (Sweden)

    2011-05-01

    Prenylation is a post-translational hydrophobic modification of proteins, important for their membrane localization and biological function. The use of inhibitors of prenylation has proven to be a useful tool in the activation of apoptotic pathways in tumor cell lines. Rab geranylgeranyl transferase (Rab GGT) is responsible for the prenylation of the Rab family. Overexpression of Rab GGTbeta has been identified in CHOP refractory diffuse large B cell lymphoma (DLBCL). Using a cell line-based model for CHOP resistant DLBCL, we show that treatment with simvastatin, which inhibits protein farnesylation and geranylgeranylation, sensitizes DLBCL cells to cytotoxic treatment. Treatment with the farnesyl transferase inhibitor FTI-277 or the geranylgeranyl transferase I inhibitor GGTI-298 indicates that the reduction in cell viability was restricted to inhibition of geranylgeranylation. In addition, treatment with BMS1, a combined inhibitor of farnesyl transferase and Rab GGT, resulted in a high cytostatic effect in WSU-NHL cells, demonstrated by reduced cell viability and decreased proliferation. Co-treatment of BMS1 or GGTI-298 with CHOP showed synergistic effects with regard to markers of apoptosis. We propose that inhibition of protein geranylgeranylation together with conventional cytostatic therapy is a potential novel strategy for treating patients with CHOP refractory DLBCL.

  5. Tumor suppressors status in cancer cell line Encyclopedia.

    Science.gov (United States)

    Sonkin, Dmitriy; Hassan, Mehedi; Murphy, Denis J; Tatarinova, Tatiana V

    2013-08-01

    Tumor suppressors play a major role in the etiology of human cancer, and typically achieve a tumor-promoting effect upon complete functional inactivation. Bi-allelic inactivation of tumor suppressors may occur through genetic mechanisms (such as loss of function mutation, copy number (CN) loss, or loss of heterozygosity (LOH)), epigenetic mechanisms (such as promoter methylation or histone modification), or a combination of the two. We report systematically derived status of 69 known or putative tumor suppressors, across 799 samples of the Cancer Cell Line Encyclopedia. In order to generate such resource we constructed a novel comprehensive computational framework for the assessment of tumor suppressor functional "status". This approach utilizes several orthogonal genomic data types, including mutation data, copy number, LOH and expression. Through correlation with additional data types (compound sensitivity and gene set activity) we show that this integrative method provides a more accurate assessment of tumor suppressor status than can be inferred by expression, copy number, or mutation alone. This approach has the potential for a more realistic assessment of tumor suppressor genes for both basic and translational oncology research.

  6. NMR metabolic fingerprints of murine melanocyte and melanoma cell lines: application to biomarker discovery

    Science.gov (United States)

    Santana-Filho, Arquimedes Paixão de; Jacomasso, Thiago; Riter, Daniel Suss; Barison, Andersson; Iacomini, Marcello; Winnischofer, Sheila Maria Brochado; Sassaki, Guilherme Lanzi

    2017-01-01

    Melanoma is the most aggressive type of skin cancer and efforts to improve the diagnosis of this neoplasia are largely based on the use of cell lines. Metabolomics is currently undergoing great advancements towards its use to screening for disease biomarkers. Although NMR metabolomics includes both 1D and 2D methodologies, there is a lack of data in the literature regarding heteronuclear 2D NMR assignments of the metabolome from eukaryotic cell lines. The present study applied NMR-based metabolomics strategies to characterize aqueous and lipid extracts from murine melanocytes and melanoma cell lines with distinct tumorigenic potential, successfully obtaining fingerprints of the metabolites from the extracts of the cell lines by means of 2D NMR HSQC correlation maps. Relative amounts of the identified metabolites were compared between the 4 cell lines. Multivariate analysis of 1H NMR data was able not only to differentiate the melanocyte cell line from the tumorigenic ones but also distinguish among the 3 tumorigenic cell lines. We also investigated the effects of mitogenic agents, and found that they can markedly influence the metabolome of the melanocyte cell line, resembling the pattern of most proliferative cell lines. PMID:28198377

  7. Glioma cells on the run – the migratory transcriptome of 10 human glioma cell lines

    Directory of Open Access Journals (Sweden)

    Holz David

    2008-01-01

    Full Text Available Abstract Background Glioblastoma multiforme (GBM is the most common primary intracranial tumor and despite recent advances in treatment regimens, prognosis for affected patients remains poor. Active cell migration and invasion of GBM cells ultimately lead to ubiquitous tumor recurrence and patient death. To further understand the genetic mechanisms underlying the ability of glioma cells to migrate, we compared the matched transcriptional profiles of migratory and stationary populations of human glioma cells. Using a monolayer radial migration assay, motile and stationary cell populations from seven human long term glioma cell lines and three primary GBM cultures were isolated and prepared for expression analysis. Results Gene expression signatures of stationary and migratory populations across all cell lines were identified using a pattern recognition approach that integrates a priori knowledge with expression data. Principal component analysis (PCA revealed two discriminating patterns between migrating and stationary glioma cells: i global down-regulation and ii global up-regulation profiles that were used in a proband-based rule function implemented in GABRIEL to find subsets of genes having similar expression patterns. Genes with up-regulation pattern in migrating glioma cells were found to be overexpressed in 75% of human GBM biopsy specimens compared to normal brain. A 22 gene signature capable of classifying glioma cultures based on their migration rate was developed. Fidelity of this discovery algorithm was assessed by validation of the invasion candidate gene, connective tissue growth factor (CTGF. siRNA mediated knockdown yielded reduced in vitro migration and ex vivo invasion; immunohistochemistry on glioma invasion tissue microarray confirmed up-regulation of CTGF in invasive glioma cells. Conclusion Gene expression profiling of migratory glioma cells induced to disperse in vitro affords discovery of genomic signatures; selected

  8. Tick cell lines for study of Crimean-Congo hemorrhagic fever virus and other arboviruses.

    Science.gov (United States)

    Bell-Sakyi, Lesley; Kohl, Alain; Bente, Dennis A; Fazakerley, John K

    2012-09-01

    Continuous cell lines derived from many of the vectors of tick-borne arboviruses of medical and veterinary importance are now available. Their role as tools in arbovirus research to date is reviewed and their potential application in studies of tick cell responses to virus infection is explored, by comparison with recent progress in understanding mosquito immunity to arbovirus infection. A preliminary study of propagation of the human pathogen Crimean-Congo hemorrhagic fever virus (CCHFV) in tick cell lines is reported; CCHFV replicated in seven cell lines derived from the ticks Hyalomma anatolicum (a known vector), Amblyomma variegatum, Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, and Ixodes ricinus, but not in three cell lines derived from Rhipicephalus appendiculatus and Ornithodoros moubata. This indicates that tick cell lines can be used to study growth of CCHFV in arthropod cells and that there may be species-specific restriction in permissive CCHFV infection at the cellular level.

  9. Phytoestrogens regulate the proliferation and expression of stem cell factors in cell lines of malignant testicular germ cell tumors.

    Science.gov (United States)

    Hasibeder, Astrid; Venkataramani, Vivek; Thelen, Paul; Radzun, Heinz-Joachim; Schweyer, Stefan

    2013-11-01

    Phytoestrogens have been shown to exert anti-proliferative effects on different cancer cells. In addition it could be demonstrated that inhibition of proliferation is associated with downregulation of the known stem cell factors NANOG, POU5F1 and SOX2 in tumor cells. We demonstrate the potential of Belamcanda chinensis extract (BCE) and tectorigenin as anticancer drugs in cell lines of malignant testicular germ cell tumor cells (TGCT) by inhibition of proliferation and regulating the expression of stem cell factors. The TGCT cell lines TCam-2 and NTera-2 were treated with BCE or tectorigenin and MTT assay was used to measure the proliferation of tumor cells. In addition, the expression of stem cell factors was analyzed by quantitative PCR and western blot analysis. Furthermore, global expression analysis was performed by microarray technique. BCE and tectorigenin inhibited proliferation and downregulated the stem cell factors NANOG and POU5F1 in TGCT cells. In addition, gene expression profiling revealed induction of genes important for the differentiation and inhibition of oncogenes. Utilizing connectivity map in an attempt to elucidate mechanism underlying BCE treatments we found highly positive association to histone deacetylase inhibitors (HDACi) amongst others. Causing no histone deacetylase inhibition, the effects of BCE on proliferation and stem cell factors may be based on histone-independent mechanisms such as direct hyperacetylation of transcription factors. Based on these findings, phytoestrogens may be useful as new agents in the treatment of TGCT.

  10. Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer.

    Science.gov (United States)

    Liu, Jun; Luo, Yan; Zheng, Liming; Liu, Qingqing; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

    2013-10-01

    This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.

  11. Identification of transporters associated with Etoposide sensitivity of stomach cancer cell lines and methotrexate sensitivity of breast cancer cell lines by quantitative targeted absolute proteomics.

    Science.gov (United States)

    Obuchi, Wataru; Ohtsuki, Sumio; Uchida, Yasuo; Ohmine, Ken; Yamori, Takao; Terasaki, Tetsuya

    2013-02-01

    Membrane transporter proteins may influence the sensitivity of cancer cells to anticancer drugs that can be recognized as substrates. The purpose of this study was to identify proteins that play a key role in the drug sensitivity of stomach and breast cancer cell lines by measuring the absolute protein expression levels of multiple transporters and other membrane proteins and examining their correlation to drug sensitivity. Absolute protein expression levels of 90 membrane proteins were examined by quantitative targeted absolute proteomics using liquid chromatography-linked tandem mass spectrometry. Among them, 11 and 14 membrane proteins, including transporters, were present in quantifiable amounts in membrane fraction of stomach cancer and breast cancer cell lines, respectively. In stomach cancer cell lines, the protein expression level of multidrug resistance-associated protein 1 (MRP1) was inversely correlated with etoposide sensitivity. MK571, an MRP inhibitor, increased both the cell-to-medium ratio of etoposide and the etoposide sensitivity of MRP1-expressing stomach cancer cell lines. In breast cancer cell lines, the protein expression level of reduced folate carrier 1 (RFC1) was directly correlated with methotrexate (MTX) sensitivity. Initial uptake rate and steady-state cell-to-medium ratio of [(3)H]MTX were correlated with both RFC1 expression level and MTX sensitivity. These results suggest that MRP1 modulates the etoposide sensitivity of stomach cancer cell lines and RFC1 modulates the MTX sensitivity of breast cancer cell lines. Our results indicate that absolute quantification of multiple membrane proteins could be a useful strategy for identification of candidate proteins involved in drug sensitivity.

  12. Generation of breast cancer stem cells by steroid hormones in irradiated human mammary cell lines.

    Directory of Open Access Journals (Sweden)

    Guillaume Vares

    Full Text Available Exposure to ionizing radiation was shown to result in an increased risk of breast cancer. There is strong evidence that steroid hormones influence radiosensitivity and breast cancer risk. Tumors may be initiated by a small subpopulation of cancer stem cells (CSCs. In order to assess whether the modulation of radiation-induced breast cancer risk by steroid hormones could involve CSCs, we measured by flow cytometry the proportion of CSCs in irradiated breast cancer cell lines after progesterone and estrogen treatment. Progesterone stimulated the expansion of the CSC compartment both in progesterone receptor (PR-positive breast cancer cells and in PR-negative normal cells. In MCF10A normal epithelial PR-negative cells, progesterone-treatment and irradiation triggered cancer and stemness-associated microRNA regulations (such as the downregulation of miR-22 and miR-29c expression, which resulted in increased proportions of radiation-resistant tumor-initiating CSCs.

  13. Ethanolic Extract Cytotoxic Effect of Zingiber Afficinale in Breast Cancer (MCF7 Cell Line

    Directory of Open Access Journals (Sweden)

    J Tavakkol Afshari

    2010-07-01

    Full Text Available Introduction & Objective: Biological activities of Zingiber afficieale plants have been reported as possessing anticancer, antibacterial, anti ulcer, antifungal, and insecticidal properties. However, its antitumor effects haven't been studied in cancer cell lines. The aim of this study was to investigate the antitumor effect of zingiber afficieale on breast cancer cell lines. Materials & Methods: This experimental study was conducted in 2010 at Mashhad University of medical Sciences. Breast cancer cell line (MCF7 and normal connective tissue cell line (L929 were cultured in DMEM medium. Ethanolic extract of Zingiber afficinale was prepared and cell lines were treated with different concentration of extract (5000 to 78 µg. Cell viability was measured by MTT assay after 24, 48, and 72 hours. The collected data were statistically analyzed by SPSS software. Results: The effects of Zingiber afficinale on cell viability were observed after 48 hours on cell lines. Ginger doses in 2500 µg concentration inhibited 50% of cell growth (IC50 in cell lines after 48 hours. Conclusion: Our study revealed that fresh ginger extract has cytotoxic effects on tumor cells, but it doesn’t have any cytotoxic effect on normal cells. It seems that ginger could be considered as a promising chemotherapeutic agent in cancer treatment.

  14. Check your cultures! A list of cross-contaminated or misidentified cell lines.

    Science.gov (United States)

    Capes-Davis, Amanda; Theodosopoulos, George; Atkin, Isobel; Drexler, Hans G; Kohara, Arihiro; MacLeod, Roderick A F; Masters, John R; Nakamura, Yukio; Reid, Yvonne A; Reddel, Roger R; Freshney, R Ian

    2010-07-01

    Continuous cell lines consist of cultured cells derived from a specific donor and tissue of origin that have acquired the ability to proliferate indefinitely. These cell lines are well-recognized models for the study of health and disease, particularly for cancer. However, there are cautions to be aware of when using continuous cell lines, including the possibility of contamination, in which a foreign cell line or microorganism is introduced without the handler's knowledge. Cross-contamination, in which the contaminant is another cell line, was first recognized in the 1950s but, disturbingly, remains a serious issue today. Many cell lines become cross-contaminated early, so that subsequent experimental work has been performed only on the contaminant, masquerading under a different name. What can be done in response-how can a researcher know if their own cell lines are cross-contaminated? Two practical responses are suggested here. First, it is important to check the literature, looking for previous work on cross-contamination. Some reports may be difficult to find and to make these more accessible, we have compiled a list of known cross-contaminated cell lines. The list currently contains 360 cell lines, drawn from 68 references. Most contaminants arise within the same species, with HeLa still the most frequently encountered (29%, 106/360) among human cell lines, but interspecies contaminants account for a small but substantial minority of cases (9%, 33/360). Second, even if there are no previous publications on cross-contamination for that cell line, it is essential to check the sample itself by performing authentication testing.

  15. Whole-exome characterization of pancreatic neuroendocrine tumor cell lines BON-1 and QGP-1.

    Science.gov (United States)

    Vandamme, Timon; Peeters, Marc; Dogan, Fadime; Pauwels, Patrick; Van Assche, Elvire; Beyens, Matthias; Mortier, Geert; Vandeweyer, Geert; de Herder, Wouter; Van Camp, Guy; Hofland, Leo J; Op de Beeck, Ken

    2015-04-01

    The human BON-1 and QGP-1 cell lines are two frequently used models in pancreatic neuroendocrine tumor (PNET) research. Data on the whole-exome genetic constitution of these cell lines is largely lacking. This study presents, to our knowledge, the first whole-exome profile of the BON-1 and QGP-1 cell lines. Cell line identity was confirmed by short tandem repeat profiling. Using GTG-banding and a CytoSNP-12v2 Beadchip array, cell line ploidy and chromosomal alterations were determined in BON-1 and QGP-1. The exomes of both cell lines were sequenced on Ilumina's HiSeq next-generation sequencing (NGS) platform. Single-nucleotide variants (SNVs) and insertions and deletions (indels) were detected using the Genome Analysis ToolKit. SNVs were validated by Sanger sequencing. Ploidy of BON-1 and QGP-1 was 3 and 4 respectively, with long stretches of loss of heterozygosity across multiple chromosomes, which is associated with aggressive tumor behavior. In BON-1, 57 frameshift indels and 1725 possible protein-altering SNVs were identified in the NGS data. In the QGP-1 cell line, 56 frameshift indels and 1095 SNVs were identified. ATRX, a PNET-associated gene, was mutated in both cell lines, while mutation of TSC2 was detected in BON-1. A mutation in NRAS was detected in BON-1, while KRAS was mutated in QGP-1, implicating aberrations in the RAS pathway in both cell lines. Homozygous mutations in TP53 with possible loss of function were identified in both cell lines. Various MUC genes, implicated in cell signaling, lubrication and chemical barriers, which are frequently expressed in PNET tissue samples, showed homozygous protein-altering SNVs in the BON-1 and QGP-1 cell lines.

  16. Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line.

    Science.gov (United States)

    Sluch, Valentin M; Davis, Chung-ha O; Ranganathan, Vinod; Kerr, Justin M; Krick, Kellin; Martin, Russ; Berlinicke, Cynthia A; Marsh-Armstrong, Nicholas; Diamond, Jeffrey S; Mao, Hai-Quan; Zack, Donald J

    2015-11-13

    Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation.

  17. Capsaicin-induced cell death in a human gastric adenocarcinoma cell line

    Institute of Scientific and Technical Information of China (English)

    Yi-Ching Lo; Yuan-Chen Yang; I-Chieh Wu; Fu-Chen Kuo; Chi-Ming Liu; Hao-Wei Wang; Chao-Hung Kuo; Jeng-Yi Wu; Deng-Chyang Wu

    2005-01-01

    AIM: Capsaicin, a pungent ingredient found in red pepper,has long been used in spices, food additives, and drugs.Cell death induced by the binding of capsaicin was examined in a human gastric adenocarcinoma cell line (AGS cells).METHODS: By using XTT-based cytotoxicityassay, flow cytometry using the TUNEL method, and quantitation of DNA fragmentation, both cell death and DNA fragmentation were detected in AGS cells treated with capsaicin. By using Western blotting methods, capsaicin reduced the expression of Bcl-2, the antiapoptotic protein, in AGS cells in a concentration-dependent manner.RESULTS: After incubation of AGS cells with capsaicin for 24 h, cell viability decreased significantly in a dose-dependent manner. After incubation of AGS cells with capsaicin for 24 h, apoptotic bodies also significantly increased, and were again correlated with the dose of capsaicin. When the concentration of capsaicin was 1 mmol/L, the amount of DNA fragments also increased. Similar results werealso in the lower traces.CONCLUSION: These results suggest that capsaicininduced cell death might be via a Bcl-2 sensitive apoptotic pathway. Therefore, capsaicin might induce protection from gastric cancer.

  18. FTIR characterization of animal lung cells: normal and precancerous modified e10 cell line

    Science.gov (United States)

    Zezell, D. M.; Pereira, T. M.; Mennecier, G.; Bachmann, L.; Govone, A. B.; Dagli, M. L. Z.

    2012-06-01

    The chemical carcinogens from tobacco are related to over 90% of lung cancers around the world. The risk of death of this kind of cancer is high because the diagnosis usually is made only in advanced stages. Therefore, it is necessary to develop new diagnostic methods for detecting the lung cancer in earlier stages. The Fourier Transform Infrared Spectroscopy (FTIR) can offer high sensibility and accuracy to detect the minimal chemical changes into the biological sample. The aim of this study is to evaluate the differences on infrared spectra between normal lung cells and precancerous lung cells transformed by NNK. Non-cancerous lung cell line e10 (ATCC) and NNK-transformed e10 cell lines were maintained in complete culture medium (1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 [DMEM/Ham's F12], supplemented with 100 ng/ml cholera enterotoxin, 10 lg/ml insulin, 0.5 lg/ml. hydrocortisol, 20 ng/ml epidermal growth factor, and 5% horse serum. The cultures were maintained in alcohol 70%. The infrared spectra were acquired on ATR-FTIR Nicolet 6700 spectrophotometer at 4 cm-1 resolution, 30 scans, in the 1800-900 cm-1 spectral range. Each sample had 3 spectra recorded, 30 infrared spectra were obtained from each cell line. The second derivate of spectra indicates that there are displacement in 1646 cm-1 (amine I) and 1255 cm-1(DNA), allowing the possibility to differentiate the two king of cells, with accuracy of 89,9%. These preliminary results indicate that ATR-FTIR is useful to differentiate normal e10 lung cells from precancerous e10 transformed by NNK.

  19. The culture of cancer cell lines as tumorspheres does not systematically result in cancer stem cell enrichment.

    Science.gov (United States)

    Calvet, Christophe Y; André, Franck M; Mir, Lluis M

    2014-01-01

    Cancer stem cells (CSC) have raised great excitement during the last decade and are promising targets for an efficient treatment of tumors without relapses and metastases. Among the various methods that enable to enrich cancer cell lines in CSC, tumorspheres culture has been predominantly used. In this report, we attempted to generate tumorspheres from several murine and human cancer cell lines: B16-F10, HT-29, MCF-7 and MDA-MB-231 cells. Tumorspheres were obtained with variable efficiencies from all cell lines except from MDA-MB-231 cells. Then, we studied several CSC characteristics in both tumorspheres and adherent cultures of the B16-F10, HT-29 and MCF-7 cells. Unexpectedly, tumorspheres-forming cells were less clonogenic and, in the case of B16-F10, less proliferative than attached cells. In addition, we did not observe any enrichment in the population expressing CSC surface markers in tumorspheres from B16-F10 (CD133, CD44 and CD24 markers) or MCF-7 (CD44 and CD24 marke