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Sample records for permissive cell lines

  1. Identification of cell lines permissive for human coronavirus NL63.

    PubMed

    Schildgen, Oliver; Jebbink, Maarten F; de Vries, Michel; Pyrc, Krzysztov; Dijkman, Ronald; Simon, Arne; Müller, Andreas; Kupfer, Bernd; van der Hoek, Lia

    2006-12-01

    Six cell lines routinely used in laboratories were tested for permissiveness to the infection with the newly identified human coronavirus NL63. Two monkey epithelial cell lines, LLC-MK2 and Vero-B4, showed a cytopathic effect (CPE) and clear viral replication, whereas no CPE or replication was observed in human lung fibroblasts MRC-5s. In Rhabdomyosarcoma cells, Madin-Darby-Canine-kidney cells and in an undefined monkey kidney cell line some replication was observed but massive exponential rise in virus yield lacked The results will lead to an improved routine diagnostic algorithm for the detection of the human coronavirus NL63.

  2. Generation and Characterization of JCV Permissive Hybrid Cell Lines

    PubMed Central

    Sariyer, Ilker K.; Safak, Mahmut; Gordon, Jennifer; Khalili, Kamel

    2009-01-01

    JC virus (JCV) is a human neurotropic polyomavirus whose replication in the central nervous system induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV particles have been detected primarily in oligodendrocytes and astrocytes of the brains of patients with PML and in the laboratory its propagation is limited to primary cultures of human fetal glial cells. In this short communication, the development of a new cell culture system is described through the fusion of primary human fetal astrocytes with the human glioblastoma cell line, U-87MG. The new hybrid cell line obtained from this fusion has the capacity to support efficiently expression of JCV and replication of viral DNA in vitro up to 16 passages. This cell line can serve as a reliable culture system to study the biology of JCV host cell interaction, determine the mechanisms involved in cell type specific replication of JCV, and provide a convenient cell culture system for high throughput screening of anti-viral agents. PMID:19442856

  3. Permissive growth of human adenovirus type 4 vaccine strain-based vector in porcine cell lines.

    PubMed

    Gao, Dong-sheng; Li, Xiao-jing; Wan, Wen-yan; Li, Hong-jie; Wang, Xiao-xue; Yang, Xia; Li, Yong-tao; Chang, Hong-tao; Chen, Lu; Wang, Chuan-qing; Zhao, Jun

    2016-02-01

    In recent years, there has been considerable interest in using adenoviruses as live vectors to develop recombinant vaccines. Previous studies have demonstrated the safety and effectiveness of HIV/SIV and influenza vaccine candidates based on human adenovirus type 4 (Ad4) replication-competent vectors in rhesus macaque and human model. To explore the possibility of human Ad4 vaccine strain used as a vector in developing porcine vaccines, the growth properties of replication-competent human Ad4 vaccine strain recombinant encoding EGFP in different porcine cell lines were investigated. All tested cell lines are permissive for Ad4 vaccine strain vector with varied replication efficiency. Thus, human Ad4 based vectors would be promising supplement to adenovirus vectors as a delivery vehicle for recombinant vaccines in swine industry.

  4. The gamma-2-herpesvirus bovine herpesvirus 4 causes apoptotic infection in permissive cell lines.

    PubMed

    Sciortino, M T; Perri, D; Medici, M A; Foti, M; Orlandella, B M; Mastino, A

    2000-11-10

    Increasing evidence suggests that regulation of apoptosis in infected cells is associated with several viral infections. The gammaherpesvirus bovine herpesvirus 4 (BHV-4) has been shown to harbor genes with antiapoptotic potentialities. However, here we have demonstrated that productive infection of adherent, permissive cell lines by BHV-4 resulted in a cytopathic effect characterized by induction of apoptosis. This phenomenon was confirmed using different techniques to detect apoptosis and using different virus strains and cell targets. Apoptosis induced by BHV-4 was inhibited by (1) treatment with doses of heparin, which completely inhibited virus attachment and infectivity; (2) UV treatment, which completely abrogated infectivity; and (3) treatment with a dose of phosphonoacetic acid, which blocked virus replication. Virus-induced apoptosis was associated with a down-regulation of Bcl-2 expression and was reduced by Z-VAD-FMK, but not by Z-DEVD-FMK (caspase-3-specific) caspase inhibitors. Inhibition of apoptosis by Z-VAD-FMK treatment during infection did not modify virus yield. Therefore, despite the presence of antiapoptotic genes in its genoma, BHV-4 could complete its cycle of productive infection while inducing apoptosis of infected cells. This finding might have implications for the pathobiology of BHV-4 and other gammaherpesviruses in vivo. Copyright 2000 Academic Press.

  5. Morphological and molecular characterization of new Drosophila cell lines established from a strain permissive for gypsy transposition.

    PubMed

    Chalvet, F; Debec, A; Marcaillou, C; Rougeau, C; Bucheton, A

    1998-01-01

    The gypsy element of Drosophila melanogaster is the first retrovirus identified in invertebrates. Its transposition is controlled by a host gene called flamenco (flam): restrictive alleles of this gene maintain the retrovirus in a repressed state while permissive alleles allow high levels of transposition. To develop a cell system to study the gypsy element, we established four independent cell lines derived from the Drosophila strain SS, which contains a permissive allele of flamenco, and which is devoid of transposing copies of gypsy. The ultrastructural analysis of three SS cell lines revealed some remarkable characteristics, such as many nuclear virus-like particles, cytoplasmic dense particles, and massive cisternae filled with a fibrous material of unknown origin. Gypsy intragenomic distribution has been compared between the three cell lines and the original SS fly strain, and revealed in two of the cell lines an increase in copy number of a restriction fragment usually present in active gypsy elements. This multiplication seems to have occurred during the passage to the cell culture. Availability of SS cell lines should assist studies of gypsy transposition and infectivity and might be useful to produce high amounts of gypsy viral particles. These new lines already allowed us to show that the Envelope-like products of gypsy can be expressed as membrane proteins.

  6. Permissivity of fish cell lines to three Chlamydia-related bacteria: Waddlia chondrophila, Estrella lausannensis and Parachlamydia acanthamoebae.

    PubMed

    Kebbi-Beghdadi, Carole; Batista, Claudia; Greub, Gilbert

    2011-12-01

    Epitheliocystis is an infectious disease affecting gills and skin of various freshwater and marine fishes, associated with high mortality and reduced growth of survivors. Candidatus Piscichlamydia salmonis and Clavochlamydia salmonicola have recently been identified as aetiological agents of epitheliocystis in Atlantic Salmon. In addition, several other members of the Chlamydiales order have been identified in other fish species. To clarify the pathogenicity of Chlamydia-like organisms towards fishes, we investigated the permissivity of two fish cell lines, EPC-175 (Fathead Minnow) and RTG-2 (rainbow trout) to three Chlamydia-related bacteria: Waddlia chondrophila, Parachlamydia acanthamoebae and Estrella lausannensis. Quantitative PCR and immunofluorescence demonstrated that W. chondrophila and, to a lesser extent, E. lausannensis were able to replicate in the two cell lines tested. Waddlia chondrophila multiplied rapidly in its host cell and a strong cytopathic effect was observed. During E. lausannensis infection, we observed a limited replication of the bacteria not followed by host cell lysis. Very limited replication of P. acanthamoebae was observed in both cell lines tested. Given its high infectivity and cytopathic effect towards fish cell lines, W. chondrophila represents the most interesting Chlamydia-related bacteria to be used to develop an in vivo model of epitheliocystis disease in fishes. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  7. Human immunodeficiency virus type 1 NL4-3 replication in four T-cell lines: rate and efficiency of entry, a major determinant of permissiveness.

    PubMed Central

    Srivastava, K K; Fernandez-Larsson, R; Zinkus, D M; Robinson, H L

    1991-01-01

    Single-cycle infections have been used to study the human immunodeficiency virus type 1 (HIV-1) life cycle in CD4+ T-cell lines that differ in their permissiveness for infection. In single-cycle infections of highly permissive C8166 cells, 50% of the infectious units escaped being blocked by a monoclonal antibody against the virus binding site on CD4 (leu3a) within 30 min. In contrast, 50% of the infectious units for three less permissive cell lines (H9, A3.01, and Jurkat) required 4 h to escape the leu3a block. Entry was also more efficient in the highly permissive cells, with NL4-3 stocks having three times more infectious units for C8166 cells than for H9, A3.01, or Jurkat cells. Postentry steps up through reverse transcription required approximately 3.5 h in each of the cell lines. The times lapsing between reverse transcription and the expression of reverse transcripts ranged from 17 to 25 h in the different cell lines. Virus production per cell was also similar in the different cell lines (within 1.5-fold of each other). These results indicate that a major determinant of the permissiveness of growing T cells for HIV-1 is the rate and efficiency of virus entry. PMID:1674969

  8. Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins.

    PubMed

    Hashimoto, Yoshi; Zhang, Sheng; Zhang, Shiying; Chen, Yun-Ru; Blissard, Gary W

    2012-04-24

    After publication we discovered an error in the identification of the origin of the cell line reported in our article in BMC Biotechnology (2010, 10:50), entitled "Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins". Upon analysis of primary A. odorata cultures, we found that they were contaminated with cells of Trichoplusia ni origin. The origin of the Ao38 cell line was determined as T. ni using three marker genes and the Ao38 cell line was renamed BTI-Tnao38. References to the origin of the cell line as Ascalapha odorata should be replaced with "a cell line of Trichoplusia ni origin". The absence of TNCL virus detection in the BTI-Tnao38 (Ao38) cell line was confirmed using a highly sensitive RT-PCR protocol capable of detecting TNCL virus RNA at approximately 0.018 copies/cell. Because of these observations, we have revised the title of the original article to "Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins" and two additional authors were added to reflect their contributions to the analysis of this cell line.

  9. Mouse fibroblast L929 cells are less permissive to infection by Nelson Bay orthoreovirus compared to other mammalian cell lines.

    PubMed

    Mok, Lawrence; Wynne, James W; Grimley, Samantha; Shiell, Brian; Green, Diane; Monaghan, Paul; Pallister, Jackie; Bacic, Antony; Michalski, Wojtek P

    2015-07-01

    In recent years, bats have been identified as a natural reservoir for a diverse range of viruses. Nelson Bay orthoreovirus (NBV) was first isolated from the heart blood of a fruit bat (Pteropus poliocephalus) in 1968. While the pathogenesis of NBV remains unknown, other related members of this group have caused acute respiratory disease in humans. Thus the potential for NBV to impact human health appears plausible. Here, to increase our knowledge of NBV, we examined the replication and infectivity of NBV using different mammalian cell lines derived from bat, human, mouse and monkey. All cell lines supported the replication of NBV; however, L929 cells showed a greater than 2 log reduction in virus titre compared with the other cell lines. Furthermore, NBV did not induce major cytopathic effects in the L929 cells, as was observed in other cell lines. Interestingly, the related Pteropine orthoreoviruses, Pulau virus (PulV) and Melaka virus (MelV) were able to replicate to high titres in L929 cells but infection resulted in reduced cytopathic effect. Our study demonstrates a unique virus-host interaction between NBV and L929 cells, where cells effectively control viral infection/replication and limit the formation of syncytia. By elucidating the molecular mechanisms that control this unique relationship, important insights will be made into the biology of this fusogenic virus.

  10. An Occult Hepatitis B-Derived Hepatoma Cell Line Carrying Persistent Nuclear Viral DNA and Permissive for Exogenous Hepatitis B Virus Infection

    PubMed Central

    Lin, Chih-Lang; Chien, Rong-Nan; Lin, Shi-Ming; Ke, Po-Yuan; Lin, Chen-Chun; Yeh, Chau-Ting

    2013-01-01

    Occult hepatitis B virus (HBV) infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg). Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV) and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2). Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection. PMID:23734258

  11. An occult hepatitis B-derived hepatoma cell line carrying persistent nuclear viral DNA and permissive for exogenous hepatitis B virus infection.

    PubMed

    Lin, Chih-Lang; Chien, Rong-Nan; Lin, Shi-Ming; Ke, Po-Yuan; Lin, Chen-Chun; Yeh, Chau-Ting

    2013-01-01

    Occult hepatitis B virus (HBV) infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg). Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV) and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2). Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection.

  12. Real-Time Expression Analysis of Selected Anticarsia gemmatalis multiple nucleopolyhedrovirus Gene Promoters during Infection of Permissive, Semipermissive and Nonpermissive Cell Lines.

    PubMed

    Morgado, Fabricio da Silva; Ardisson-Araújo, Daniel Mendes Pereira; Ribeiro, Bergmann Morais

    2017-06-01

    Baculovirus infection follows a transcriptionally controlled sequence of gene expression that occurs by activation of different viral gene promoter sequences during infection. This sequence of promoter activation may be disrupted by cellular defenses against viral infection, which might interfere with viral progeny formation. In this work, the activity of the ie1, gp64, lef-1, vp39, p6.9 and polh promoters of the Anticarsia gemmatalis multiple nucleopolyhedrovirus was assessed during infection of permissive, semipermissive and nonpermissive cell lines by a novel methodology that detects reporter protein luminescence in real-time. This technique allowed us to characterize in rich detail the AgMNPV promoters in permissive cell lines and revealed differential profiles of expression in cells with limited permissivity that correlate well with limitations in viral DNA replication. Semipermissive and nonpermissive cell lines presented delays and restrictions in late and very late promoter expression. Cells undergoing apoptosis did not inhibit late gene expression; however, viral progeny formation is severely affected. This work demonstrates the application of the real-time luminescence detection methodology and how the promoter expression profile may be used to diagnose cellular permissivity to baculovirus infection.

  13. Real-Time Expression Analysis of Selected Anticarsia gemmatalis multiple nucleopolyhedrovirus Gene Promoters during Infection of Permissive, Semipermissive and Nonpermissive Cell Lines

    PubMed Central

    Morgado, Fabricio da Silva; Ardisson-Araújo, Daniel Mendes Pereira; Ribeiro, Bergmann Morais

    2017-01-01

    Baculovirus infection follows a transcriptionally controlled sequence of gene expression that occurs by activation of different viral gene promoter sequences during infection. This sequence of promoter activation may be disrupted by cellular defenses against viral infection, which might interfere with viral progeny formation. In this work, the activity of the ie1, gp64, lef-1, vp39, p6.9 and polh promoters of the Anticarsia gemmatalis multiple nucleopolyhedrovirus was assessed during infection of permissive, semipermissive and nonpermissive cell lines by a novel methodology that detects reporter protein luminescence in real-time. This technique allowed us to characterize in rich detail the AgMNPV promoters in permissive cell lines and revealed differential profiles of expression in cells with limited permissivity that correlate well with limitations in viral DNA replication. Semipermissive and nonpermissive cell lines presented delays and restrictions in late and very late promoter expression. Cells undergoing apoptosis did not inhibit late gene expression; however, viral progeny formation is severely affected. This work demonstrates the application of the real-time luminescence detection methodology and how the promoter expression profile may be used to diagnose cellular permissivity to baculovirus infection. PMID:28587184

  14. Expression of a viral polymerase-bound host factor turns human cell lines permissive to a plant- and insect-infecting virus

    PubMed Central

    de Medeiros, Ricardo B.; Figueiredo, Juliana; Resende, Renato de O.; De Avila, Antonio C.

    2005-01-01

    Tospoviruses are the only plant-infecting members of the Bunyaviridae family of ambisense ssRNA viruses. Tomato spotted wilt tospovirus (TSWV), the type-member, also causes mild infection on its main insect vector, Frankliniella occidentalis. Herein, we identified an F. occidentalis putative transcription factor (FoTF) that binds to the TSWV RNA-dependent RNA polymerase and to viral RNA. Using in vitro RNA synthesis assays, we show that addition of purified FoTF improves viral replication, but not transcription. Expression of FoTF deletion mutants, unable to bind the RNA-dependent RNA polymerase or viral RNA, blocks TSWV replication in F. occidentalis cells. Finally, expression of FoTF wild-type turns human cell lines permissive to TSWV replication. These data indicate that FoTF is a host factor required for TSWV replication in vitro and in vivo, provide an experimental system that could be used to compare molecular defense mechanisms in plant, insect, and human cells against the same pathogen (TSWV), and could lead to a better understanding of evolutionary processes of ambisense RNA viruses. PMID:15657123

  15. Functional characterization of Bombyx mori nucleopolyhedrovirus late gene transcription and genome replication factors in the non-permissive insect cell line SF-21

    SciTech Connect

    Berretta, Marcelo F.; Deshpande, Mandar; Crouch, Erin A.; Passarelli, A. Lorena . E-mail: lpassar@ksu.edu

    2006-04-25

    We compared the abilities of late gene transcription and DNA replication machineries of the baculoviruses Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV) in SF-21 cells, an insect-derived cell line permissive for AcMNPV infection. It has been well established that 19 AcMNPV late expression factors (lefs) stimulate substantial levels of late gene promoter activity in SF-21 cells. Thus, we constructed a set of clones containing the BmNPV homologs of the AcMNPV lefs under control of the constitutive Drosophila heat shock 70 protein promoter and tested their ability to activate an AcMNPV late promoter-reporter gene cassette in SF-21 cells. We tested the potential of individual or predicted functional groups of BmNPV lefs to successfully replace the corresponding AcMNPV gene(s) in transient late gene expression assays. We found that most, but not all, BmNPV lefs were able to either fully or partially substitute for the corresponding AcMNPV homolog in the context of the remaining AcMNPV lefs with the exception of BmNPV p143, ie-2, and p35. BmNPV p143 was unable to support late gene expression or be imported into the nucleus of cells in the presence of the AcMNPV or the BmNPV LEF-3, a P143 nuclear shuttling factor. Our results suggest that host-specific factors may affect the function of homologous proteins.

  16. Permissive and restricted virus infection of murine embryonic stem cells

    PubMed Central

    Wash, Rachael; Calabressi, Sabrina; Franz, Stephanie; Griffiths, Samantha J.; Goulding, David; Tan, E-Pien; Wise, Helen; Digard, Paul; Haas, Jürgen; Efstathiou, Stacey

    2012-01-01

    Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA ‘off-target’ effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host–pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host–virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host–virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence. PMID:22815272

  17. Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins

    PubMed Central

    2010-01-01

    Background The insect cell line is a critical component in the production of recombinant proteins in the baculovirus expression system and new cell lines hold the promise of increasing both quantity and quality of protein production. Results Seventy cell lines were established by single-cell cloning from a primary culture of cells derived from eggs of the black witch moth (Ascalapha odorata; Lepidoptera, Noctuidae). Among 8 rapidly growing lines, cell line 38 (Ao38) was selected for further analysis, based on susceptibility to AcMNPV infection and production of secreted alkaline phosphatase (SEAP) from a baculovirus expression vector. In comparisons with low-passage High Five (BTI-Tn-5B1-4) cells, infected Ao38 cells produced β-galactosidase and SEAP at levels higher (153% and 150%, respectively) than those measured from High Five cells. Analysis of N-glycans of SEAP produced in Ao38 cells revealed two N-glycosylation sites and glycosylation patterns similar to those reported for High Five and Sf9 cells. Glycopeptide isoforms consisted of pauci- or oligomannose, with and without fucose on N-acetylglucosamine(s) linked to asparagine residues. Estimates of Ao38 cell volume suggest that Ao38 cells are approximately 2.5× larger than Sf9 cells but only approximately 74% of the size of High Five cells. Ao38 cells were highly susceptible to AcMNPV infection, similar to infectivity of Sf9 cells. Production of infectious AcMNPV budded virions from Ao38 cells peaked at approximately 4.5 × 107 IU/ml, exceeding that from High Five cells while lower than that from Sf9 cells. Ao38 cells grew rapidly in stationary culture with a population doubling time of 20.2 hr, and Ao38 cells were readily adapted to serum-free medium (Sf-900III) and to a suspension culture system. Analysis of Ao38 and a parental Ascalapha odorata cell line indicated that these lines were free of the alphanodavirus that was recently identified as an adventitious agent in High Five cell lines. Conclusions Ao

  18. HSC70 interactions with SV40 viral proteins differ between permissive and nonpermissive mammalian cells

    PubMed Central

    Sainis, Ioannis; Angelidis, Charalambos; Pagoulatos, Gerasimos N.; Lazaridis, Ioannis

    2000-01-01

    SV40 belongs to a group of DNA tumor viruses which induce the expression of the 70 Kd heat shock proteins, but the meaning of this induction remains unclear. Investigating the role of hsc70 in the SV40 life cycle, we found that the protein translocates to the nucleus late in infection of permissive CV1 cells, in contrast to infected nonpermissive BALB/3T3 and NIH/3T3 cells in which hsc70 remains cytoplasmic. Moreover, the pattern of hsc70 nuclear staining was diffused and clearly distinguishable from that observed after heat shock. In addition hsc70 late in infection coimmunoprecipitated with the viral capsid protein VP1, suggesting a role in the process of viral packaging. Interactions of hsc70 with the early viral oncoprotein T antigen were observed only in nonpermissive cells, indicating that the binding of the above proteins is specific to cells that do not support viral propagation. Finally, treatment of permissive CV1 cells with interferon γ, a known antiviral cytokine, resulted in hsc70 binding to T antigen. Our results suggest that the role of hsc70 in the process of SV40 infection is directly related to the ability of the host cells to support viral propagation and is clearly different between permissive and nonpermissive cell lines. PMID:11147964

  19. HSV-2- and HIV-1- permissive cell lines co-infected by HSV-2 and HIV-1 co-replicate HSV-2 and HIV-1 without production of HSV-2/HIV-1 pseudotype particles

    PubMed Central

    LeGoff, Jérôme; Bouhlal, Hicham; Lecerf, Maxime; Klein, Christophe; Hocini, Hakim; Si-Mohamed, Ali; Muggeridge, Martin; Bélec, Laurent

    2007-01-01

    Background Herpes simplex virus type 2 (HSV-2) is a major cofactor of human immunodeficiency virus type 1 (HIV-1) sexual acquisition and transmission. In the present study, we investigated whether HIV-1 and HSV-2 may interact at the cellular level by forming HIV-1 hybrid virions pseudotyped with HSV-2 envelope glycoproteins, as was previously reported for HSV type 1. Methods We evaluated in vitro the production of HSV-2/HIV-1 pseudotypes in mononuclear CEM cells and epithelial HT29 and P4P cells. We analyzed the incorporation into the HIV-1 membrane of HSV-2 gB and gD, two major HSV-2 glycoproteins required for HSV-2 fusion with the cell membrane, in co-infected cells and in HIV-1-infected P4P cells transfected by plasmids coding for gB or gD. Results We show that HSV-2 and HIV-1 co-replicated in dually infected cells, and gB and gD were co-localized with gp160. However, HIV-1 particles, produced in HIV-1-infected cells expressing gB or gD after transfection or HSV-2 superinfection, did not incorporate either gB or gD in the viral membrane, and did not have the capacity to infect cells normally non-permissive for HIV-1, such as epithelial cells. Conclusion Our results do not support the hypothesis of HSV-2/HIV-1 pseudotype formation and involvement in the synergistic genital interactions between HIV-1 and HSV-2. PMID:17207276

  20. Human hepatic stellate cells are not permissive for hepatitis C virus entry and replication.

    PubMed

    Florimond, Alexandre; Chouteau, Philippe; Bruscella, Patrice; Le Seyec, Jacques; Mérour, Emilie; Ahnou, Nazim; Mallat, Ariane; Lotersztajn, Sophie; Pawlotsky, Jean-Michel

    2015-06-01

    Chronic HCV infection is associated with the development of hepatic fibrosis. The direct role of HCV in the fibrogenic process is unknown. Specifically, whether HCV is able to infect hepatic stellate cells (HSCs) is debated. To assess whether human HSCs are susceptible to HCV infection. We combined a set of original HCV models, including the infectious genotype 2a JFH1 model (HCVcc), retroviral pseudoparticles expressing the folded HCV genotype 1b envelope glycoproteins (HCVpp) and a subgenomic genotype 1b HCV replicon, and two relevant cellular models, primary human HSCs from different patients and the LX-2 cell line, to assess whether HCV can infect/replicate in HSCs. In contrast with the hepatocyte cell line Huh-7, neither infectious HCVcc nor HCVpp infected primary human HSCs or LX-2 cells. The cellular expression of host cellular factors required for HCV entry was high in Huh-7 cells but low in HSCs and LX-2 cells, with the exception of CD81. Finally, replication of a genotype 2a full-length RNA genome and a genotype 1b subgenomic replicon was impaired in primary human HSCs and LX-2 cells, which expressed low levels of cellular factors known to play a key role in the HCV life-cycle, suggesting that human HSCs are not permissive for HCV replication. Human HSCs are refractory to HCV infection. Both HCV entry and replication are deficient in these cells, regardless of the HCV genotype and origin of the cells. Thus, HCV infection of HSCs does not play a role in liver fibrosis. These results do not rule out a direct role of HCV infection of hepatocytes in the fibrogenic process. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  1. Pseudorabies virus infection inhibits autophagy in permissive cells in vitro

    PubMed Central

    Sun, Mingxia; Hou, Linlin; Tang, Yan-dong; Liu, Yonggang; Wang, Shujie; Wang, Jingfei; Shen, Nan; An, Tongqing; Tian, Zhijun; Cai, Xuehui

    2017-01-01

    A large number of studies have demonstrated that autophagy is involved in the infection processes of different pathogens. Autophagy is now recognized as an essential component of innate and adaptive immunity. Several herpesviruses have developed various strategies to evade this antiviral mechanism. Pseudorabies virus (PRV) is a swine herpesvirus with a broad host range that causes devastating disease in infected pigs. In this study, we described the interaction between PRV and autophagy for the first time. PRV infection had a dual effect on the cell autophagy response; during the early period of infection, PRV virions induced autophagy without viral replication, and with viral protein expression, PRV reduced the basal level of autophagy in several permissive cells. We observed that inhibit the level of autophagy could increase the titer of infectious PRV. We also found that the conserved alphaherpesvirus US3 tegument protein may reduce the level of autophagy via activation of the AKT/mTOR pathways in PRV infected cells. These findings suggest that autophagy likely contributes to clearance of PRV, and that the virus has evolved strategies to antagonize this pathway. PMID:28059118

  2. Heat shock protein 72 expression allows permissive replication of oncolytic adenovirus dl1520 (ONYX-015) in rat glioblastoma cells

    PubMed Central

    Madara, Jonathan; Krewet, James A; Shah, Maulik

    2005-01-01

    In this study we have made novel observations with regards to potentiation of the tumoricidal activity of the oncolytic adenovirus, dl1520 (ONYX-015) in rat glioblastoma cell lines expressing heat shock protein 72 (HSP72) due to permissive virus replication. ONYX-015 is a conditionally replicating adenovirus that is deleted for the E1B 55 kDA gene product whose normal function is to interact with cell-cycle regulatory proteins to permit virus replication. However, many murine and rodent cell lines are not permissive for adenovirus replication. Previously, it has been reported that the heat shock response is necessary for adenovirus replication and that induction of heat shock proteins is mediated by E1 region gene products. Therefore, we hypothesized that HSP72 expression may allow for permissive replication of ONYX-015 in previously non-permissive cells. Rat glioma cell lines 9L and RT2 were transfected with a plasmids expressing HSP72 or GFP. After infection with ONYX-015, no tumoricidal activity is observed in GFP expressing cell lines despite adequate transduction. In contrast, HSP72 transfected cells show cytopathic effects by 72 hours and greater than 75% loss of viability by 96 hours. Burst assays show active virus replication in the HSP72 expressing cell lines. Therefore, 9L-HSP72 and RT2-HSP72 are ideal models to evaluate the efficacy of ONYX-015 in an immunocompetent rat model. Our study has implications for creating rodent tumor models for pre-clinical studies with E1 region deleted conditionally replicating adenovirus. PMID:15762988

  3. Permissiveness of bovine epithelial cells from lung, intestine, placenta and udder for infection with Coxiella burnetii.

    PubMed

    Sobotta, Katharina; Bonkowski, Katharina; Liebler-Tenorio, Elisabeth; Germon, Pierre; Rainard, Pascal; Hambruch, Nina; Pfarrer, Christiane; Jacobsen, Ilse D; Menge, Christian

    2017-04-12

    Ruminants are the main source of human infections with the obligate intracellular bacterium Coxiella (C.) burnetii. Infected animals shed high numbers of C. burnetii by milk, feces, and birth products. In goats, shedding by the latter route coincides with C. burnetii replication in epithelial (trophoblast) cells of the placenta, which led us to hypothesize that epithelial cells are generally implicated in replication and shedding of C. burnetii. We therefore aimed at analyzing the interactions of C. burnetii with epithelial cells of the bovine host (1) at the entry site (lung epithelium) which govern host immune responses and (2) in epithelial cells of gut, udder and placenta decisive for the quantity of pathogen excretion. Epithelial cell lines [PS (udder), FKD-R 971 (small intestine), BCEC (maternal placenta), F3 (fetal placenta), BEL-26 (lung)] were inoculated with C. burnetii strains Nine Mile I (NMI) and NMII at different cultivation conditions. The cell lines exhibited different permissiveness for C. burnetii. While maintaining cell viability, udder cells allowed the highest replication rates with formation of large cell-filling Coxiella containing vacuoles. Intestinal cells showed an enhanced susceptibility to invasion but supported C. burnetii replication only at intermediate levels. Lung and placental cells also internalized the bacteria but in strikingly smaller numbers. In any of the epithelial cells, both Coxiella strains failed to trigger a substantial IL-1β, IL-6 and TNF-α response. Epithelial cells, with mammary epithelial cells in particular, may therefore serve as a niche for C. burnetii replication in vivo without alerting the host's immune response.

  4. Recombinant pestivirus E2 glycoproteins prevent viral attachment to permissive and non permissive cells with different efficiency.

    PubMed

    Asfor, A S; Wakeley, P R; Drew, T W; Paton, D J

    2014-08-30

    Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen, which like other pestiviruses has similar molecular biological features to hepaciviruses, including human Hepatitis C virus. The pestivirus E2 glycoproteins are the major target for virus-neutralising antibodies, as well as playing a role in receptor binding and host range restriction. In this study, recombinant E2 glycoproteins (rE2) derived from three different pestivirus species were examined for their inhibitory effects on pestivirus infectivity in cell culture. Histidine-tagged rE2 glycoproteins of BVDV type 2 strain 178003, BVDV type 1 strain Oregon C24V and CSFV strain Alfort 187 were produced in Spodoptera frugiperda insect cells and purified under native conditions. The ability of rE2 glycoprotein to inhibit the infection of permissive cells by both homologous and heterologous virus was compared, revealing that the inhibitory effects of rE2 glycoproteins correlated with the predicted similarity of the E2 structures in the recombinant protein and the test virus. This result suggests that the sequence and structure of E2 are likely to be involved in the host specificity of pestiviruses at their point of uptake into cells.

  5. SAMHD1 is active in cycling cells permissive to HIV-1 infection.

    PubMed

    Badia, Roger; Pujantell, Maria; Torres-Torronteras, Javier; Menéndez-Arias, Luis; Martí, Ramón; Ruzo, Albert; Pauls, Eduardo; Clotet, Bonaventura; Ballana, Ester; Esté, José A; Riveira-Muñoz, Eva

    2017-06-01

    SAMHD1 is a triphosphohydrolase that restricts HIV-1 by limiting the intracellular dNTP pool required for reverse transcription. Although SAMHD1 is expressed and active/unphosphorylated in most cell lines, its restriction activity is thought to be relevant only in non-cycling cells. However, an in depth evaluation of SAMHD1 function and relevance in cycling cells is required. Here, we show that SAMHD1-induced degradation by HIV-2 Vpx affects the dNTP pool and HIV-1 replication capacity in the presence of the 3'-azido-3'-deoxythymidine (AZT) in cycling cells. Similarly, in SAMHD1 knockout cells, HIV-1 showed increased replicative capacity in the presence of nucleoside inhibitors, especially AZT, that was reverted by re-expression of wild type SAMHD1. Sensitivity to non-nucleoside inhibitors (nevirapine and efavirenz) or the integrase inhibitor raltegravir was not affected by SAMHD1. Combination of three mutations (S18A, T21A, T25A) significantly prevented SAMHD1 phosphorylation but did not significantly affect HIV-1 replication in the presence of AZT. Our results demonstrate that SAMHD1 is active in HIV-1 permissive cells, does not modify susceptibility to HIV-1 infection but strongly affects sensitivity to nucleoside inhibitors. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Isolation and characterization of mouse-human microcell hybrid cell clones permissive for infectious HIV particle release

    PubMed Central

    Coskun, Ayse K.; van Maanen, Marc; Janka, David; Stockton, David; Stankiewicz, Pawel; Yatsenko, Svetlana; Sutton, Richard E.

    2007-01-01

    Mouse cells are non-permissive to human immunodeficiency virus type 1 (HIV) in that there is a pronounced post-integration block to viral replication. We have recently demonstrated that mouse-human somatic cell hybrids that contain human chromosome 2 increase both HIV Capsid (CA) production and infectious virus release. Here we report on the isolation of three mouse-human microcell hybrids (MCHs) that behave similarly, starting from a pool of 500 MCH clones. Release of virus was specific to HIV and cell revertants that no longer contained any human chromosome fragments did not release CA or infectious virus. Two of the three cell clones were identical as judged by PCR STS content and fluorescence in situ hybridization (FISH) and contained a single 2-12 human chromosome chimera. The third cell clone only contained human chromosome 12, as determined by PCR, FISH, and microarray analyses. There were no consistent differences in Gag protein and spliced/unspliced viral RNA levels between mouse cell lines. CMV promoter-driven, codon-optimized gag-pol had no effect on infectious HIV release from these mouse cells, despite allowing Gag targeting and increasing CA production. These permissive mouse-human MCHs and their corresponding non-permissive revertants may prove useful for mechanistic studies and also for identifying the responsible gene(s) or factor(s) involved in the production of HIV. PMID:17270231

  7. Human Endometrial Stromal Cells Are Highly Permissive To Productive Infection by Zika Virus.

    PubMed

    Pagani, Isabel; Ghezzi, Silvia; Ulisse, Adele; Rubio, Alicia; Turrini, Filippo; Garavaglia, Elisabetta; Candiani, Massimo; Castilletti, Concetta; Ippolito, Giuseppe; Poli, Guido; Broccoli, Vania; Panina-Bordignon, Paola; Vicenzi, Elisa

    2017-03-10

    Zika virus (ZIKV) is a recently re-emerged flavivirus transmitted to humans by mosquito bites but also from mother to fetus and by sexual intercourse. We here show that primary human endometrial stromal cells (HESC) are highly permissive to ZIKV infection and support its in vitro replication. ZIKV envelope expression was detected in the endoplasmic reticulum whereas double-stranded viral RNA colocalized with vimentin filaments to the perinuclear region. ZIKV productive infection also occurred in the human T-HESC cell line together with the induction of interferon-β (IFN-β) and of IFN-stimulated genes. Notably, in vitro decidualization of T-HESC with cyclic AMP and progesterone upregulated the cell surface expression of the ZIKV entry co-receptor AXL and boosted ZIKV replication by ca. 100-fold. Thus, endometrial stromal cells, particularly if decidualized, likely represent a crucial cell target of ZIKV reaching them, either via the uterine vasculature in the viremic phase of the infection or by sexual viral transmission, and a potential source of virus spreading to placental trophoblasts during pregnancy.

  8. Human Endometrial Stromal Cells Are Highly Permissive To Productive Infection by Zika Virus

    PubMed Central

    Pagani, Isabel; Ghezzi, Silvia; Ulisse, Adele; Rubio, Alicia; Turrini, Filippo; Garavaglia, Elisabetta; Candiani, Massimo; Castilletti, Concetta; Ippolito, Giuseppe; Poli, Guido; Broccoli, Vania; Panina-Bordignon, Paola; Vicenzi, Elisa

    2017-01-01

    Zika virus (ZIKV) is a recently re-emerged flavivirus transmitted to humans by mosquito bites but also from mother to fetus and by sexual intercourse. We here show that primary human endometrial stromal cells (HESC) are highly permissive to ZIKV infection and support its in vitro replication. ZIKV envelope expression was detected in the endoplasmic reticulum whereas double-stranded viral RNA colocalized with vimentin filaments to the perinuclear region. ZIKV productive infection also occurred in the human T-HESC cell line together with the induction of interferon-β (IFN-β) and of IFN-stimulated genes. Notably, in vitro decidualization of T-HESC with cyclic AMP and progesterone upregulated the cell surface expression of the ZIKV entry co-receptor AXL and boosted ZIKV replication by ca. 100-fold. Thus, endometrial stromal cells, particularly if decidualized, likely represent a crucial cell target of ZIKV reaching them, either via the uterine vasculature in the viremic phase of the infection or by sexual viral transmission, and a potential source of virus spreading to placental trophoblasts during pregnancy. PMID:28281680

  9. Comparison of egg and high yielding MDCK cell-derived live attenuated influenza virus for commercial production of trivalent influenza vaccine: in vitro cell susceptibility and influenza virus replication kinetics in permissive and semi-permissive cells.

    PubMed

    Hussain, Althaf I; Cordeiro, Melissa; Sevilla, Elizabeth; Liu, Jonathan

    2010-05-14

    Currently MedImmune manufactures cold-adapted (ca) live, attenuated influenza vaccine (LAIV) from specific-pathogen free (SPF) chicken eggs. Difficulties in production scale-up and potential exposure of chicken flocks to avian influenza viruses especially in the event of a pandemic influenza outbreak have prompted evaluation and development of alternative non-egg based influenza vaccine manufacturing technologies. As part of MedImmune's effort to develop the live attenuated influenza vaccine (LAIV) using cell culture production technologies we have investigated the use of high yielding, cloned MDCK cells as a substrate for vaccine production by assessing host range and virus replication of influenza virus produced from both SPF egg and MDCK cell production technologies. In addition to cloned MDCK cells the indicator cell lines used to evaluate the impact of producing LAIV in cells on host range and replication included two human cell lines: human lung carcinoma (A549) cells and human muco-epidermoid bronchiolar carcinoma (NCI H292) cells. The influenza viruses used to infect the indicators cell lines represented both the egg and cell culture manufacturing processes and included virus strains that composed the 2006-2007 influenza seasonal trivalent vaccine (A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/05 (H3N2) and B/Malaysia/2506/04). Results from this study demonstrate remarkable similarity between influenza viruses representing the current commercial egg produced and developmental MDCK cell produced vaccine production platforms. MedImmune's high yielding cloned MDCK cells used for the cell culture based vaccine production were highly permissive to both egg and cell produced ca attenuated influenza viruses. Both the A549 and NCI H292 cells regardless of production system were less permissive to influenza A and B viruses than the MDCK cells. Irrespective of the indicator cell line used the replication properties were similar between egg and the cell produced

  10. Acute Myeloid Leukemia Targeting by Myxoma Virus In Vivo Depends on Cell Binding But Not Permissiveness to Infection In Vitro

    PubMed Central

    Madlambayan, Gerard J.; Bartee, Eric; Kim, Manbok; Rahman, Masmudur M.; Meacham, Amy; Scott, Edward W.; McFadden, Grant; Cogle, Christopher R.

    2012-01-01

    Some oncolytic viruses, such as myxoma virus (MYXV), can selectively target malignant hematopoietic cells, while sparing normal hematopoietic cells. This capacity for discrimination creates an opportunity to use oncolytic viruses as ex vivo purging agents of autologous hematopoietic cell grafts in patients with hematologic malignancies. However, the mechanisms by which oncolytic viruses select malignant hematopoietic cells are poorly understood. In this study, we investigated how MYXV specifically targets human AML cells. MYXV prevented chloroma formation and bone marrow engraftment of two human AML cell lines, KG-1 and THP-1. The reduction in human leukemia engraftment after ex vivo MYXV treatment was dose-dependent and required a minimum MOI of 3. Both AML cell lines demonstrated MYXV binding to leukemia cell membranes following co-incubation: however, evidence of productive MYXV infection was observed only in THP-1 cells. This observation, that KG-1 can be targeted in vivo even in the absence of in vitro permissive viral infection, contrasts with the current understanding of oncolytic virotherapy, which assumes that virus infection and productive replication is a requirement. Preventing MYXV binding to AML cells with heparin abrogated the purging capacity of MYXV, indicating that binding of infectious virus particles is a necessary step for effective viral oncolysis. Our results challenge the current dogma of oncolytic virotherapy and show that in vitro permissiveness to an oncolytic virus is not necessarily an accurate predictor of oncolytic potency in vivo. PMID:22341701

  11. Acute myeloid leukemia targeting by myxoma virus in vivo depends on cell binding but not permissiveness to infection in vitro.

    PubMed

    Madlambayan, Gerard J; Bartee, Eric; Kim, Manbok; Rahman, Masmudur M; Meacham, Amy; Scott, Edward W; McFadden, Grant; Cogle, Christopher R

    2012-05-01

    Some oncolytic viruses, such as myxoma virus (MYXV), can selectively target malignant hematopoietic cells, while sparing normal hematopoietic cells. This capacity for discrimination creates an opportunity to use oncolytic viruses as ex vivo purging agents of autologous hematopoietic cell grafts in patients with hematologic malignancies. However, the mechanisms by which oncolytic viruses select malignant hematopoietic cells are poorly understood. In this study, we investigated how MYXV specifically targets human AML cells. MYXV prevented chloroma formation and bone marrow engraftment of two human AML cell lines, KG-1 and THP-1. The reduction in human leukemia engraftment after ex vivo MYXV treatment was dose-dependent and required a minimum MOI of 3. Both AML cell lines demonstrated MYXV binding to leukemia cell membranes following co-incubation: however, evidence of productive MYXV infection was observed only in THP-1 cells. This observation, that KG-1 can be targeted in vivo even in the absence of in vitro permissive viral infection, contrasts with the current understanding of oncolytic virotherapy, which assumes that virus infection and productive replication is a requirement. Preventing MYXV binding to AML cells with heparin abrogated the purging capacity of MYXV, indicating that binding of infectious virus particles is a necessary step for effective viral oncolysis. Our results challenge the current dogma of oncolytic virotherapy and show that in vitro permissiveness to an oncolytic virus is not necessarily an accurate predictor of oncolytic potency in vivo.

  12. The adenovirus type 12 - mouse cell system: permissivity and analysis of integration patterns of viral DNA in tumor cells.

    PubMed Central

    Starzinski-Powitz, A; Schulz, M; Esche, H; Mukai, N; Doerfler, W

    1982-01-01

    The integration patterns of persisting adenovirus type 12 (Ad12) DNA were analyzed in two Ad12-induced tumors of Balb/c and CBA/J mice and in one tumor cell line derived from an Ad12-induced retinoblastoma of C3H origin. In all three tumors the Ad12 genome was integrated colinearly and various copy numbers of viral DNA were found. Analysis of the Ad12 integration patterns revealed relatively simple offsize band patterns regardless of Ad12 copy numbers. The degree of methylation at the 5'-CCGG-3' sites in the inserted Ad12 genome was determined using the isoschizomeric restriction endonuclease pair HpaII and MspI. Methylation was rather incomplete in the primary tumor tissues but almost complete in the retinoblastoma line carried in culture for many passages. The levels of expression of the viral genome in the Balb/c tumor and in the retinoblastoma line were determined by in vitro translation of RNA isolated from these cells and selected with appropriate restriction endonuclease fragments of Ad12 DNA. In both instances the 59 K, 19 K, and 17 K proteins of the E1b region were expressed. Proteins of the E1a region appeared very faint in the size class between 22 K and 42 K. The permissivity of Ad12 and the replication of Ad12 DNA in mouse cells were investigated by blotting restricted DNA from cells soon after, and a long time after, infection and by hybridization with 32P-labeled Ad12 DNA. Neither primary mouse kidney cells nor the established L929 mouse cell line supported viral DNA replication. These results raise the question to what extent host cell factors determine Ad12 DNA replication in mammalian cells. Images Fig. 2. Fig. 3. Fig. 4. PMID:7188349

  13. Cell line provenance.

    PubMed

    Freshney, R Ian

    2002-07-01

    Cultured cell lines have become an extremely valuable resource, both in academic research and in industrial biotechnology. However, their value is frequently compromised by misidentification and undetected microbial contamination. As detailed elsewhere in this volume, the technology, both simple and sophisticated, is available to remedy the problems of misidentification and contamination, given the will to apply it. Combined with proper records of the origin and history of the cell line, assays for authentication and contamination contribute to the provenance of the cell line. Detailed records should start from the initiation or receipt of the cell line, and should incorporate data on the donor as well as the tissue from which the cell line was derived, should continue with details of maintenance, and include any accidental as well as deliberate deviations from normal maintenance. Records should also contain details of authentication and regular checks for contamination. With this information, preferably stored in a database, and suitable backed up, the provenance of the cell line so created makes the cell line a much more valuable resource, fit for validation in industrial applications and more likely to provide reproducible experimental results when disseminated for research in other laboratories.

  14. The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells

    PubMed Central

    Leisi, Remo; Von Nordheim, Marcus; Ros, Carlos; Kempf, Christoph

    2016-01-01

    Parvovirus B19 (B19V) is a small non-enveloped virus and known as the causative agent for the mild childhood disease erythema infectiosum. B19V has an extraordinary narrow tissue tropism, showing only productive infection in erythroid precursor cells in the bone marrow. We recently found that the viral protein 1 unique region (VP1u) contains an N-terminal receptor-binding domain (RBD), which mediates the uptake of the virus into cells of the erythroid lineage. To further investigate the role of the RBD in connection with a B19V-unrelated capsid, we chemically coupled the VP1u of B19V to the bacteriophage MS2 capsid and tested the internalization capacity of the bioconjugate on permissive cells. In comparison, we studied the cellular uptake and infection of B19V along the erythroid differentiation. The results showed that the MS2-VP1u bioconjugate mimicked the specific internalization of the native B19V into erythroid precursor cells, which further coincides with the restricted infection profile. The successful mimicry of B19V uptake demonstrates that the RBD in the VP1u is sufficient for the endocytosis of the viral capsid. Furthermore, the recombinant VP1u competed with B19V uptake into permissive cells, thus excluding a significant alternative uptake mechanism by other receptors. Strikingly, the VP1u receptor appeared to be expressed only on erythropoietin-dependent erythroid differentiation stages that also provide the necessary intracellular factors for a productive infection. Taken together, these findings suggest that the VP1u binds to a yet-unknown erythroid-specific cellular receptor and thus restricts the virus entry to permissive cells. PMID:27690083

  15. High permissivity of human HepG2 hepatoma cells for influenza viruses.

    PubMed

    Ollier, Laurence; Caramella, Anne; Giordanengo, Valérie; Lefebvre, Jean-Claude

    2004-12-01

    Human HepG2 hepatoma cells are highly permissive for influenza virus type A and type B, even without the addition of trypsin, and they exhibit a marked cytopathic effect. This property greatly facilitates the primary isolation of influenza viruses. Virus replication was significantly reduced by the plasmin(ogen)-specific inhibitor tranexamic acid, and this suggests a potential role played by the plasminogen/tissue plasminogen activator complex at the surface of HepG2 cells. This might represent a new approach for study of the interrelations of this complex with influenza viruses.

  16. High Permissivity of Human HepG2 Hepatoma Cells for Influenza Viruses

    PubMed Central

    Ollier, Laurence; Caramella, Anne; Giordanengo, Valérie; Lefebvre, Jean-Claude

    2004-01-01

    Human HepG2 hepatoma cells are highly permissive for influenza virus type A and type B, even without the addition of trypsin, and they exhibit a marked cytopathic effect. This property greatly facilitates the primary isolation of influenza viruses. Virus replication was significantly reduced by the plasmin(ogen)-specific inhibitor tranexamic acid, and this suggests a potential role played by the plasminogen/tissue plasminogen activator complex at the surface of HepG2 cells. This might represent a new approach for study of the interrelations of this complex with influenza viruses. PMID:15583326

  17. Telomere-surrounding regions are transcription-permissive 3D nuclear compartments in human cells

    SciTech Connect

    Quina, Ana Sofia; Parreira, Leonor . E-mail: lparreir@igc.gulbenkian.pt

    2005-07-01

    Positioning of genes relative to nuclear heterochromatic compartments is thought to help regulate their transcriptional activity. Given that human subtelomeric regions are rich in highly expressed genes, we asked whether human telomeres are related to transcription-permissive nuclear compartments. To address this question, we investigated in the nuclei of normal human lymphocytes the spatial relations of two constitutively expressed genes (ACTB and RARA) and three nuclear transcripts (ACTB, IL2RA and TCRB) to telomeres and centromeres, as a function of gene activity and transcription levels. We observed that genes and gene transcripts locate close to telomere clusters and away from chromocenters upon activation of transcription. These findings, together with the observation that SC35 domains, which are enriched in pre-mRNA processing factors, are in close proximity to telomeres, indicate that telomere-neighboring regions are permissive to gene expression in human cells. Therefore, the associations of telomeres observed in the interphase nucleus might contribute, as opposed to chromocenters, for the establishment of transcription-permissive 3D nuclear compartments.

  18. Goat umbilical cord cells are permissive to small ruminant lentivirus infection in vitro.

    PubMed

    Martins, Gabrielle R; Marinho, Rebeca C; Junior, Rosivaldo Q Bezerra; Alves, Antoniel de O; Câmara, Lilia M C; Albuquerque-Pinto, Luiz C; Teixeira, Maria F da S

    Small ruminant lentiviruses isolated from peripheral blood leukocytes and target organs can be propagated in vitro in fibroblasts derived from goat synovial membrane cells. These cells are obtained from tissues collected from embryos or fetuses and are necessary for the establishment of the fibroblast primary culture. A new alternative type of host cells, derived from goat umbilical cord, was isolated and characterized phenotypically with its main purpose being to obtain cell monolayers that could be used for the diagnosis and isolation of small ruminant lentiviruses in cell culture. To accomplish this goal, cells were isolated from umbilical cords; characterized phenotypically by flow cytometry analysis; differentiate into osteogenic, chondrogenic and adipogenic lineage; and submitted to viral challenge. The proliferation of goat umbilical cord cells was fast and cell monolayers formed after 15 days. These cells exhibited morphology, immunophenotype, growth characteristics, and lineage differentiation potential similar to mesenchymal stem cells of other origins. The goat umbilical cord derived cells stained positive for vimentin and CD90, but negative for cytokeratin, CD34 and CD105 markers. Syncytia and cell lysis were observed in cell monolayers infected by CAEV-Cork and MVV-K1514, showing that the cells are permissive to small ruminant lentivirus infection in vitro. These data demonstrate the proliferative competence of cells derived from goat umbilical cords and provide a sound basis for future research to standardize this cell lineage.

  19. Myeloid-derived suppressor cells are implicated in regulating permissiveness for tumor metastasis during mouse gestation

    PubMed Central

    Mauti, Laetitia A.; Le Bitoux, Marie-Aude; Baumer, Karine; Stehle, Jean-Christophe; Golshayan, Dela; Provero, Paolo; Stamenkovic, Ivan

    2011-01-01

    Metastasis depends on the ability of tumor cells to establish a relationship with the newly seeded tissue that is conducive to their survival and proliferation. However, the factors that render tissues permissive for metastatic tumor growth have yet to be fully elucidated. Breast tumors arising during pregnancy display early metastatic proclivity, raising the possibility that pregnancy may constitute a physiological condition of permissiveness for tumor dissemination. Here we have shown that during murine gestation, metastasis is enhanced regardless of tumor type, and that decreased NK cell activity is responsible for the observed increase in experimental metastasis. Gene expression changes in pregnant mouse lung and liver were shown to be similar to those detected in premetastatic sites and indicative of myeloid cell infiltration. Indeed, myeloid-derived suppressor cells (MDSCs) accumulated in pregnant mice and exerted an inhibitory effect on NK cell activity, providing a candidate mechanism for the enhanced metastatic tumor growth observed in gestant mice. Although the functions of MDSCs are not yet understood in the context of pregnancy, our observations suggest that they may represent a shared mechanism of immune suppression occurring during gestation and tumor growth. PMID:21646719

  20. Meningeal cells and glia establish a permissive environment for axon regeneration after spinal cord injury in newts

    PubMed Central

    2011-01-01

    Background Newts have the remarkable ability to regenerate their spinal cords as adults. Their spinal cords regenerate with the regenerating tail after tail amputation, as well as after a gap-inducing spinal cord injury (SCI), such as a complete transection. While most studies on newt spinal cord regeneration have focused on events occurring after tail amputation, less attention has been given to events occurring after an SCI, a context that is more relevant to human SCI. Our goal was to use modern labeling and imaging techniques to observe axons regenerating across a complete transection injury and determine how cells and the extracellular matrix in the injury site might contribute to the regenerative process. Results We identify stages of axon regeneration following a spinal cord transection and find that axon regrowth across the lesion appears to be enabled, in part, because meningeal cells and glia form a permissive environment for axon regeneration. Meningeal and endothelial cells regenerate into the lesion first and are associated with a loose extracellular matrix that allows axon growth cone migration. This matrix, paradoxically, consists of both permissive and inhibitory proteins. Axons grow into the injury site next and are closely associated with meningeal cells and glial processes extending from cell bodies surrounding the central canal. Later, ependymal tubes lined with glia extend into the lesion as well. Finally, the meningeal cells, axons, and glia move as a unit to close the gap in the spinal cord. After crossing the injury site, axons travel through white matter to reach synaptic targets, and though ascending axons regenerate, sensory axons do not appear to be among them. This entire regenerative process occurs even in the presence of an inflammatory response. Conclusions These data reveal, in detail, the cellular and extracellular events that occur during newt spinal cord regeneration after a transection injury and uncover an important role for

  1. Claudin-1, miR-122 and apolipoprotein E transductions improve the permissivity of SNU-182, SNU-398 and SNU-449 hepatoma cells to hepatitis C virus.

    PubMed

    Fournier, C; Hoffmann, T W; Morel, V; Descamps, V; Dubuisson, J; Brochot, E; Francois, C; Duverlie, G; Castelain, S; Helle, F

    2017-08-03

    Hepatitis C virus (HCV) is a human hepatotropic virus, but many hepatoma cell lines are not permissive to this virus. In a previous study, we observed that SNU-182, SNU-398 and SNU-449 hepatoma cell lines were nonpermissive to HCV. To understand the nonpermissivity, we evaluated the ability of each cell line to support the different steps of HCV life cycle (entry, replication and production of infectious particles). Using retroviral pseudoparticles pseudotyped with HCV envelope proteins and recombinant HCV produced in cell culture, we observed that low level or absence of claudin-1 (CLDN1) expression limited the viral entry process in SNU-182 and SNU-398 cells, respectively. Our results also showed that supplementation of the three cell lines with miR-122 partly restored the replication of a JFH1 HCV replicon. Finally, we observed that expression of apolipoprotein E (ApoE) was very low or undetectable in the three cell lines and that its ectopic expression permits the production of infectious viral particles in SNU-182 and SNU-398 cells but not in SNU-449 cells. Nevertheless, the supplementation of SNU-182, SNU-398 and SNU-449 cells with CLDN1, miR-122 and ApoE was not sufficient to render these cells as permissive as HuH-7 cells. Thus, these cell lines could serve as cell culture models for functional studies on the role of CLDN1, miR-122 and ApoE in HCV life cycle but also for the identification of new restriction and/or dependency host factors essential for HCV infection. © 2017 John Wiley & Sons Ltd.

  2. Crayfish hematopoietic tissue cells but not hemocytes are permissive for white spot syndrome virus replication.

    PubMed

    Wu, Junjun; Li, Fang; Huang, Jiajun; Xu, Limei; Yang, Feng

    2015-03-01

    Hemocytes are the major immune cells of crustaceans which are believed to be essential for the pathogenesis of white spot syndrome virus (WSSV) infection. Crayfish hemocytes and hematopoietic tissue (HPT) cells have been found to be susceptible to WSSV infection, but the procedure of WSSV infection to both cell types has not yet been carefully investigated. In this study, we analyzed the infection and proliferation of WSSV in crayfish hemocytes as well as HPT cells in detail through transmission electronic microscopy (TEM). The results showed that WSSV could enter both hemocytes and HPT cells through endocytosis, but the production of progeny virus was only achieved in HPT cells. Further investigation demonstrated that although WSSV could transcribe its genes in both cell types, viral genome replication and structural protein expression were unsuccessful in hemocytes, which may be responsible for the failure of progeny production. Therefore, we propose that both hemocytes and HPT cells are susceptible to WSSV infection but only HPT cells are permissive to WSSV replication. These findings will extend our knowledge of the interaction between WSSV and the host immune system.

  3. Epigenetic bivalent marking is permissive to the synergy of HDAC and PARP inhibitors on TXNIP expression in breast cancer cells.

    PubMed

    Baldan, Federica; Mio, Catia; Lavarone, Elisa; Di Loreto, Carla; Puglisi, Fabio; Damante, Giuseppe; Puppin, Cinzia

    2015-05-01

    Studies on stem cell differentiation led to the identification of paused genes, characterized by the contemporary presence of both activator and repressor epigenetic markers (bivalent marking). TXNIP is an oncosuppressor gene the expression of which was reduced in breast cancer. In the present study, we evaluated whether the concept of epigenetic bivalent marking can be applied to TXNIP gene in breast cancer cells. Using chromatin immunoprecipitation (ChIP), three histone modifications were investigated: two associated with transcriptional activation, lysines 9-14 acetylation of H3 histone (H3K9K14ac) and lysine 4 trimethylation of H3 histone (H3K4me3), and one associated with transcriptional silencing, lysine 27 trimethylation of H3 histone (H3K27me3). According to the bivalent marking model, TXNIP gene appears to be paused in MDA157 cells (markers of active and repressed transcription are present), but are definitively silenced in MDA468 cells (presence of only markers of transcription repression). This was proven by evaluating TXNIP mRNA and protein levels after the treatment of cell lines with a histone deacetylase inhibitor (SAHA) and a poly-ADP-ribose polymerases inhibitor (PJ34). In MDA157 cells, SAHA and PJ34 showed a synergistic effect: a large increment was observed in TXNIP mRNA and protein levels. By contrast, in MDA468 cells, synergy between the two compounds was not observed. Therefore, the pausing epigenetic signature was permissive for synergy between SAHA and PJ34 on TXNIP gene expression. The synergy between SAHA and PJ34 on TXNIP expression was associated with variation in cell viability and apoptosis. In MDA157 cells, but not in MDA468 cells, combined treatment of SAHA and PJ34 induced a decrease in cell viability and an increase of apoptosis. Thus, our data support the hypothesis that TXNIP is an effective target for the treatment of breast cancer.

  4. Human cytomegalovirus renders cells non-permissive for replication of herpes simplex viruses

    SciTech Connect

    Cockley, K.D.

    1988-01-01

    The herpes simplex virus (HSV) genome during production infection in vitro may be subject to negative regulation which results in modification of the cascade of expression of herpes virus macromolecular synthesis leading to establishment of HSV latency. In the present study, human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of HSV type-1 (HSV-1). A delay in HSV replication of 15 hr as well as a consistent, almost 1000-fold inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 hr after superinfection were observed compared with controls infected with HSV alone. HSV type-2 (HSV-2) replication was similarly inhibited in HCMV-infected HEL cells. Prior ultraviolet-irradiation (UV) of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HCMV deoxyribonucleic acid (DNA) negative temperature-sensitive (ts) mutants inhibited HSV replications as efficiently as wild-type (wt) HCMV at the non-permissive temperature. Evidence for penetration and replication of superinfecting HSV into HCMV-infected cells was provided by blot hybridization of HSV DNA synthesized in HSV-superinfected cell cultures and by cesium chloride density gradient analysis of ({sup 3}H)-labeled HSV-1-superinfected cells.

  5. Beclin 1 is involved in regulation of apoptosis and autophagy during replication of ectromelia virus in permissive L929 cells.

    PubMed

    Martyniszyn, Lech; Szulc, Lidia; Boratyńska, Anna; Niemiałtowski, Marek G

    2011-12-01

    Several reports have brought to light new and interesting findings on the involvement of autophagy and apoptosis in pathogenesis of viral and bacterial diseases, as well as presentation of foreign antigens. Our model studies focused on the involvement of apoptosis during replication of highly virulent Moscow strain of ectromelia virus (ECTV-MOS). Here, we show evidence that autophagy is induced during mousepox replication in a cell line. Fluorescence microscopy revealed increase of LC3 (microtubule-associated protein 1 light chain 3) aggregation in infected as opposed to non-infected control L929 cells. Furthermore, Western blot analysis showed that replication of ECTV-MOS in L929 cells led to the increase in LC3-II (marker of autophagic activity) expression. Beclin 1 strongly colocalized with extranuclear viral replication centers in infected cells, whereas expression of Bcl-2 decreased in those centers as shown by fluorescence microscopy. Loss of Beclin 1-Bcl-2 interaction may lead to autophagy in virus-infected L929 cells. To assess if Beclin 1 has a role in regulation of apoptosis during ECTV-MOS infection, we used small interfering RNA directed against beclin 1 following infection. Early and late apoptotic cells were analyzed by flow cytometry after AnnexinV and propidium iodide staining. Silencing of beclin 1 resulted in decreased percentage of early and late apoptotic cells in the late stage of ECTV-MOS infection in L929 cells. We conclude that Beclin 1 plays an important role in regulation of both, autophagy and apoptosis, during ECTV-MOS replication in L929 permissive cells.

  6. Changes in the mitochondrial network during ectromelia virus infection of permissive L929 cells.

    PubMed

    Gregorczyk, Karolina P; Szulc-Dąbrowska, Lidia; Wyżewski, Zbigniew; Struzik, Justyna; Niemiałtowski, Marek

    2014-01-01

    Mitochondria are extremely important organelles in the life of a cell. Recent studies indicate that mitochondria also play a fundamental role in the cellular innate immune mechanisms against viral infections. Moreover, mitochondria are able to alter their shape continuously through fusion and fission. These tightly regulated processes are activated or inhibited under physiological or pathological (e.g. viral infection) conditions to help restore homeostasis. However, many types of viruses, such as orthopoxviruses, have developed various strategies to evade the mitochondrial-mediated antiviral innate immune responses. Moreover, orthopoxviruses exploit the mitochondria for their survival. Such viral activity has been reported during vaccinia virus (VACV) infection. Our study shows that the Moscow strain of ectromelia virus (ECTV-MOS), an orthopoxvirus, alters the mitochondrial network in permissive L929 cells. Upon infection, the branching structure of the mitochondrial network collapses and becomes disorganized followed by destruction of mitochondrial tubules during the late stage of infection. Small, discrete mitochondria co-localize with progeny virions, close to the cell membrane. Furthermore, clustering of mitochondria is observed around viral factories, particularly between the nucleus and viroplasm. Our findings suggest that ECTV-MOS modulates mitochondrial cellular distribution during later stages of the replication cycle, probably enabling viral replication and/or assembly as well as transport of progeny virions inside the cell. However, this requires further investigation.

  7. Stat5-deficient hematopoiesis is permissive for Myc-induced B-cell leukemogenesis.

    PubMed

    Wang, Zhengqi; Medrzycki, Magdalena; Bunting, Silvia T; Bunting, Kevin D

    2015-10-06

    Despite being an attractive molecular target for both lymphoid and myeloid leukemias characterized by activated tyrosine kinases, the molecular and physiological consequences of reduced signal transducer and activator of transcription-5 (Stat5) during leukemogenesis are not well known. Stat5 is a critical regulator of mouse hematopoietic stem cell (HSC) self-renewal and is essential for normal lymphocyte development. We report that pan-hematopoietic deletion in viable adult Vav1-Cre conditional knockout mice as well as Stat5ab(null/null) fetal liver transplant chimeras generated HSCs with reduced expression of quiescence regulating genes (Tie2, Mpl, Slamf1, Spi1, Cited2) and increased expression of B-cell development genes (Satb1, Dntt, Btla, Flk2). Using a classical murine B-cell acute lymphoblastic leukemia (B-ALL) model, we demonstrate that these HSCs were also poised to produce a burst of B-cell precursors upon expression of Bcl-2 combined with oncogenic Myc. This strong selective advantage for leukemic transformation in the background of Stat5 deficient hematopoiesis was permissive for faster initiation of Myc-induced transformation to B-ALL. However, once established, the B-ALL progression in secondary transplant recipients was Stat5-independent. Overall, these studies suggest that Stat5 can play multiple important roles that not only preserve the HSC compartment but can limit accumulation of potential pre-leukemic lymphoid populations.

  8. CLO: The cell line ontology

    PubMed Central

    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 consortium, new cell line cells, upper level alignment with the Cell Ontology (CL) and the Ontology for Biomedical Investigation, and logical extensions. Construction and content Collaboration among the CLO, CL, and OBI has established consensus definitions of cell line-specific terms such as ‘cell line’, ‘cell line cell’, ‘cell line culturing’, and ‘mortal’ vs. ‘immortal cell line cell’. A cell line is a genetically stable cultured cell population that contains individual cell line cells. The hierarchical structure of the CLO is built based on the hierarchy of the in vivo cell types defined in CL and tissue types (from which cell line cells are derived) defined in the UBERON cross-species anatomy ontology. The new hierarchical structure makes it easier to browse, query, and perform automated classification. We have recently added classes representing more than 2,000 cell line cells from the RIKEN BRC Cell Bank to CLO. Overall, the CLO now contains ~38,000 classes of specific cell line cells derived from over 200 in vivo cell types from various organisms. Utility and discussion The CLO has been applied to different biomedical research studies. Example case studies include annotation and analysis of EBI ArrayExpress data, bioassays, and host-vaccine/pathogen interaction. CLO’s utility goes beyond a catalogue of cell line types. The alignment of the CLO with related ontologies combined with the use of ontological reasoners will support sophisticated inferencing to advance translational informatics development. PMID:25852852

  9. Identification of a target cell permissive factor required for contact-dependent growth inhibition (CDI)

    PubMed Central

    Diner, Elie J.; Beck, Christina M.; Webb, Julia S.; Low, David A.; Hayes, Christopher S.

    2012-01-01

    Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CdiA effector proteins are exported onto the surface of CDI+ inhibitor cells, where they interact with susceptible bacteria and deliver effectors/toxins derived from their C-terminal regions (CdiA-CT). CDI+ cells also produce an immunity protein that binds the CdiA-CT and blocks its activity to prevent autoinhibition. Here, we show that the CdiA-CT from uropathogenic Escherichia coli strain 536 (UPEC536) is a latent tRNase that requires activation by the biosynthetic enzyme CysK (O-acetylserine sulfhydrylase A). UPEC536 CdiA-CT exhibits no nuclease activity in vitro, but cleaves within transfer RNA (tRNA) anti-codon loops when purified CysK is added. CysK and CdiA-CT form a stable complex, and their binding interaction appears to mimic that of the CysK/CysE cysteine synthase complex. CdiA-CT activation is also required for growth inhibition. Synthesis of CdiA-CT in E. coli cysK+ cells arrests cell growth, whereas the growth of ΔcysK mutants is unaffected by the toxin. Moreover, E. coli ΔcysK cells are completely resistant to inhibitor cells expressing UPEC536 CdiA, indicating that CysK is required to activate the tRNase during CDI. Thus, CysK acts as a permissive factor for CDI, providing a potential mechanism to modulate growth inhibition in target cells. PMID:22333533

  10. Embryonic stem cell lines of nonhuman primates.

    PubMed

    Nakatsuji, Norio; Suemori, Hirofumi

    2002-06-26

    Human embryonic stem (ES) cell lines have opened great potential and expectation for cell therapy and regenerative medicine. Monkey and human ES cell lines, which are very similar to each other, have been established from monkey blastocysts and surplus human blastocysts from fertility clinics. Nonhuman primate ES cell lines provide important research tools for basic and applicative research. Firstly, they provide wider aspects of investigation of the regulative mechanisms of stem cells and cell differentiation among primate species. Secondly, their usage does not need clearance or permission from the regulative rules in many countries that are associated with the ethical aspects of human ES cells, although human and nonhuman embryos and fetuses are very similar to each other. Lastly and most importantly, they are indispensable for animal models of cell therapy to test effectiveness, safety, and immunological reaction of the allogenic transplantation in a setting similar to the treatment of human diseases. So far, ES cell lines have been established from rhesus monkey (Macaca mulatta), common marmoset (Callithrix jacchus), and cynomolgus monkey (Macaca fascicularis), using blastocysts produced naturally or by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). These cell lines seem to have very similar characteristics. They express alkaline phosphatase activity and stage-specific embryonic antigen (SSEA)-4 and, in most cases, SSEA-3. Their pluripotency was confirmed by the formation of embryoid bodies and differentiation into various cell types in culture and also by the formation of teratomas that contained many types of differentiated tissues including derivatives of three germ layers after transplantation into the severe combined immunodeficiency (SCID) mice. The noneffectiveness of the leukemia inhibitory factor (LIF) signal makes culture of primate and human ES cell lines prone to undergo spontaneous differentiation and thus it is

  11. Multipotent mesenchymal stromal cells are fully permissive for human cytomegalovirus infection.

    PubMed

    Qiao, Guan-Hua; Zhao, Fei; Cheng, Shuang; Luo, Min-Hua

    2016-06-01

    Congenital human cytomegalovirus (HCMV) infection is a leading infectious cause of birth defects. Previous studies have reported birth defects with multiple organ maldevelopment in congenital HCMV-infected neonates. Multipotent mesenchymal stromal cells (MSCs) are a group of stem/progenitor cells that are multi-potent and can self-renew, and they play a vital role in multi-organ formation. Whether MSCs are susceptible to HCMV infection is unclear. In this study, MSCs were isolated from Wharton's jelly of the human umbilical cord and identified by their plastic adherence, surface marker pattern, and differentiation capacity. Then, the MSCs were infected with the HCMV Towne strain, and infection status was assessed via determination of viral entry, replication initiation, viral protein expression, and infectious virion release using western blotting, immunofluorescence assays, and plaque forming assays. The results indicate that the isolated MSCs were fully permissive for HCMV infection and provide a preliminary basis for understanding the pathogenesis of HCMV infection in non-nervous system diseases, including multi-organ malformation during fetal development.

  12. Permission Forms

    ERIC Educational Resources Information Center

    Zirkel, Perry A.

    2005-01-01

    The prevailing practice in public schools is to routinely require permission or release forms for field trips and other activities that pose potential for liability. The legal status of such forms varies, but they are generally considered to be neither rock-solid protection nor legally valueless in terms of immunity. The following case and the…

  13. Permission Forms

    ERIC Educational Resources Information Center

    Zirkel, Perry A.

    2005-01-01

    The prevailing practice in public schools is to routinely require permission or release forms for field trips and other activities that pose potential for liability. The legal status of such forms varies, but they are generally considered to be neither rock-solid protection nor legally valueless in terms of immunity. The following case and the…

  14. Epigenetic silencing of antiviral genes renders clones of Huh-7 cells permissive for hepatitis C virus replication.

    PubMed

    Chen, Qiuyue; Denard, Bray; Huang, Hua; Ye, Jin

    2013-01-01

    Hepatitis C virus (HCV) does not replicate efficiently in wild-type human hepatoma Huh-7 cells, but it replicates robustly in certain subclones of Huh-7 cells. Previously, we demonstrated that silencing of cyclic AMP (cAMP) response element binding protein 3-like 1 (CREB3L1), a cellular transcription factor that inhibits HCV replication, allows HCV to replicate in HRP1 cells, a subclone of Huh-7 cells permissive for HCV replication. Here we show that silencing of myxovirus resistant 1 (MX1), a known interferon-induced antiviral gene, is responsible for HRP4 cells, another subclone of Huh-7 cells, being permissive for HCV replication. Both CREB3L1 and MX1 are epigenetically silenced through DNA methylation in HRP1 and HRP4 cells, respectively. We further demonstrate that Huh-7 cells exist as a mixed population of cells with distinct patterns of gene methylation and HCV replicates in subpopulations of Huh-7 cells that have antiviral genes epigenetically silenced by DNA hypermethylation. Our results demonstrate that understanding the mechanism through which subclones of Huh-7 cells become permissive for HCV replication is crucial for studying their interaction with HCV.

  15. Permissive hypercapnia.

    PubMed

    Thome, Ulrich H; Carlo, Waldemar A

    2002-10-01

    Although lifesaving, mechanical ventilation can result in lung injury and contribute to the development of bronchopulmonary dysplasia. The most critical determinants of lung injury are tidal volume and end-inspiratory lung volume. Permissive hypercapnia offers to maintain gas exchange with lower tidal volumes and thus decrease lung injury. Further physiologic benefits include improved oxygen delivery and neuroprotection, the latter through both avoidance of accidental hypocapnia, which is associated with a poor neurologic outcome, and direct cellular effects. Clinical trials in adults with acute respiratory failure indicated improved survival and reduced incidence of organ failure in subjects managed with low tidal volumes and permissive hypercapnia. Retrospective studies in low birth weight infants found an association of bronchopulmonary dysplasia with low PaCO(2). Randomized clinical trials of low birth weight infants did not achieve sufficient statistical power to demonstrate a reduction of BPD by permissive hypercapnia, but strong trends indicated the possibility of important benefits without increased adverse events. Herein, we review the mechanisms leading to lung injury, the physiologic effects of hypercapnia, the dangers of hypocapnia, and the available clinical data.

  16. In vitro permissivity of bovine cells for wild-type and vaccinal myxoma virus strains.

    PubMed

    Pignolet, Béatrice; Duteyrat, Jean-Luc; Allemandou, Aude; Gelfi, Jacqueline; Foucras, Gilles; Bertagnoli, Stéphane

    2007-09-27

    Myxoma virus (MYXV), a leporide-specific poxvirus, represents an attractive candidate for the generation of safe, non-replicative vaccine vector for non-host species. However, there is very little information concerning infection of non-laboratory animals species cells with MYXV. In this study, we investigated interactions between bovine cells and respectively a wild type strain (T1) and a vaccinal strain (SG33) of MYXV. We showed that bovine KOP-R, BT and MDBK cell lines do not support MYXV production. Electron microscopy observations of BT-infected cells revealed the low efficiency of viral entry and the production of defective virions. In addition, infection of bovine peripheral blood mononuclear cells (PBMC) occurred at a very low level, even following non-specific activation, and was always abortive. We did not observe significant differences between the wild type strain and the vaccinal strain of MYXV, indicating that SG33 could be used for new bovine vaccination strategies.

  17. Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci

    PubMed Central

    Philippe, Claude; Vargas-Landin, Dulce B; Doucet, Aurélien J; van Essen, Dominic; Vera-Otarola, Jorge; Kuciak, Monika; Corbin, Antoine; Nigumann, Pilvi; Cristofari, Gaël

    2016-01-01

    LINE-1 (L1) retrotransposons represent approximately one sixth of the human genome, but only the human-specific L1HS-Ta subfamily acts as an endogenous mutagen in modern humans, reshaping both somatic and germline genomes. Due to their high levels of sequence identity and the existence of many polymorphic insertions absent from the reference genome, the transcriptional activation of individual genomic L1HS-Ta copies remains poorly understood. Here we comprehensively mapped fixed and polymorphic L1HS-Ta copies in 12 commonly-used somatic cell lines, and identified transcriptional and epigenetic signatures allowing the unambiguous identification of active L1HS-Ta copies in their genomic context. Strikingly, only a very restricted subset of L1HS-Ta loci - some being polymorphic among individuals - significantly contributes to the bulk of L1 expression, and these loci are differentially regulated among distinct cell lines. Thus, our data support a local model of L1 transcriptional activation in somatic cells, governed by individual-, locus-, and cell-type-specific determinants. DOI: http://dx.doi.org/10.7554/eLife.13926.001 PMID:27016617

  18. Efficient Hepatitis Delta Virus RNA Replication in Avian Cells Requires a Permissive Factor(s) from Mammalian Cells

    PubMed Central

    Liu, Yu-Tsueng; Brazas, Rob; Ganem, Don

    2001-01-01

    Hepatitis delta virus (HDV) is a highly pathogenic human RNA virus whose genome is structurally related to those of plant viroids. Although its spread from cell to cell requires helper functions supplied by hepatitis B virus (HBV), intracellular HDV RNA replication can proceed in the absence of HBV proteins. As HDV encodes no RNA-dependent RNA polymerase, the identity of the (presumably cellular) enzyme responsible for this reaction remains unknown. Here we show that, in contrast to mammalian cells, avian cells do not support efficient HDV RNA replication and that this defect cannot be rescued by provision of HDV gene products in trans. Contrary to earlier assertions, this defect is not due to enhanced apoptosis triggered in avian cells by HDV. Fusion of avian cells to mammalian cells rescues HDV replication in avian nuclei, indicating that the nonpermissive phenotype of avian cells is not due to the presence of dominantly acting inhibitors of replication. Rather, avian cells lack one or more essential permissive factors present in mammalian cells. These results set the stage for the identification of such factors and also explain the failure of earlier efforts to transmit HDV infection to avian hosts harboring indigenous hepadnaviruses. PMID:11462021

  19. Pediatric brain tumor cell lines.

    PubMed

    Xu, Jingying; Margol, Ashley; Asgharzadeh, Shahab; Erdreich-Epstein, Anat

    2015-02-01

    Pediatric brain tumors as a group, including medulloblastomas, gliomas, and atypical teratoid rhabdoid tumors (ATRT) are the most common solid tumors in children and the leading cause of death from childhood cancer. Brain tumor-derived cell lines are critical for studying the biology of pediatric brain tumors and can be useful for initial screening of new therapies. Use of appropriate brain tumor cell lines for experiments is important, as results may differ depending on tumor properties, and can thus affect the conclusions and applicability of the model. Despite reports in the literature of over 60 pediatric brain tumor cell lines, the majority of published papers utilize only a small number of these cell lines. Here we list the approximately 60 currently-published pediatric brain tumor cell lines and summarize some of their central features as a resource for scientists seeking pediatric brain tumor cell lines for their research.

  20. Drug/Cell-line Browser: interactive canvas visualization of cancer drug/cell-line viability assay datasets.

    PubMed

    Duan, Qiaonan; Wang, Zichen; Fernandez, Nicolas F; Rouillard, Andrew D; Tan, Christopher M; Benes, Cyril H; Ma'ayan, Avi

    2014-11-15

    Recently, several high profile studies collected cell viability data from panels of cancer cell lines treated with many drugs applied at different concentrations. Such drug sensitivity data for cancer cell lines provide suggestive treatments for different types and subtypes of cancer. Visualization of these datasets can reveal patterns that may not be obvious by examining the data without such efforts. Here we introduce Drug/Cell-line Browser (DCB), an online interactive HTML5 data visualization tool for interacting with three of the recently published datasets of cancer cell lines/drug-viability studies. DCB uses clustering and canvas visualization of the drugs and the cell lines, as well as a bar graph that summarizes drug effectiveness for the tissue of origin or the cancer subtypes for single or multiple drugs. DCB can help in understanding drug response patterns and prioritizing drug/cancer cell line interactions by tissue of origin or cancer subtype. DCB is an open source Web-based tool that is freely available at: http://www.maayanlab.net/LINCS/DCB CONTACT: avi.maayan@mssm.edu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. Baculovirus Infection Influences Host Protein Expression in Two Established Insect Cell Lines

    USDA-ARS?s Scientific Manuscript database

    We identified host proteins that changed in response to host cell susceptibility to baculovirus infection. We used three baculovirus–host cell systems utilizing two cell lines derived from pupal ovaries, Hz-AM1 (from Helicoverpa zea) and Hv-AM1 (from Heliothis virescens). Hv-AM1 cells are permissive...

  2. Host natural killer immunity is a key indicator of permissiveness for donor cell engraftment in patients with severe combined immunodeficiency.

    PubMed

    Hassan, Amel; Lee, Pamela; Maggina, Paraskevi; Xu, Jin Hua; Moreira, Diana; Slatter, Mary; Nademi, Zohreh; Worth, Austen; Adams, Stuart; Jones, Alison; Cale, Catherine; Allwood, Zoe; Rao, Kanchan; Chiesa, Robert; Amrolia, Persis; Gaspar, Hubert; Davies, E Graham; Veys, Paul; Gennery, Andrew; Qasim, Waseem

    2014-06-01

    Severe combined immunodeficiency (SCID) can be cured by using allogeneic hematopoietic stem cell transplantation, and the absence of host immunity often obviates the need for preconditioning. Depending on the underlying genetic defect and when blocks in differentiation occur during lymphocyte ontogeny, infants with SCID have absent or greatly reduced numbers of functional T cells. Natural killer (NK) cell populations are usually absent in the SCID-X1 and Janus kinase 3 forms of SCID and greatly reduced in adenosine deaminase deficiency SCID but often present in other forms of the disorder. To determine if SCID phenotypes indicate host permissiveness to donor cell engraftment. A retrospective data analysis considered whether host NK cells influenced donor T-cell engraftment, immune reconstitution, and long-term outcomes in children who had undergone nonconditioned allogeneic stem cell transplantation between 1990 and 2011 in the United Kingdom. Detailed analysis of T- and B-cell immune reconstitution and donor chimerism was compared between the NK(+) (n = 24) and NK(-) (n = 53) forms of SCID. Overall, 77 children underwent transplantation, with survival of 90% in matched sibling donor/matched family donor transplants compared with 60% when alternative donors were used. Infants with NK(-)SCID were more likely to survive than NK(+) recipients (87% vs 62%, P < .01) and had high-level donor T-cell chimerism with superior long-term recovery of CD4 T-cell immunity. Notably, 33% of children with NK(+)SCID required additional transplantation procedures compared with only 8% of children with NK(-)SCID (P < .005). NK(-)SCID disorders are highly permissive for donor T-cell engraftment without preconditioning, whereas the presence of NK cells is a strong indicator that preparative conditioning is required for engraftment of T-cell precursors capable of supporting robust T-cell reconstitution. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by

  3. Host natural killer immunity is a key indicator of permissiveness for donor cell engraftment in patients with severe combined immunodeficiency☆

    PubMed Central

    Hassan, Amel; Lee, Pamela; Maggina, Paraskevi; Xu, Jin Hua; Moreira, Diana; Slatter, Mary; Nademi, Zohreh; Worth, Austen; Adams, Stuart; Jones, Alison; Cale, Catherine; Allwood, Zoe; Rao, Kanchan; Chiesa, Robert; Amrolia, Persis; Gaspar, Hubert; Davies, E. Graham; Veys, Paul; Gennery, Andrew; Qasim, Waseem

    2014-01-01

    Background Severe combined immunodeficiency (SCID) can be cured by using allogeneic hematopoietic stem cell transplantation, and the absence of host immunity often obviates the need for preconditioning. Depending on the underlying genetic defect and when blocks in differentiation occur during lymphocyte ontogeny, infants with SCID have absent or greatly reduced numbers of functional T cells. Natural killer (NK) cell populations are usually absent in the SCID-X1 and Janus kinase 3 forms of SCID and greatly reduced in adenosine deaminase deficiency SCID but often present in other forms of the disorder. Objective To determine if SCID phenotypes indicate host permissiveness to donor cell engraftment. Methods A retrospective data analysis considered whether host NK cells influenced donor T-cell engraftment, immune reconstitution, and long-term outcomes in children who had undergone nonconditioned allogeneic stem cell transplantation between 1990 and 2011 in the United Kingdom. Detailed analysis of T- and B-cell immune reconstitution and donor chimerism was compared between the NK+ (n = 24) and NK− (n = 53) forms of SCID. Results Overall, 77 children underwent transplantation, with survival of 90% in matched sibling donor/matched family donor transplants compared with 60% when alternative donors were used. Infants with NK−SCID were more likely to survive than NK+ recipients (87% vs 62%, P < .01) and had high-level donor T-cell chimerism with superior long-term recovery of CD4 T-cell immunity. Notably, 33% of children with NK+SCID required additional transplantation procedures compared with only 8% of children with NK−SCID (P < .005). Conclusions NK−SCID disorders are highly permissive for donor T-cell engraftment without preconditioning, whereas the presence of NK cells is a strong indicator that preparative conditioning is required for engraftment of T-cell precursors capable of supporting robust T-cell reconstitution. PMID:24794685

  4. Molecular detection of Leishmania infection in sand flies in border line of Iran-Turkmenistan: restricted and permissive vectors.

    PubMed

    Bakhshi, H; Oshaghi, M A; Abai, M R; Rassi, Y; Akhavan, A A; Sheikh, Z; Mohtarami, F; Saidi, Z; Mirzajani, H; Anjomruz, M

    2013-10-01

    A molecular study was carried out to incriminate sand fly vectors of cutaneous leishmaniasis (CL) in rural areas of Sarakhs district, Khorassane-Razavi Province, northeastern Iran, in 2011. Sand flies of Sergentomyia with three species and Phlebotomus with six species respectively comprised 73.3% and 26.7% of the specimens. Phlebotomus papatasi was the most common Phlebotomine species in outdoor and indoor resting places. Leishmania infection was found at least in 17 (22%) specimens including Ph. papatasi (n=9 pool samples), Phlebotomus caucasicus (n=6), Phlebotomus alexandri (n=1), and Sergentomyia sintoni (n=1). The parasites were found comprised Leishmania major (n=5), Leishmania turanica (n=10), and Leishmania gerbilli (n=4). Infection of Ph. papatasi with both L. major and L. turanica supporting the new suggestion indicating that it is not restricted only with L. major. Circulation of L. major by Ph. alexandri, and both L. gerbilli and L. turanica by Ph. caucasicus, in addition to previous data indicating the ability of Ph. alexandri to circulate Leishmania infantum and Leishmania donovani, and Ph. caucasicus to circulate L. major, suggests that these two species can be permissive vectors. The results suggest that Ph. papatasi and Ph. alexandri are the primary and secondary vectors of CL where circulating L. major between human and reservoirs, whereas Ph. caucasicus is circulating L. turanica and L. gerbilli between the rodents in the region.

  5. Characterization of resistance to rhabdovirus and retrovirus infection in a human myeloid cell line.

    PubMed

    Boso, Guney; Somia, Nikunj V

    2015-01-01

    Viruses interact with various permissive and restrictive factors in host cells throughout their replication cycle. Cell lines that are non-permissive to viral infection have been particularly useful in discovering host cell proteins involved in viral life cycles. Here we describe the characterization of a human myeloid leukemia cell line, KG-1, that is resistant to infection by retroviruses and a Rhabdovirus. We show that KG-1 cells are resistant to infection by Vesicular Stomatits Virus as well as VSV Glycoprotein (VSVG) pseudotyped retroviruses due to a defect in binding. Moreover our results indicate that entry by xenotropic retroviral envelope glycoprotein RD114 is impaired in KG-1 cells. Finally we characterize a post- entry block in the early phase of the retroviral life cycle in KG-1 cells that renders the cell line refractory to infection. This cell line will have utility in discovering proteins involved in infection by VSV and HIV-1.

  6. Optimization of cell lines as tumour models by integrating multi-omics data.

    PubMed

    Zhao, Ning; Liu, Yongjing; Wei, Yunzhen; Yan, Zichuang; Zhang, Qiang; Wu, Cheng; Chang, Zhiqiang; Xu, Yan

    2017-05-01

    Cell lines are widely used as in vitro models of tumorigenesis. However, an increasing number of researchers have found that cell lines differ from their sourced tumour samples after long-term cell culture. The application of unsuitable cell lines in experiments will affect the experimental accuracy and the treatment of patients. Therefore, it is imperative to identify optimal cell lines for each cancer type. Here, we review the methods used to evaluate cell lines since 2005. Furthermore, gene expression, copy number and mutation profiles from The Cancer Genome Atlas and the Cancer Cell Line Encyclopedia are used to calculate similarity between tumours and cell lines. Then, the ideal cell lines to use for experiments for eight types of cancers are found by combining the results with Gene Ontology functional similarity. After verification, the optimal cell lines have the same genomic characteristics as their homologous tumour samples. The contaminated cell lines identified in previous research are also determined to be unsuitable in vitro cancer models here. Moreover, our study suggests that some of the commonly used cell lines are not suitable cancer models. In summary, we provide a reference for ideal cell lines to use in in vitro experiments and contribute to improving the accuracy of future cancer research. Furthermore, this research provides a foundation for identifying more effective treatment strategies. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Human Th17 Cells Lack HIV-Inhibitory RNases and Are Highly Permissive to Productive HIV Infection

    PubMed Central

    Christensen-Quick, Aaron; Lafferty, Mark; Sun, Lingling; Marchionni, Luigi; DeVico, Anthony

    2016-01-01

    ABSTRACT Human immunodeficiency virus (HIV) infects and depletes CD4+ T cells, but subsets of CD4+ T cells vary in their susceptibility and permissiveness to infection. For example, HIV preferentially depletes interleukin-17 (IL-17)-producing T helper 17 (Th17) cells and T follicular helper (Tfh) cells. The preferential loss of Th17 cells during the acute phase of infection impairs the integrity of the gut mucosal barrier, which drives chronic immune activation—a key determinant of disease progression. The preferential loss of Th17 cells has been attributed to high CD4, CCR5, and CXCR4 expression. Here, we show that Th17 cells also exhibit heightened permissiveness to productive HIV infection. Primary human CD4+ T cells were sorted, activated under Th17- or Th0-polarizing conditions and infected, and then analyzed by flow cytometry. Th17-polarizing cytokines increased HIV infection, and HIV infection was disproportionately higher among Th17 cells than among IL-17− or gamma interferon-positive (IFN-γ+) cells, even upon infection with a replication-defective HIV vector with a pseudotype envelope. Further, Th17-polarized cells produced more viral capsid protein. Our data also reveal that Th17-polarized cells have diminished expression of RNase A superfamily proteins, and we report for the first time that RNase 6 inhibits HIV. Thus, our findings link Th17 polarization to increased HIV replication. IMPORTANCE Our study compares the intracellular replicative capacities of several different HIV isolates among different T cell subsets, providing a link between the differentiation of Th17 cells and HIV replication. Th17 cells are of key importance in mucosal integrity and in the immune response to certain pathogens. Based on our findings and the work of others, we propose a model in which HIV replication is favored by the intracellular environment of two CD4+ T cell subsets that share several requirements for their differentiation: Th17 and Tfh cells. Characterizing

  8. Human Th17 Cells Lack HIV-Inhibitory RNases and Are Highly Permissive to Productive HIV Infection.

    PubMed

    Christensen-Quick, Aaron; Lafferty, Mark; Sun, Lingling; Marchionni, Luigi; DeVico, Anthony; Garzino-Demo, Alfredo

    2016-09-01

    Human immunodeficiency virus (HIV) infects and depletes CD4(+) T cells, but subsets of CD4(+) T cells vary in their susceptibility and permissiveness to infection. For example, HIV preferentially depletes interleukin-17 (IL-17)-producing T helper 17 (Th17) cells and T follicular helper (Tfh) cells. The preferential loss of Th17 cells during the acute phase of infection impairs the integrity of the gut mucosal barrier, which drives chronic immune activation-a key determinant of disease progression. The preferential loss of Th17 cells has been attributed to high CD4, CCR5, and CXCR4 expression. Here, we show that Th17 cells also exhibit heightened permissiveness to productive HIV infection. Primary human CD4(+) T cells were sorted, activated under Th17- or Th0-polarizing conditions and infected, and then analyzed by flow cytometry. Th17-polarizing cytokines increased HIV infection, and HIV infection was disproportionately higher among Th17 cells than among IL-17(-) or gamma interferon-positive (IFN-γ(+)) cells, even upon infection with a replication-defective HIV vector with a pseudotype envelope. Further, Th17-polarized cells produced more viral capsid protein. Our data also reveal that Th17-polarized cells have diminished expression of RNase A superfamily proteins, and we report for the first time that RNase 6 inhibits HIV. Thus, our findings link Th17 polarization to increased HIV replication. Our study compares the intracellular replicative capacities of several different HIV isolates among different T cell subsets, providing a link between the differentiation of Th17 cells and HIV replication. Th17 cells are of key importance in mucosal integrity and in the immune response to certain pathogens. Based on our findings and the work of others, we propose a model in which HIV replication is favored by the intracellular environment of two CD4(+) T cell subsets that share several requirements for their differentiation: Th17 and Tfh cells. Characterizing cells that

  9. Thyroid cell lines in research on goitrogenesis.

    PubMed

    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. HLA-C expression levels define permissible mismatches in hematopoietic cell transplantation

    PubMed Central

    Gooley, Theodore A.; Malkki, Mari; Bacigalupo, Andrea P.; Cesbron, Anne; Du Toit, Ernette; Ehninger, Gerhard; Egeland, Torstein; Fischer, Gottfried F.; Gervais, Thibaut; Haagenson, Michael D.; Horowitz, Mary M.; Hsu, Katharine; Jindra, Pavel; Madrigal, Alejandro; Oudshoorn, Machteld; Ringdén, Olle; Schroeder, Marlis L.; Spellman, Stephen R.; Tiercy, Jean-Marie; Velardi, Andrea; Witt, Campbell S.; O’Huigin, Colm; Apps, Richard; Carrington, Mary

    2014-01-01

    Life-threatening graft-versus-host disease (GVHD) limits the use of HLA-C-mismatched unrelated donors in transplantation. Clinicians lack criteria for donor selection when HLA-C-mismatched donors are a patient’s only option for cure. We examined the role for HLA-C expression levels to identify permissible HLA-C mismatches. The median fluorescence intensity, a proxy of HLA-C expression, was assigned to each HLA-C allotype in 1975 patients and their HLA-C-mismatched unrelated transplant donors. The association of outcome with the level of expression of patients’ and donors’ HLA-C allotypes was evaluated in multivariable models. Increasing expression level of the patient’s mismatched HLA-C allotype was associated with increased risks of grades III to IV acute GVHD, nonrelapse mortality, and mortality. Increasing expression level among HLA-C mismatches with residue 116 or residue 77/80 mismatching was associated with increased nonrelapse mortality. The immunogenicity of HLA-C mismatches in unrelated donor transplantation is influenced by the expression level of the patient’s mismatched HLA-C allotype. HLA-C expression levels provide new information on mismatches that should be avoided and extend understanding of HLA-C-mediated immune responses in human disease. PMID:25323824

  11. Porphyromonas gingivalis induces CCR5-dependent transfer of infectious HIV-1 from oral keratinocytes to permissive cells

    PubMed Central

    Giacaman, Rodrigo A; Asrani, Anil C; Gebhard, Kristin H; Dietrich, Elizabeth A; Vacharaksa, Anjalee; Ross, Karen F; Herzberg, Mark C

    2008-01-01

    Background Systemic infection with HIV occurs infrequently through the oral route. The frequency of occurrence may be increased by concomitant bacterial infection of the oral tissues, since co-infection and inflammation of some cell types increases HIV-1 replication. A putative periodontal pathogen, Porphyromonas gingivalis selectively up-regulates expression of the HIV-1 coreceptor CCR5 on oral keratinocytes. We, therefore, hypothesized that P. gingivalis modulates the outcome of HIV infection in oral epithelial cells. Results Oral and tonsil epithelial cells were pre-incubated with P. gingivalis, and inoculated with either an X4- or R5-type HIV-1. Between 6 and 48 hours post-inoculation, P. gingivalis selectively increased the infectivity of R5-tropic HIV-1 from oral and tonsil keratinocytes; infectivity of X4-tropic HIV-1 remained unchanged. Oral keratinocytes appeared to harbor infectious HIV-1, with no evidence of productive infection. HIV-1 was harbored at highest levels during the first 6 hours after HIV exposure and decreased to barely detectable levels at 48 hours. HIV did not appear to co-localize with P. gingivalis, which increased selective R5-tropic HIV-1 trans infection from keratinocytes to permissive cells. When CCR5 was selectively blocked, HIV-1 trans infection was reduced. Conclusion P. gingivalis up-regulation of CCR5 increases trans infection of harbored R5-tropic HIV-1 from oral keratinocytes to permissive cells. Oral infections such as periodontitis may, therefore, increase risk for oral infection and dissemination of R5-tropic HIV-1. PMID:18371227

  12. Cytotoxic T-lymphocyte precursor frequency (CTLp-f) as a tool for distinguishing permissible from non-permissible class I mismatches in T-cell-depleted allogeneic bone marrow transplantation.

    PubMed

    van der Meer, A; Joosten, I; Schattenberg, A V; de Witte, T J; Allebes, W A

    2000-11-01

    Matching for HLA has been the gold standard in bone marrow donor selection. But, with the ever increasing number of identified HLA alleles, it is becoming more difficult to find a fully HLA-identical donor other than a sibling. Retrospective analysis revealed that HLA mismatches do not necessarily give rise to acute graft-versus-host-disease (GVHD). However, we have no means of defining these 'permissible' mismatches before bone marrow transplantation (BMT). Thus, we set out to establish whether functional matching by means of helper and cytotoxic T-lymphocyte precursor frequency analysis (HTLp-f and CTLp-f respectively) can be applied to this end. Fifty-five recipient-donor pairs other than HLA-identical siblings, the recipient of which received a T-cell-depleted graft, were analysed by high-resolution HLA typing and/or HTLp-f/CTLp-f analysis. The predictive value of the CTLp-f assay for development of acute GVHD was confirmed. More importantly, our data indicate that the CTLp-f assay was able to discriminate permissible from non-permissible HLA-A, -B or -Cw mismatches, but not for DRB/DQB mismatches. The absolute number of alloreactive CTLs present in the graft correlated with the risk of acute GVHD. Although HTLp-f and CTLp-f together had a high negative predictive value, HTLp-f outcome by itself was not correlated with acute GVHD. As we have no evidence yet that HTLp-f or CTLp-f can identify permissible DRB/DQB mismatches, high-resolution matching for these antigens remains the best option. The combination of high-resolution DRB/DQB typing and the CTLp-f assay would enable the accurate prediction of the risk of acute GVHD while extending the pool of potential donors. Furthermore, it would enable adjustment of the number of T- cells in the graft accordingly to improve clinical outcome.

  13. Red Blood Cells Preconditioned with Hemin Are Less Permissive to Plasmodium Invasion In Vivo and In Vitro.

    PubMed

    Gaudreault, Véronique; Wirbel, Jakob; Jardim, Armando; Rohrbach, Petra; Scorza, Tatiana

    2015-01-01

    Malaria is a parasitic disease that causes severe hemolytic anemia in Plasmodium-infected hosts, which results in the release and accumulation of oxidized heme (hemin). Although hemin impairs the establishment of Plasmodium immunity in vitro and in vivo, mice preconditioned with hemin develop lower parasitemia when challenged with Plasmodium chabaudi adami blood stage parasites. In order to understand the mechanism accounting for this resistance as well as the impact of hemin on eryptosis and plasma levels of scavenging hemopexin, red blood cells were labeled with biotin prior to hemin treatment and P. c. adami infection. This strategy allowed discriminating hemin-treated from de novo generated red blood cells and to follow the infection within these two populations of cells. Fluorescence microscopy analysis of biotinylated-red blood cells revealed increased P. c. adami red blood cells selectivity and a decreased permissibility of hemin-conditioned red blood cells for parasite invasion. These effects were also apparent in in vitro P. falciparum cultures using hemin-preconditioned human red blood cells. Interestingly, hemin did not alter the turnover of red blood cells nor their replenishment during in vivo infection. Our results assign a function for hemin as a protective agent against high parasitemia, and suggest that the hemolytic nature of blood stage human malaria may be beneficial for the infected host.

  14. TFH cells accumulate in mucosal tissues of humanized-DRAG mice and are highly permissive to HIV-1

    PubMed Central

    Allam, Atef; Majji, Sai; Peachman, Kristina; Jagodzinski, Linda; Kim, Jiae; Ratto-Kim, Silvia; Wijayalath, Wathsala; Merbah, Melanie; Kim, Jerome H.; Michael, Nelson L.; Alving, Carl R.; Casares, Sofia; Rao, Mangala

    2015-01-01

    CD4+ T follicular helper cells (TFH) in germinal centers are required for maturation of B-cells. While the role of TFH-cells has been studied in blood and lymph nodes of HIV-1 infected individuals, its role in the mucosal tissues has not been investigated. We show that the gut and female reproductive tract (FRT) of humanized DRAG mice have a high level of human lymphocytes and a high frequency of TFH (CXCR5+PD-1++) and precursor-TFH (CXCR5+PD-1+) cells. The majority of TFH-cells expressed CCR5 and CXCR3 and are the most permissive to HIV-1 infection. A single low-dose intravaginal HIV-1 challenge of humanized DRAG mice results in 100% infectivity with accumulation of TFH-cells mainly in the Peyer’s patches and FRT. The novel finding of TFH-cells in the FRT may contribute to the high susceptibility of DRAG mice to HIV-1 infection. This mouse model thus provides new opportunities to study TFH-cells and to evaluate HIV-1 vaccines. PMID:26034905

  15. TFH cells accumulate in mucosal tissues of humanized-DRAG mice and are highly permissive to HIV-1.

    PubMed

    Allam, Atef; Majji, Sai; Peachman, Kristina; Jagodzinski, Linda; Kim, Jiae; Ratto-Kim, Silvia; Wijayalath, Wathsala; Merbah, Melanie; Kim, Jerome H; Michael, Nelson L; Alving, Carl R; Casares, Sofia; Rao, Mangala

    2015-06-02

    CD4(+) T follicular helper cells (TFH) in germinal centers are required for maturation of B-cells. While the role of TFH-cells has been studied in blood and lymph nodes of HIV-1 infected individuals, its role in the mucosal tissues has not been investigated. We show that the gut and female reproductive tract (FRT) of humanized DRAG mice have a high level of human lymphocytes and a high frequency of TFH (CXCR5(+)PD-1(++)) and precursor-TFH (CXCR5(+)PD-1(+)) cells. The majority of TFH-cells expressed CCR5 and CXCR3 and are the most permissive to HIV-1 infection. A single low-dose intravaginal HIV-1 challenge of humanized DRAG mice results in 100% infectivity with accumulation of TFH-cells mainly in the Peyer's patches and FRT. The novel finding of TFH-cells in the FRT may contribute to the high susceptibility of DRAG mice to HIV-1 infection. This mouse model thus provides new opportunities to study TFH-cells and to evaluate HIV-1 vaccines.

  16. Red Blood Cells Preconditioned with Hemin Are Less Permissive to Plasmodium Invasion In Vivo and In Vitro

    PubMed Central

    Gaudreault, Véronique; Wirbel, Jakob; Jardim, Armando; Rohrbach, Petra; Scorza, Tatiana

    2015-01-01

    Malaria is a parasitic disease that causes severe hemolytic anemia in Plasmodium-infected hosts, which results in the release and accumulation of oxidized heme (hemin). Although hemin impairs the establishment of Plasmodium immunity in vitro and in vivo, mice preconditioned with hemin develop lower parasitemia when challenged with Plasmodium chabaudi adami blood stage parasites. In order to understand the mechanism accounting for this resistance as well as the impact of hemin on eryptosis and plasma levels of scavenging hemopexin, red blood cells were labeled with biotin prior to hemin treatment and P. c. adami infection. This strategy allowed discriminating hemin-treated from de novo generated red blood cells and to follow the infection within these two populations of cells. Fluorescence microscopy analysis of biotinylated-red blood cells revealed increased P. c. adami red blood cells selectivity and a decreased permissibility of hemin-conditioned red blood cells for parasite invasion. These effects were also apparent in in vitro P. falciparum cultures using hemin-preconditioned human red blood cells. Interestingly, hemin did not alter the turnover of red blood cells nor their replenishment during in vivo infection. Our results assign a function for hemin as a protective agent against high parasitemia, and suggest that the hemolytic nature of blood stage human malaria may be beneficial for the infected host. PMID:26465787

  17. Peripheral blood mononuclear cells from field cattle immune to bovine viral diarrhea virus (BVDV) are permissive in vitro to BVDV.

    PubMed

    Gupta, V; Mishra, N; Pateriya, A; Behera, S P; Rajukumar, K

    2014-01-01

    The aim of this study was to determine the in vitro permissivity of peripheral blood mononuclear cells (PBMCs) from bovine viral diarrhea virus (BVDV)-immune field cattle to homologous and heterologous BVDVs. PBMCs from seventeen BVDV-naïve and sixteen BVDV-immune animals were infected with noncytopathic BVDV-1 or BVDV-2. The immune status of cattle was indicated by the presence of virus neutralizing antibodies, while viral load of PBMCs was determined by real-time RT-PCR. The results revealed that the PBMCs from naïve or immune animals were permissive to either BVDV-1 or BVDV-2, but the viral load was significantly higher for the naïve than for the immune animals. Furthermore, the load of homologous virus in PBMCs from immune animals was lower than that of heterologous virus. Our results provide evidence that the PBMCs from BVDV-immune cattle in field are susceptible to reinfection with homologous or heterologous BVDV, albeit to a lower extent in the former case.

  18. Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment

    PubMed Central

    Sekinaka, Tamotsu; Hayashi, Yohei; Noce, Toshiaki; Niwa, Hitoshi; Matsui, Yasuhisa

    2016-01-01

    Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs. PMID:27608931

  19. Oligodendrocyte precursor cells differentially expressing Nogo-A but not MAG are more permissive to neurite outgrowth than mature oligodendrocytes.

    PubMed

    Ma, Zhengwen; Cao, Qilin; Zhang, Liqun; Hu, Jianguo; Howard, Russell M; Lu, Peihua; Whittemore, Scott R; Xu, Xiao-Ming

    2009-05-01

    Grafting oligodendrocyte precursor cells (OPCs) has been used as a strategy to repair demyelination of the central nervous system (CNS). Whether OPCs can promote CNS axonal regeneration remains to be tested. If so, they should be permissive to axonal growth and may express less inhibitory molecules on their surface. Here we examined the expression of two oligodendrocyte-associated myelin inhibitors Nogo-A and myelin-associated glycoprotein (MAG) during oligodendrogliogenesis and tested their abilities to promote neurite outgrowth in vitro. Whereas the intracellular domain of Nogo-A was consistently expressed throughout oligodendrocyte differentiation, MAG was expressed only at later stages. Furthermore, the membrane-associated extracellular domain of Nogo-A was not expressed in OPCs but expressed in mature oligodendrocytes. In a dorsal root ganglion (DRG) and OPC/oligodendrocyte co-culture model, significantly greater DRG neurite outgrowth onto OPC monolayer than mature oligodendrocyte was found (1042+/-123 vs. 717+/-342 micrometer; p=0.011). Moreover, DRG neurites elongated as fasciculated fiber tracts and contacted directly on OPCs (133+/-37 cells/fascicle). In contrast, few, if any, direct contacts were found between DRG neurites and mature oligodendrocytes (5+/-3 cells/fascicle, p<0.001). In fact, acellular spaces were found between neurites and surrounding mature oligodendrocytes in contrast to the lack of such spaces in OPC/DRG coculture (51.1+/-16.5 vs. 2.4+/-3.9 micrometer; p<0.001). Thus, OPCs expressing neither extracellular domain of Nogo-A nor MAG are significantly more permissive than mature oligodendrocytes expressing both. Grafting OPCs may thus represent a feasible strategy to foster CNS axonal regeneration.

  20. Permissive effect of dexamethasone on the increase of proenkephalin mRNA induced by depolarization of chromaffin cells

    SciTech Connect

    Naranjo, J.R.; Mocchetti, I.; Schwartz, J.P.; Costa, E.

    1986-03-01

    In cultured bovine chromaffin cells, changes in the dynamic state of enkephalin stores elicited experimentally were studied by measuring cellular proenkephalin mRNA, as well as enkephalin precursors and authentic enkephalin content of cells and culture media. In parallel, tyrosine hydroxylase mRNA and catecholamine cell content were also determined. Low concentrations (0.5-100 pM) of dexamethasone increased the cell contents of proenkephalin mRNA and enkephalin-containing peptides. High concentrations of the hormone(1 ..mu..M) were required to increase the cell contents of tyrosine hydroxylase mRNA and catecholamines. Depolarization of the cells with 10 ..mu..M veratridine resulted in a depletion of enkephalin and catecholamine stores after 24 hr. The enkephalin, but not the catecholamine, content was restored by 48 hr. An increase in proenkephalin mRNA content might account for the recovery; this increase was curtailed by tetrodotoxin and enhanced by 10 pM dexamethasone. Tyrosine hydroxylase mRNA content was not significantly modified by depolarization, even in the presence of 1 ..mu..M dexamethasone. Aldosterone, progesterone, testosterone, or estradiol (1 ..mu..M) failed to change proenkephalin mRNA. Hence, dexamethasone appears to exert a specific permissive action on the stimulation of the proenkephalin gene elicited by depolarization. Though the catecholamines and enkephalins are localized in the same chromaffin granules and are coreleased by depolarization, the genes coding for the processes that are rate limiting in the production of these neuromodulators can be differentially regulated.

  1. Creating permissive microenvironments for stem cell transplantation into the central nervous system.

    PubMed

    Kim, Howard; Cooke, Michael J; Shoichet, Molly S

    2012-01-01

    Traumatic injury to the central nervous system (CNS) is highly debilitating, with the clinical need for regenerative therapies apparent. Neural stem/progenitor cells (NSPCs) are promising because they can repopulate lost or damaged cells and tissues. However, the adult CNS does not provide an optimal milieu for exogenous NSPCs to survive, engraft, differentiate, and integrate with host tissues. This review provides an overview of tissue engineering strategies to improve stem cell therapies by providing a defined microenvironment during transplantation. The use of biomaterials for physical support, growth factor delivery, and cellular co-transplantation are discussed. Providing the proper environment for stem cell survival and host tissue integration is crucial in realizing the full potential of these cells in CNS repair strategies.

  2. Formation of Viral Ribonucleic Acid and Virus in Cells that are Permissive or Nonpermissive for Murine Encephalomyelitis Virus (GDVII) 1

    PubMed Central

    Sturman, Lawrence S.; Tamm, Igor

    1969-01-01

    GDVII virus growth in BHK-21 cells, a permissive host for the virus, resembled productive infections with other picornaviruses. Virus yields ranged from 100 to 600 plaque-forming units (PFU)/cell. Virus replication in HeLa cells, a nonpermissive host for GDVII virus, was characterized by virus yields of only 0.1 to 5 PFU/cell. Similar low yields of virus have been obtained from HeLa cells at all multiplicities of input up to 6,000 per cell. The progeny particles from HeLa cells were, like the infecting particles, restricted in the HeLa cell host. Despite the great difference in final yields of virus from BHK-21 and HeLa cells, the times when maximal yields were reached were similar. GDVII virus stock grown in BHK-21 cells was designated HeLa-. A variant of GDVII virus which is capable of extensive growth in HeLa cells was obtained. This variant, designated HeLa+ GDVII virus, was passaged serially in HeLa cells. Virus yields of 50 to 150 infective virus particles per cell were obtained from infection of HeLa cells with HeLa+ GDVII virus. The major species of HeLa+ virus-specific ribonucleic acid (RNA) produced was single stranded and sedimented with an S value of 35S. The rate of accumulation of HeLa+ virus-specific RNA in HeLa cell cultures was about four times that of HeLa- RNA. The amount of virus-specific HeLa+ RNA formed in HeLa cells was several-fold greater than that of HeLa- RNA. With HeLa- parent GDVII virus undergoing productive replication in BHK-21 cells or abortive replication in HeLa cells, the major species of virus-specific RNA produced was single stranded and sedimented with an approximate S value of 35S. The amount of HeLa- virus-specific RNA extracted from BHK-21 cells was several-fold greater than the amount obtained from HeLa cells. PMID:4306304

  3. Host cis-Mediated Extinction of a Retrovirus Permissive for Expression in Embryonal Stem Cells during Differentiation

    PubMed Central

    Laker, Christine; Meyer, Johann; Schopen, Arndt; Friel, Jutta; Heberlein, Christoph; Ostertag, Wolfram; Stocking, Carol

    1998-01-01

    The use of retroviral vectors for gene transfer into animals has been severely hampered by the lack of provirus transcription in the early embryo and embryonic stem (ES) cells. This primary block in provirus expression is maintained in differentiated cells by a cis-acting mechanism that is not well characterized. Retroviral vectors based on the murine embryonal stem cell virus (MESV), which overcome the transcriptional block in ES cells, were constructed to investigate this secondary mechanism. These vectors transferred G418 resistance to ES cells with the same efficiency as to fibroblasts, but overall transcript levels were greatly reduced. A mosaic but stable expression pattern was observed when single cells from G418-resistant clones were replated in G418 or assayed for expression of LacZ or interleukin-3. The expression levels in independent clones were variable and correlated inversely with methylation. However, a second, more pronounced, block to transcription was found upon differentiation induction. Differentiation of the infected ES cells to cells permissive for retroviral expression resulted in repression and complete extinction of provirus expression. Extinction was not accompanied by increased levels of methylation. Provirus expression is thus regulated by two independent cis-acting mechanisms: (i) partial repression in the undifferentiated state, accompanied by increased methylation but compatible with long-term, low expression of retroviral genes, and (ii) total repression and extinction during early stages of differentiation, apparently independent of changes in methylation. These results indicate a time window early during the transition from an undifferentiated to a differentiated stage in which provirus expression is silenced. The mechanisms are presently unknown, but elucidation of these events will have an important impact on vector development for targeting stem cells and for gene therapy. PMID:9420232

  4. Germinal Center T Follicular Helper Cells (GC TFH) are Highly Permissive to HIV-1 and Alter Their Phenotype During Virus Replication

    PubMed Central

    Kohler, Stephanie L.; Pham, Michael N.; Folkvord, Joy M.; Arends, Tessa; Miller, Shannon M.; Miles, Brodie; Meditz, Amie L.; McCarter, Martin; Levy, David N.; Connick, Elizabeth

    2016-01-01

    HIV-1 replication is concentrated within CD4+ T cells in B-cell follicles of secondary lymphoid tissues during asymptomatic disease. Limited data suggest that a subset of T follicular helper cells (TFH) within germinal centers (GC) is highly permissive to HIV-1. Whether GC TFH are the major HIV-1 virus-producing cells in vivo has not been established. Here, we investigated TFH permissivity to HIV-1 ex vivo by spinoculating and culturing tonsil cells with HIV-1 GFP reporter viruses. Using flow cytometry, higher percentages of GC TFH (CXCR5highPD-1high) and CXCR5+PD-1low cells were GFP+ than non-GC TFH (CXCR5+PD-1intermediate) or extrafollicular (CXCR5-) cells. When sorted prior to spinoculation, however, GC TFH were substantially more permissive than CXCR5+PD-1low or extrafollicular cells, suggesting that many GC TFH transition to a CXCR5+PD-1low phenotype during productive infection. In situ hybridization on inguinal lymph node sections from untreated HIV-1-infected individuals without AIDS revealed higher frequencies of HIV-1 RNA+ cells in GC than non-GC regions of follicle or extrafollicular regions. Superinfection of HIV-1-infected individuals’ lymph node cells with GFP reporter virus confirmed the permissivity of follicular cells ex vivo. Lymph node immunostaining revealed 96% of CXCR5+CD4+ cells were located in follicles. Within sorted lymph node cells from four HIV-infected individuals, CXCR5+ subsets harbored 11- to 66-fold more HIV-1 RNA than CXCR5-subsets, as determined by RT PCR. Thus, GC TFH are highly permissive to HIV-1, but downregulate PD-1 and to a lesser extent CXCR5 during HIV-1 replication. These data further implicate GC TFH as the major HIV-1-producing cells in chronic asymptomatic HIV-1 infection. PMID:26873986

  5. Differential autophagic effects triggered by five different vertebrate iridoviruses in a common, highly permissive mandarinfish fry (MFF-1) cell model.

    PubMed

    Qi, Hemei; Yi, Yang; Weng, Shaoping; Zou, Weibing; He, Jianguo; Dong, Chuanfu

    2016-02-01

    Autophagy of five vertebrate iridoviruses, including one megalocytivirus (infectious spleen and kidney necrosis virus, ISKNV) and four ranaviruses (Chinese giant salamander iridovirus, CGSIV; Tiger frog virus, TFV; Grouper iridovirus, GIV; and Largemouth bass virus, LMBV) were investigated in a common, highly permissive mandarinfish fry (MFF-1) cell model. The results showed marked autophagosome formation in GIV- and LMBV-infected cells but not in ISKNV-, CGSIV- and TFV-infected MFF-1 cells. Strong evidence for the autophagosomes was provided by transmission electron microscopy, the detection of mandarinfish microtubule-associated protein 1 light chain 3B (mLC3)-based fluorescent dot formation and mLC3-I/mLC3-II conversion was provided by Western blotting. Pharmacological tests indicated that autophagy plays an antiviral role during GIV or LMBV infection. Collectively, our data are the first to show that antiviral autophagic effects can be triggered by GIV and LMBV but not by ISKNV, TFV and CGSIV in a common susceptible cell model. These results suggest that differential host-virus interaction strategies may be utilized against different vertebrate iridoviruses; they also indicate the potential effectiveness of an antiviral treatment that modulates autophagy to control iridoviral infections, such as GIV and LMBV.

  6. Circulating endothelial giant cells permissive for human cytomegalovirus (HCMV) are detected in disseminated HCMV infections with organ involvement.

    PubMed Central

    Percivalle, E; Revello, M G; Vago, L; Morini, F; Gerna, G

    1993-01-01

    Giant cells fully permissive for human cytomegalovirus (HCMV) were found to circulate, at a variable proportion, in peripheral blood of 21 out of 25 immunocompromised patients with disseminated HCMV infection. Circulating endothelial giant cells (EGC) were identified by a specific monoclonal antibody of endothelial origin and shown to express immediate-early, early, and late viral proteins. Immunostaining patterns of different viral proteins were comparable to those detected in vitro in cultured human umbilical vein endothelial cells. EGC counts > 10 were associated with high levels (> 100) of HCMV viremia and antigenemia, as well as with an overt clinical syndrome in transplanted patients, and to an untreated long lasting organ localization in AIDS patients. On the other hand, EGC counts were < 10 during disseminated HCMV infections of both transplant recipients with no apparent organ syndrome and AIDS patients with recent organ involvement. In tissue sections from AIDS patients, infected endothelial cells were found to progressively enlarge till detaching from the small vessel wall and entering blood stream. HCMV-infected EGC represent a new systemic parameter suitable for the diagnosis of HCMV organ involvement and for the study of the pathogenesis of disseminated infections. Images PMID:8394385

  7. Chapter 6. available lepidopteran insect cell lines

    USDA-ARS?s Scientific Manuscript database

    This chapter lists the known cell lines from Lepidoptera, largely based on previous compilations of insect cell lines published by W. Fred Hink. More than 320 lines from 65 species are listed. The official designation is given for each cell line as well as the species, tissue source, and, when kno...

  8. The impact of amino acid variability on alloreactivity defines a functional distance predictive of permissive HLA-DPB1 mismatches in hematopoietic stem cell transplantation.

    PubMed

    Crivello, Pietro; Zito, Laura; Sizzano, Federico; Zino, Elisabetta; Maiers, Martin; Mulder, Arend; Toffalori, Cristina; Naldini, Luigi; Ciceri, Fabio; Vago, Luca; Fleischhauer, Katharina

    2015-02-01

    A major challenge in unrelated hematopoietic stem cell transplantation (HSCT) is the prediction of permissive HLA mismatches, ie, those associated with lower clinical risks compared to their nonpermissive counterparts. For HLA-DPB1, a clinically prognostic model has been shown to be matching for T cell epitope (TCE) groups assigned by cross reactivity of T cells alloreactive to HLA-DPB1∗09:01; however, the molecular basis of this observation is not fully understood. Here, we have mutated amino acids (aa) in 10 positions of HLA-DPB1∗09:01 to other naturally occurring variants, expressed them by lentiviral vectors in B cell lines, and quantitatively measured allorecognition by 17 CD4(+) T cell effectors from 6 unrelated individuals. A significant impact on the median alloresponse was observed for peptide contact positions 9, 11, 35, 55, 69, 76, and 84, but not for positions 8, 56, and 57 pointing away from the groove. A score for the "functional distance" (FD) from HLA-DPB1∗09:01 was defined as the sum of the median impact of polymorphic aa in a given HLA-DPB1 allele on T cell alloreactivity. Established TCE group assignment of 23 alleles correlated with FD scores of ≤0.5, 0.6 to 1.9 and ≥2 for TCE groups 1, 2, and 3, respectively. Based on this, prediction of TCE group assignment will be possible for any given HLA-DPB1 allele, including currently 367 alleles encoding distinct proteins for which T cell cross reactivity patterns are unknown. Experimental confirmation of the in silico TCE group classification was successfully performed for 7 of 7 of these alleles. Our findings have practical implications for the applicability of TCE group matching in unrelated HSCT and provide new insights into the molecular mechanisms underlying this model. The innovative concept of FD opens new potential avenues for risk prediction in unrelated HSCT.

  9. Novel hepatitis C virus reporter replicon cell lines enable efficient antiviral screening against genotype 1a.

    PubMed

    Robinson, Margaret; Yang, Huiling; Sun, Siu-Chi; Peng, Betty; Tian, Yang; Pagratis, Nikos; Greenstein, Andrew E; Delaney, William E

    2010-08-01

    The hepatitis C virus (HCV) subgenomic replicon is the primary tool for evaluating the activity of anti-HCV compounds in drug discovery research. Despite the prevalence of HCV genotype 1a (approximately 70% of U.S. HCV patients), few genotype 1a reporter replicon cell lines have been described; this is presumably due to the low replication capacity of such constructs in available Huh-7 cells. In this report, we describe the selection of highly permissive Huh-7 cell lines that support robust replication of genotype 1a subgenomic replicons harboring luciferase reporter genes. These novel cell lines support the replication of multiple genotype 1a replicons (including the H77 and SF9 strains), are significantly more permissive to genotype 1a HCV replication than parental Huh7-Lunet cells, and maintain stable genotype 1a replication levels suitable for antiviral screening. We found that the sensitivity of genotype 1a luciferase replicons to known antivirals was highly consistent between individual genotype 1a clonal cell lines but could vary significantly between genotypes 1a and 1b. Sequencing of the nonstructural region of 12 stable replicon cell clones suggested that the enhanced permissivity is likely due to cellular component(s) in these new cell lines rather than the evolution of novel adaptive mutations in the replicons. These new reagents will enhance drug discovery efforts targeting genotype 1a and facilitate the profiling of compound activity among different HCV genotypes and subtypes.

  10. Permissive Schwann cell graft/spinal cord interfaces for axon regeneration.

    PubMed

    Williams, Ryan R; Henao, Martha; Pearse, Damien D; Bunge, Mary Bartlett

    2015-01-01

    The transplantation of autologous Schwann cells (SCs) to repair the injured spinal cord is currently being evaluated in a clinical trial. In support, this study determined properties of spinal cord/SC bridge interfaces that enabled regenerated brainstem axons to cross them, possibly leading to improvement in rat hindlimb movement. Fluid bridges of SCs and Matrigel were placed in complete spinal cord transections. Compared to pregelled bridges of SCs and Matrigel, they improved regeneration of brainstem axons across the rostral interface. The regenerating brainstem axons formed synaptophysin(+) bouton-like terminals and contacted MAP2A(+) dendrites at the caudal interface. Brainstem axon regeneration was directly associated with glial fibrillary acidic protein (GFAP(+)) astrocyte processes that elongated into the SC bridge. Electron microscopy revealed that axons, SCs, and astrocytes were enclosed together within tunnels bounded by a continuous basal lamina. Neuroglycan (NG2) expression was associated with these tunnels. One week after injury, the GFAP(+) processes coexpressed nestin and brain lipid-binding protein, and the tips of GFAP(+)/NG2(+) processes extended into the bridges together with the regenerating brainstem axons. Both brainstem axon regeneration and number of GFAP(+) processes in the bridges correlated with improvement in hindlimb locomotion. Following SCI, astrocytes may enter a reactive state that prohibits axon regeneration. Elongation of astrocyte processes into SC bridges, however, and formation of NG2(+) tunnels enable brainstem axon regeneration and improvement in function. It is important for spinal cord repair to define conditions that favor elongation of astrocytes into lesions/transplants.

  11. Identification of a permissible HLA mismatch in hematopoietic stem cell transplantation

    PubMed Central

    Fernandez-Viña, Marcelo A.; Wang, Tao; Lee, Stephanie J.; Haagenson, Michael; Aljurf, Mahmoud; Askar, Medhat; Battiwalla, Minoo; Baxter-Lowe, Lee-Ann; Gajewski, James; Jakubowski, Ann A.; Marino, Susana; Oudshoorn, Machteld; Marsh, Steven G. E.; Petersdorf, Effie W.; Schultz, Kirk; Turner, E. Victoria; Waller, Edmund K.; Woolfrey, Ann; Umejiego, John; Spellman, Stephen R.; Setterholm, Michelle

    2014-01-01

    In subjects mismatched in the HLA alleles C*03:03/C*03:04 no allogeneic cytotoxic T-lymphocyte responses are detected in vitro. Hematopoietic stem cell transplantation (HSCT) with unrelated donors (UDs) showed no association between the HLA-C allele mismatches (CAMMs) and adverse outcomes; antigen mismatches at this and mismatches other HLA loci are deleterious. The absence of effect of the CAMM may have resulted from the predominance of the mismatch C*03:03/C*03:04. Patients with hematologic malignancies receiving UD HSCT matched in 8/8 and 7/8 HLA alleles were examined. Transplants mismatched in HLA-C antigens or mismatched in HLA-A, -B, or -DRB1 presented significant differences (P < .0001) in mortality (hazard ratio [HR] = 1.37, 1.30), disease-free survival (HR = 1.33, 1.27), treatment-related mortality (HR = 1.54, 1.54), and grade 3-4 acute graft-versus-host disease (HR = 1.49, 1.77) compared with the 8/8 group; transplants mismatched in other CAMMs had similar outcomes with HR ranging from 1.34 to 172 for these endpoints. The C*03:03/C*03:04 mismatched and the 8/8 matched groups had identical outcomes (HR ranging from 0.96-1.05). The previous finding that CAMMs do not associate with adverse outcomes is explained by the predominance (69%) of the mismatch C*03:03/03:04 in this group that is better tolerated than other HLA mismatches. PMID:24408320

  12. A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

    USDA-ARS?s Scientific Manuscript database

    Classical scrapie is a form of transmissible spongiform encephalopathies (TSE) affecting domestic goats and sheep and disease is characterized by the accumulation of abnormal conformational isoform (PrP-Sc) of normal cellular prion protein (PrP-C) in the central nervous system and, in most cases, ly...

  13. Autographa caljfornica nuclear polyhedrosis virus replication in non-permissive Lymantria dispar cell lines

    Treesearch

    Edward M. Dougherty; David Guzo; Kathleen S. Shields; Dwight E. Lynn; Susan K. Braun

    1991-01-01

    Autographa californica nuclear polyhedrosis virus (AcNPV) the prototypic group A baculovirus, has the widest reported host range of the baculoviruses and is considered to be one of the most virulent baculoviruses studied. The gypsy moth Lymantria dispar is not considered a natural host of AcNPV, however. To determine the factors...

  14. Porcine Endogenous Retrovirus Infects but Does Not Replicate in Nonhuman Primate Primary Cells and Cell Lines

    PubMed Central

    Ritzhaupt, Armin; van der Laan, Luc J. W.; Salomon, Daniel R.; Wilson, Carolyn A.

    2002-01-01

    Porcine endogenous retroviruses (PERV) can infect human cell lines in vitro; hence, there is a presumed risk of viral exposure to a recipient when pig cells are transplanted into humans (xenotransplantation). Nonhuman primates (NHP) are considered a potential permissive animal model to study the risk of in vivo infection of PERV after xenotransplantation. We set out to determine whether PERV can infect and replicate in NHP primary cells or established cell lines from African green monkey, rhesus macaque, and baboon. We confirm that the NHP cell lines under investigation were infected with PERV as measured by detection of viral DNA and RNA by PCR and reverse transcription (RT)-PCR, respectively, indicating that a functional receptor must be present on the cell surface. However, the load of detectable viral DNA in infected NHP cells declined over time, and the cells never had detectable reverse transcriptase activity. Utilizing quantitative real-time TaqMan PCR we found detectable levels of unintegrated DNA intermediates, but the levels were approximately 100-fold lower compared to HEK 293 cells infected with PERV. Virions released from infected NHP cells could productively infect naïve human cell lines, HEK 293 and HeLa, as shown by RT-PCR and RT assay. However, naïve NHP cells remained negative in RT-PCR and RT assay after exposure to virions from infected NHP cells. Together our data demonstrate that NHP cells are not permissive to productive replication by PERV, presumably due to inefficient cell entry and replication. In light of these observations, the appropriateness of NHP as suitable animal models to study PERV infection in vivo needs to be reevaluated. PMID:12388691

  15. Reduced expression of the immediate-early protein IE0 enables efficient replication of Autographa californica multiple nucleopolyhedrovirus in poorly permissive Spodoptera littoralis cells.

    PubMed

    Lu, Liqun; Du, Quansheng; Chejanovsky, Nor

    2003-01-01

    Infection of Spodoptera littoralis SL2 cells with the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) results in apoptosis and low yields of viral progeny, in contrast to infection with S. littoralis nucleopolyhedrovirus (SlNPV). By cotransfecting SL2 cells with AcMNPV genomic DNA and a cosmid library representing the complete SlNPV genome, we were able to rescue AcMNPV replication and to isolate recombinant virus vAcSL2, which replicated efficiently in SL2 cells. Moreover, vAcSL2 showed enhanced infectivity for S. littoralis larvae compared to AcMNPV. The genome of vAcSL2 carried a 519-bp insert fragment that increased the distance between the TATA element and the transcriptional initiation site (CAGT) of immediate-early gene ie0. This finding correlated with low steady-state levels of IE0 and higher steady-state levels of IE1 (the product of the ie1 gene, a major AcMNPV transactivator, and a multifunctional protein) than of IE0. Mutagenesis of the ie0 promoter locus by insertion of the chloramphenical acetyltransferase (cat) gene yielded a new recombinant AcMNPV with replication properties identical to those of vAcSL2. Thus, the analysis indicated that increasing the steady-state levels of IE1 relative to IE0 should enable AcMNPV replication in SL2 cells. This suggestion was confirmed by constructing a recombinant AcMNPV bearing an extra copy of the ie1 gene under the control of the Drosophila hsp70 promoter. These results suggest that IE0 plays a role in the regulation of AcMNPV infection and show, for the first time, that significant improvement in the ability of AcMNPV to replicate in a poorly permissive cell line and organism can be achieved by increasing the expression of the main multiple functional protein, IE1.

  16. Cell-dependent gag mutants of HIV-1 are crucially defective at the stage of uncoating/reverse transcription in non-permissive cells.

    PubMed

    Koh, K; Miyaura, M; Yoshida, A; Sakurai, A; Fujita, M; Adachi, A

    2000-10-01

    We have previously shown that some of the human immunodeficiency virus type 1 (HIV-1) gag matrix (MA), capsid (CA), and nucleocapsid (NC) mutants display host-cell-dependent replication potential, and that they are defective at the early phase of the virus replication cycle in non-permissive cells. To determine the defective replication stage of the cell-dependent mutants precisely, the processes of virus entry into cells and virus DNA synthesis were monitored by the highly sensitive enzyme-linked immunosorbent assay and polymerase chain reaction amplification analysis. The results obtained indicated that all the cell-dependent MA, CA and NC mutants are defective at the stage of uncoating/reverse transcription, and that a cellular factor(s) is involved in this process.

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

    PubMed Central

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

    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 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. PMID:24198436

  18. Tick Cell Lines for Study of Crimean-Congo Hemorrhagic Fever Virus and Other Arboviruses

    PubMed Central

    Kohl, Alain; Bente, Dennis A.; Fazakerley, John K.

    2012-01-01

    Abstract 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. PMID:21955214

  19. Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

    SciTech Connect

    Kawata, Shigehisa; Suzuki, Jun; Maruoka, Masahiro; Mizutamari, Megumi; Ishida-Kitagawa, Norihiro; Yogo, Keiichiro; Jat, Parmjit S.; Shishido, Tomoyuki . E-mail: shishid@bs.naist.jp

    2006-11-10

    Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 deg. C, but stopped growing at the non-permissive temperature of 39 deg. C. In the presence of receptor activator of NF{kappa}B ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 deg. C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.

  20. LINE-1 Cultured Cell Retrotransposition Assay.

    PubMed

    Kopera, Huira C; Larson, Peter A; Moldovan, John B; Richardson, Sandra R; Liu, Ying; Moran, John V

    2016-01-01

    The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells.

  1. Saffold virus is able to productively infect primate and rodent cell lines and induces apoptosis in these cells

    PubMed Central

    Xu, Yishi; Victorio, Carla Bianca Luena; Ng, Qimei; Tan, Yee Joo; Chua, Kaw Bing

    2014-01-01

    Saffold virus (SAFV), a newly discovered human cardiovirus of the Picornaviridae family, causes widespread infection among children, as shown by previous seroprevalence studies. To determine the host cell range of SAFV and its cytopathogenicity, eight mammalian cell lines that were available in the laboratory were screened for productive SAFV infection by a laboratory-adapted SAFV of genotype 3. Five of the cell lines (Neuro2A, CHO-K1, NIH/3T3, Vero and HEp-2) were found to be permissible. The time required for SAFV to induce complete lysis as a cytopathic effect (CPE) in these permissibly infected cells and the resultant end point virus titer differed for each cell type. HEp-2 exhibited the shortest time frame to reach full CPE compared to the others. All infected cell lines produced a high virus titer at 72 h post-infection. In addition to causing lytic cell death, SAFV also induced apoptotic cell death in host cells through both extrinsic and intrinsic pathways, although the apoptotic events in HEp-2 cells appeared to have been blocked between the early and late stages. In conclusion, laboratory-adapted SAFV is able to productively infect a number of mammalian cell lines and induce apoptosis in the infected host cells. However, apoptosis in HEp-2 cells is blocked before the end stage. PMID:26038510

  2. Saffold virus is able to productively infect primate and rodent cell lines and induces apoptosis in these cells.

    PubMed

    Xu, Yishi; Victorio, Carla Bianca Luena; Ng, Qimei; Tan, Yee Joo; Chua, Kaw Bing

    2014-02-01

    Saffold virus (SAFV), a newly discovered human cardiovirus of the Picornaviridae family, causes widespread infection among children, as shown by previous seroprevalence studies. To determine the host cell range of SAFV and its cytopathogenicity, eight mammalian cell lines that were available in the laboratory were screened for productive SAFV infection by a laboratory-adapted SAFV of genotype 3. Five of the cell lines (Neuro2A, CHO-K1, NIH/3T3, Vero and HEp-2) were found to be permissible. The time required for SAFV to induce complete lysis as a cytopathic effect (CPE) in these permissibly infected cells and the resultant end point virus titer differed for each cell type. HEp-2 exhibited the shortest time frame to reach full CPE compared to the others. All infected cell lines produced a high virus titer at 72 h post-infection. In addition to causing lytic cell death, SAFV also induced apoptotic cell death in host cells through both extrinsic and intrinsic pathways, although the apoptotic events in HEp-2 cells appeared to have been blocked between the early and late stages. In conclusion, laboratory-adapted SAFV is able to productively infect a number of mammalian cell lines and induce apoptosis in the infected host cells. However, apoptosis in HEp-2 cells is blocked before the end stage.

  3. HOLDING THE LINE WITH A WATCHFUL EYE: THE IMPACT OF PERCEIVED PARENTAL PERMISSIVENESS AND PARENTAL MONITORING ON RISKY SEXUAL BEHAVIOR AMONG ADOLESCENTS IN PSYCHIATRIC CARE

    PubMed Central

    Donenberg, Geri R.; Wilson, Helen W.; Emerson, Erin; Bryant, Fred B.

    2005-01-01

    Adolescents in psychiatric care are at increased risk of HIV, yet little is known about the family factors related to sexual risk taking among these youth. We explored whether perceived parental monitoring and perceived parental permissiveness were linked to high-risk sexual behavior in 169 ethnically diverse urban youth seeking mental health services in Chicago, and we tested whether adolescent gender moderated these associations. We evaluated sexual risk taking at a global level and for specific risk behaviors (e.g., sex without a condom, sex while using drugs and alcohol). Girls reported more risky sex overall than boys, and girls were more likely than boys to report having sex without a condom. At low levels of parental permissiveness, rates of risky sex among boys and girls’ did not differ, but at high levels of permissiveness girls reported more sexual risk taking than boys, and girls were more likely than boys to report having sex while using drugs and alcohol and having sex without a condom. Findings highlight the complexity of adolescent sexual behavior and the need for multilevel assessment of risk taking. Results suggest that parental monitoring and permissiveness are more strongly associated with sexual risk taking in troubled girls than troubled boys, and they underscore a need for gender-sensitive, family-focused HIV-prevention programs. PMID:12000232

  4. Research Resource: Transcriptome Profiling of Genes Regulated by RXR and Its Permissive and Nonpermissive Partners in Differentiating Monocyte-Derived Dendritic Cells

    PubMed Central

    Széles, Lajos; Póliska, Szilárd; Nagy, Gergely; Szatmari, Istvan; Szanto, Attila; Pap, Attila; Lindstedt, Malin; Santegoets, Saskia J.A.M.; Rühl, Ralph; Dezsö, Balázs; Nagy, László

    2010-01-01

    Retinoid X receptors (RXRs) are heterodimerization partners for many nuclear receptors and also act as homodimers. Heterodimers formed by RXR and a nonpermissive partner, e.g. retinoic acid receptor (RAR) and vitamin D receptor (VDR), can be activated only by the agonist of the partner receptor. In contrast, heterodimers that contain permissive partners, e.g. liver X receptor (LXR) and peroxisome proliferator-activated receptor (PPAR), can be activated by agonists for either the partner receptor or RXR, raising the possibility of pleiotropic RXR signaling. However, it is not known to what extent the receptor’s activation results in triggering mechanisms dependent or independent of permissive heterodimers. In this study, we systematically and quantitatively characterized all probable RXR-signaling pathways in differentiating human monocyte-derived dendritic cells (Mo-DCs). Using pharmacological, microarray and quantitative RT-PCR techniques, we identified and characterized gene sets regulated by RXR agonists (LG100268 and 9-cis retinoic acid) and agonists for LXRs, PPARs, RARα, and VDR. Our results demonstrated that permissiveness was partially impaired in Mo-DCs, because a large number of genes regulated by PPAR or LXR agonists was not affected by RXR-specific agonists or was regulated to a lesser extent. As expected, we found that RXR agonists regulated only small portions of RARα or VDR targets. Importantly, we could identify and characterize PPAR- and LXR-independent pathways in Mo-DCs most likely mediated by RXR homodimers. These data suggested that RXR signaling in Mo-DCs was mediated via multiple permissive heterodimers and also by mechanism(s) independent of permissive heterodimers, and it was controlled in a cell-type and gene-specific manner. PMID:20861222

  5. Persistent use of false myeloma cell lines.

    PubMed

    Drexler, Hans G; Matsuo, Yoshinobu; MacLeod, Roderick A E

    2003-09-01

    Multiple myeloma (MM) is a neoplasm of a terminally differentiated B-cell. Human myeloma cell lines were shown to be suitable model systems for use in various fields of the biological sciences. Within the last 20 years more than 100 cell lines have been established. So-called 'myeloma cell lines' have been previously reported and are still widely used which are in reality Epstein-Barr virus (EBV)-positive B-lymphoblastoid cell lines. The presence of the EBV-genome in residual normal B-cells provides them with a selective growth advantage after explantation. 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 material, it is crucial that the cells in the culture flasks faithfully correspond to the purported objects of study. On closer examination, the use of false cell lines may be seen to invalidate a significant percentage of scientific work, or at least cast doubts on the relevance of these in vitro results to the cell type or tumor in vivo. Ultimately, use of cross-contaminated cell lines is a waste of human and material resources. Henceforth, it should be mandatory to prove the proper derivation of each new cell line by comparing DNA fingerprints or karyotypes of the patient's primary cells and the cultured cells. The availability of well characterized and authenticated bona fide MM cell lines is of great importance for the study of the biology, etiology and treatment of the disease.

  6. A bovine cell line that can be infected by natural sheep scrapie prions.

    PubMed

    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.

  7. A Bovine Cell Line That Can Be Infected by Natural Sheep Scrapie Prions

    PubMed Central

    Oelschlegel, Anja M.; Geissen, Markus; Lenk, Matthias; Riebe, Roland; Angermann, Marlies; 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. PMID:25565633

  8. Cell line fingerprinting using retroelement insertion polymorphism.

    PubMed

    Ustyugova, Svetlana V; Amosova, Anna L; Lebedev, Yuri B; Sverdlov, Eugene D

    2005-04-01

    Human cell lines are an indispensable tool for functional studies of living entities in their numerous manifestations starting with integral complex systems such as signal pathways and networks, regulation of gene ensembles, epigenetic factors, and finishing with pathological changes and impact of artificially introduced elements, such as various transgenes, on the behavior of the cell. Therefore, it is highly desirable to have reliable cell line identification techniques to make sure that the cell lines to be used in experiments are exactly what is expected. To this end, we developed a set of informative markers based on insertion polymorphism of human retroelements (REs). The set includes 47 pairs of PCR primers corresponding to introns of the human genes with dimorphic LINE1 (L1) and Alu insertions. Using locus-specific PCR assays, we have genotyped 10 human cell lines of various origins. For each of these cell lines, characteristic fingerprints were obtained. An estimated probability that two different cell lines possess the same marker genotype is about 10-18. Therefore, the proposed set of markers provides a reliable tool for cell line identification.

  9. uPAR induces expression of transforming growth factor β and interleukin-4 in cancer cells to promote tumor-permissive conditioning of macrophages.

    PubMed

    Hu, Jingjing; Jo, Minji; Eastman, Boryana M; Gilder, Andrew S; Bui, Jack D; Gonias, Steven L

    2014-12-01

    Cancer cells condition macrophages and other inflammatory cells in the tumor microenvironment so that these cells are more permissive for cancer growth and metastasis. Conditioning of inflammatory cells reflects, at least in part, soluble mediators (such as transforming growth factor β and IL-4) that are released by cancer cells and alter the phenotype of cells of the innate immune system. Signaling pathways in cancer cells that potentiate this activity are incompletely understood. The urokinase receptor (uPAR) is a cell-signaling receptor known to promote cancer cell survival, proliferation, metastasis, and cancer stem cell-like properties. The present findings show that uPAR expression in diverse cancer cells, including breast cancer, pancreatic cancer, and glioblastoma cells, promotes the ability of these cells to condition co-cultured bone marrow-derived macrophages so that the macrophages express significantly increased levels of arginase 1, a biomarker of the alternatively activated M2 macrophage phenotype. Expression of transforming growth factor β was substantially increased in uPAR-expressing cancer cells via a mechanism that requires uPA-initiated cell signaling. uPAR also controlled expression of IL-4 in cancer cells via a mechanism that involves activation of ERK1/2. The ability of uPAR to induce expression of factors that condition macrophages in the tumor microenvironment may constitute an important mechanism by which uPAR promotes cancer progression.

  10. Standards for Cell Line Authentication and Beyond

    PubMed Central

    Cole, Kenneth D.; Plant, Anne L.

    2016-01-01

    Different genomic technologies have been applied to cell line authentication, but only one method (short tandem repeat [STR] profiling) has been the subject of a comprehensive and definitive standard (ASN-0002). Here we discuss the power of this document and why standards such as this are so critical for establishing the consensus technical criteria and practices that can enable progress in the fields of research that use cell lines. We also examine other methods that could be used for authentication and discuss how a combination of methods could be used in a holistic fashion to assess various critical aspects of the quality of cell lines. PMID:27300367

  11. Thyroid cancer cell lines: an overview

    PubMed Central

    Saiselet, Manuel; Floor, Sébastien; Tarabichi, Maxime; Dom, Geneviève; Hébrant, Aline; van Staveren, Wilma C. G.; Maenhaut, Carine

    2012-01-01

    Human thyroid cancer cell lines are the most used models for thyroid cancer studies. They must be used with detailed knowledge of their characteristics. These in vitro cell lines originate from differentiated and dedifferentiated in vivo human thyroid tumors. However, it has been shown that mRNA expression profiles of these cell lines were closer to dedifferentiated in vivo thyroid tumors (anaplastic thyroid carcinoma, ATC) than to differentiated ones. Here an overview of the knowledge of these models was made. The mutational status of six human thyroid cancer cell lines (WRO, FTC133, BCPAP, TPC1, K1, and 8505C) was in line with previously reported findings for 10 genes frequently mutated in thyroid cancer. However, the presence of a BRAF mutation (T1799A: V600E) in WRO questions the use of this cell line as a model for follicular thyroid carcinoma (FTC). Next, to investigate the biological meaning of the modulated mRNAs in these cells, a pathway analysis on previously obtained mRNA profiles was performed on five cell lines. In five cell lines, the MHC class II pathway was down-regulated and in four of them, ribosome biosynthesis and translation pathways were up-regulated. mRNA expression profiles of the cell lines were also compared to those of the different types of thyroid cancers. Three datasets originating from different microarray platforms and derived from distinct laboratories were used. This meta-analysis showed a significant higher correlation between the profiles of the thyroid cancer cell lines and ATC, than to differentiated thyroid tumors (i.e., PTC or FTC) specifically for DNA replication. This already observed higher correlation was obtained here with an increased number of in vivo tumors and using different platforms. In summary, this would suggest that some papillary thyroid carcinoma or follicular thyroid carcinoma (PTC or FTC) cell lines (i.e., TPC-1) might have partially lost their original DNA synthesis/replication regulation mechanisms during

  12. Restriction of human adenovirus replication in Chinese hamster cell lines and their hybrids with human cells.

    PubMed

    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.

  13. Prostaglandin Actions in Established Insect Cell Lines

    USDA-ARS?s Scientific Manuscript database

    Prostaglandins (PGs) are oxygenated metabolites of arachidonic acid (AA) and two other C20 polyunsaturated fatty acids that serve as biochemical signals that mediate a wide range of physiological functions in animal cells. For example, PGs influence protein expression in establish insect cell lines ...

  14. Virus Discovery Using Tick Cell Lines

    PubMed Central

    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

  15. Establishment, characterization and viral susceptibility of two cell lines derived from leopard wrasse Macropharyngodon geoffroy.

    PubMed

    Ma, J; Sun, S; Zeng, L; Lu, Y

    2013-09-01

    Two new fish cell lines were established from skin (LWSK) and fin (LWFN) of leopard wrasse Macropharyngodon geoffroy. These cells grew optimally at 25° C in Leibovitz-15 medium supplemented with 10% foetal bovine serum. Proliferation of M. geoffroy cells remained serum dependent up to cell passage 16, and cell-plating efficiency ranged from 12 to 16%. Karyotypic analysis of these new cell lines at cell passage 8 indicated that both cell lines remained diploid with a peak chromosomal count of 144. PCR amplification of 16S mitochondrial DNA and the subsequent analysis confirmed that these cell lines were indeed derived from M. geoffroy. Results of viral challenge assays revealed that both LWSK and LWFN shared patterns of viral susceptibility similar to that of six fish viruses tested: LWSK and LWFN cells were highly permissive to channel catfish virus, spring viremia carp virus and snakehead rhabdovirus with high-yield virus production ranging from 10(7·18±0·17) to 10(8·37±0·16) TCID50  ml(-1) (mean ± s.d.). These newly established cell lines would be useful in attempts to isolate and study aquatic viruses, particularly the viral aetiology of green turtle fibropapilloma as M. geoffroy is known to be one of the common cleaner fish of green sea turtles. © 2013 The Fisheries Society of the British Isles.

  16. A Transcriptionally Permissive Epigenetic Landscape at the Vasoactive Intestinal Peptide Receptor-1 Promoter Suggests an Euchromatin Nuclear Position in Murine CD4 T Cells

    PubMed Central

    Benton, K.D.; Hermann, R.J.; Vomhof-DeKrey, E.E.; Haring, J.S.; Van der Steen, T.; Smith, J; Dovat, S; Dorsam, G.P.

    2009-01-01

    Summary T cells express receptors for neuropeptides that mediate immunological activities. Vasoactive intestinal peptide receptor – 1 (VPAC1), the prototypical group II G protein coupled receptor, binds two neuropeptides with high affinity, called vasoactive intestinal peptide and pituitary adenylate cyclase activating peptide. During T cell signaling, VPAC1 mRNA expression levels are significantly downregulated through a Src kinase dependent mechanism, thus altering the sensitivity for these neuropeptides during an immune reaction. Presently, it is unknown whether the mechanism that regulates VPAC1 during T cell signaling involves epigenetic changes. Therefore, we hypothesized that the epigenetic landscape consisting of diacetylation at H3K9/14 and trimethylation at H3K4, two transcriptionally permissive histone modifications, would parallel VPAC1 expression showing high enrichment in untreated T cells, but lower enrichment in α-CD3 treated T cells. To this end, quantitative chromatin immunoprecipitation (ChIP) analysis of H3K9/14ac and H3K4me3 was conducted using purified CD4+ T cells, with CD45R+ B cells as a negative control. Our data revealed that these histone modifications at the VPAC1 promoter did indeed parallel its mRNA levels between T and B lymphocytes, but did not decrease during T cell signaling. Collectively, these data strongly imply a euchromatin nuclear position for the VPAC1 locus irrespective of the activation status of T cells. PMID:19729043

  17. Development of improved vaccine cell lines against rotavirus

    PubMed Central

    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

  18. Refractory lining for electrochemical cell

    DOEpatents

    Blander, Milton; Cook, Glenn M.

    1987-01-01

    Apparatus for processing a metallic fluid containing iron oxide, container for a molten metal including an electrically conductive refractory disposed for contact with the molten metal which contains iron oxide, an electrolyte in the form of a basic slag on top of the molten metal, an electrode in the container in contcat with the slag electrically separated from the refractory, and means for establishing a voltage across the refractory and the electrode to reduce iron oxide to iron at the surface of the refractory in contact with the iron oxide containing fluid. A process is disclosed for refining an iron product containing not more than about 10% by weight oxygen and not more than about 10% by weight sulfur, comprising providing an electrolyte of a slag containing one or more of calcium oxide, magnesium oxide, silica or alumina, providing a cathode of the iron product in contact with the electrolyte, providing an anode in contact with the electrolyte electrically separated from the cathode, and operating an electrochemical cell formed by the anode, the cathode and the electrolyte to separate oxygen or sulfur present in the iron product therefrom.

  19. Using Surplus Embryos and Research Embryos in Stem Cell Research: Ethical Viewpoints of Buddhist, Hindu and Catholic Leaders in Malaysia on the Permissibility of Research.

    PubMed

    Sivaraman, Mathana Amaris Fiona

    2017-03-09

    The sources of embryos for Embryonic Stem Cell Research (ESCR) include surplus embryos from infertility treatments, and research embryos which are created solely for an ESCR purpose. The latter raises more ethical concerns. In a multi-religious country like Malaysia, ethical discussions on the permissibility of ESCR with regard to the use surplus and research embryos are diversified. Malaysia has formulated guidelines influenced by the national fatwa ruling which allows the use of surplus embryos in ESCR. Input from other main religions is yet to be documented. In light of this, this study addresses (i) the ethical viewpoints of Buddhist, Hindu and Catholic leaders on the permissibility of using surplus and research embryos; and (ii) the moral standpoints of religious leaders towards attaining a consensus on the practice of ESCR in Malaysia. Responses from the religious leaders were obtained via semi-structured, face-to-face interviews. The findings show that generally the Buddhist and Hindu leaders approve the use of surplus embryos. Their responses on the creation of research embryos for ESCR are varied. Meanwhile, the Catholic leaders distinctively objected to ESCR regardless of the embryo sources, referring to it as the destruction of life. Taking into account the diverse views, this study explores the response of the religious leaders for a general consensus wherever possible. The ethical discourse surrounding ESCR in a multi-religious setting offers new perspective, which needs to be explored in a broader global community.

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

    PubMed

    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.

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

    PubMed Central

    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. PMID:18927105

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

    PubMed

    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 basic and preclinical research. Yet, international hESC usage patterns, in both permissive and restrictive political environments, do not correlate with a specific type of stem cell policy. Here we conducted a descriptive analysis of hESC line usage and compared the ability of policy-driven processes and collaborative processes inherent to biomedical research to recapitulate global hESC usage patterns. We find that current global hESC usage can be modelled as a cumulative advantage process, independent of restrictive or permissive policy influence, suggesting a primarily innovation-driven (rather than policy-driven) mechanism underlying human pluripotent stem cell usage in preclinical research.

  3. Relevance, Derogation and Permission

    NASA Astrophysics Data System (ADS)

    Stolpe, Audun

    We show that a recently developed theory of positive permission based on the notion of derogation is hampered by a triviality result that indicates a problem with the underlying full-meet contraction operation. We suggest a solution that presupposes a particular normal form for codes of norms, adapted from the theory of relevance through propositional letter sharing. We then establish a correspondence between contractions on sets of norms in input/output logic (derogations), and AGM-style contractions on sets of formulae, and use it as a bridge to migrate results on propositional relevance from the latter to the former idiom. Changing the concept accordingly we show that positive permission now incorporates a relevance requirement that wards off triviality.

  4. Heterogeneity of alpha-fetoprotein(AFP) and albumin containing cells in normal and pathological permissive states for AFP production: AFP containing cells induced in adult rats recapitulate the appearance of AFP containing hepatocytes in fetal rats.

    PubMed

    Sell, S

    1980-01-01

    The cellular localization of alpha-fetoprotein (AFP) and albumin (ALB) in permissive states for AFP synthesis has been examined. The cells containing AFP associated with permissive states in the adult are similar in appearance to cells that are present during the development of fetal liver. In fetal liver, AFP is seen in most developing hepatocytes ranging from small 'oval' like cells to larger dividing hepatocytes and in cells organized in glandular structures. Following exposure to some chemical hepatocarcinogens, AFP can also be seen in small 'oval' cells, ductal-like cells, and larger atypical hepatocytes that form glandular-like structures. Following partial hepatectomy or galactosamine-induced live injury, AFP is seen in a few large parenchymal cells usually containing identifiable chromatin Cells which contain AFP almost always contain ALB as well, but for each cell type there are many more ALB containing cells than AFP containing cells. ALB and AFP containing hepatoma cells are more frequently located adjacent to tumor vessels and AFP production by hepatoma 777 in vitro is associated with the growth state of the tumor. The AFP containing cells that are seen during restitutive proliferation most likely arise from deregulation of proliferating adult hepatocytes. The non-hepatoma AFP containing cells that appear early during carcinogenesis may arise in the adult by retrodifferentiation of hepatocytes or by proliferation of stem cells. These morphologically different AFP containing cells may or may not be precursors of the hepatocellular carcinomas which develop later.

  5. Effects of teicoplanin on cell number of cultured cell lines

    PubMed Central

    Kashkolinejad-Koohi, Tahere; Saadat, Iraj

    2015-01-01

    Teicoplanin is a glycopeptide antibiotic with a wide variation in human serum half-life. It is also a valuable alternative of vancomycin. There is however no study on its effect on cultured cells. The aim of the present study was to test the effect of teicoplanin on cultured cell lines CHO, Jurkat E6.1 and MCF-7. The cultured cells were exposed to teicoplanin at final concentrations of 0–11000 μg/ml for 24 hours. To determine cell viability, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was performed. At low concentrations of teicoplanin the numbers of cultured cells (due to cell proliferation) were increased in the three cell lines examined. The maximum cell proliferation rates were observed at concentrations of 1000, 400, and 200 μg/ml of teicoplanin for CHO, MCF-7 and Jurkat cell lines, respectively. Cell toxicity was observed at final concentrations over 2000, 6000, and 400 μg/ml of teicoplanin for CHO, MCF-7 and Jurkat cell lines, respectively. A dose-dependent manner of cell toxicity was observed. Our present findings indicated that teicoplanin at clinically used concentrations induced cell proliferation. It should therefore be used cautiously, particularly in children, pregnant women and patients with cancer. PMID:27486356

  6. Radiation sensitivity of Merkel cell carcinoma cell lines

    SciTech Connect

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W.

    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. Hepatocyte-stellate cell cross-talk in the liver engenders a permissive inflammatory microenvironment that drives progression in hepatocellular carcinoma.

    PubMed

    Coulouarn, Cédric; Corlu, Anne; Glaise, Denise; Guénon, Isabelle; Thorgeirsson, Snorri S; Clément, Bruno

    2012-05-15

    Many solid malignant tumors arise on a background of inflamed and/or fibrotic tissues, features that are found in more than 80% hepatocellular carcinomas (HCC). Activated hepatic stellate cells (HSC) play a critical role in fibrogenesis associated with HCC onset and progression, yet their functional impact on hepatocyte fate remains largely unexplored. Here, we used a coculture model to investigate the cross-talk between hepatocytes (human hepatoma cells) and activated human HSCs. Unsupervised genome-wide expression profiling showed that hepatocyte-HSC cross-talk is bidirectional and results in the deregulation of functionally relevant gene networks. Notably, coculturing increased the expression of proinflammatory cytokines and modified the phenotype of hepatocytes toward motile cells. Hepatocyte-HSC cross-talk also generated a permissive proangiogenic microenvironment, particularly by inducing VEGFA and matrix metalloproteinase (MMP)9 expression in HSCs. An integrative genomic analysis revealed that the expression of genes associated with hepatocyte-HSC cross-talk correlated with HCC progression in mice and was predictive of a poor prognosis and metastasis propensity in human HCCs. Interestingly, the effects of cross-talk on migration and angiogenesis were reversed by the histone deacetylase inhibitor trichostatin A. Our findings, therefore, indicate that the cross-talk between hepatoma cells and activated HSCs is an important feature of HCC progression, which may be targeted by epigenetic modulation.

  8. Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

    SciTech Connect

    Felthaus, O.; Ettl, T.; Gosau, M.; Driemel, O.; Brockhoff, G.; Reck, A.; Zeitler, K.; Hautmann, M.; Reichert, T.E.; Schmalz, G.; Morsczeck, C.

    2011-04-01

    Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

  9. Investigating citrullinated proteins in tumour cell lines

    PubMed Central

    2013-01-01

    Background The conversion of arginine into citrulline, termed citrullination, has important consequences for the structure and function of proteins. Studies have found PADI4, an enzyme performing citrullination, to be highly expressed in a variety of malignant tumours and have shown that PADI4 participates in the process of tumorigenesis. However, as citrullinated proteins have not been systematically investigated in tumours, the present study aimed to identify novel citrullinated proteins in tumours by 2-D western blotting (2-D WB). Methods Two identical two-dimensional electrophoresis (2-DE) gels were prepared using extracts from ECA, H292, HeLa, HEPG2, Lovo, MCF-7, PANC-1, SGC, and SKOV3 tumour cell lines. The expression profiles on a 2-DE gel were trans-blotted to PVDF membranes, and the blots were then probed with an anti-citrulline antibody. By comparing the 2-DE profile with the parallel 2-D WB profile at a global level, protein spots with immuno-signals were collected from the second 2-DE gel and identified using mass spectrometry. Immunoprecipitation was used to verify the expression and citrullination of the targeted proteins in tumour cell lines. Results 2-D WB and mass spectrometry identified citrullinated α-enolase (ENO1), heat shock protein 60 (HSP60), keratin 8 (KRT8), tubulin beta (TUBB), T cell receptor chain and vimentin in these cell lines. Immunoprecipitation analyses verified the expression and citrullination of ENO1, HSP60, KRT8, and TUBB in the total protein lysates of the tumour cell lines. Conclusions The citrullination of these proteins suggests a new mechanism in the tumorigenic process. PMID:24099319

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

    PubMed Central

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

    2016-01-01

    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. PMID:27213425

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

    PubMed

    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. Spontaneous Cell Competition in Immortalized Mammalian Cell Lines

    PubMed Central

    Penzo-Méndez, Alfredo I.; Chen, Yi-Ju; Li, Jinyang; Witze, Eric S.; Stanger, Ben Z.

    2015-01-01

    Cell competition is a form of cell-cell interaction by which cells compare relative levels of fitness, resulting in the active elimination of less-fit cells, “losers,” by more-fit cells, “winners.” Here, we show that in three routinely-used mammalian cell lines – U2OS, 3T3, and MDCK cells – sub-clones arise stochastically that exhibit context-dependent competitive behavior. Specifically, cell death is elicited when winner and loser sub-clones are cultured together but not alone. Cell competition and elimination in these cell lines is caspase-dependent and requires cell-cell contact but does not require de novo RNA synthesis. Moreover, we show that the phenomenon involves differences in cellular metabolism. Hence, our study demonstrates that cell competition is a common feature of immortalized mammalian cells in vitro and implicates cellular metabolism as a mechanism by which cells sense relative levels of “fitness.” PMID:26200654

  13. The potential of GMP-compliant platelet lysate to induce a permissive state for cardiovascular transdifferentiation in human mediastinal adipose tissue-derived mesenchymal stem cells.

    PubMed

    Siciliano, Camilla; Chimenti, Isotta; Bordin, Antonella; Ponti, Donatella; Iudicone, Paola; Peruzzi, Mariangela; Rendina, Erino Angelo; Calogero, Antonella; Pierelli, Luca; Ibrahim, Mohsen; De Falco, Elena

    2015-01-01

    Human adipose tissue-derived mesenchymal stem cells (ADMSCs) are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL), a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs.

  14. Functional features of cancer stem cells in melanoma cell lines.

    PubMed

    Zimmerer, Rüdiger M; Korn, Philippe; Demougin, Philippe; Kampmann, Andreas; Kokemüller, Horst; Eckardt, André M; Gellrich, Nils-Claudius; Tavassol, Frank

    2013-08-06

    Recent evidence suggests a subset of cells within a tumor with "stem-like" characteristics. These cells are able to transplant tumors in immunodeficient hosts. Distinct from non-malignant stem cells, cancer stem cells (CSC) show low proliferative rates, high self-renewing capacity, propensity to differentiate into actively proliferating tumor cells, and resistance to chemotherapy or radiation. They are often characterized by elevated expression of stem cell surface markers, in particular CD133, and sets of differentially expressed stem cell-associated genes. CSC are usually rare in clinical specimens and hardly amenable to functional studies and gene expression profiling. In this study, a panel of heterogenous melanoma cell lines was screened for typical CSC features. Nine heterogeneous metastatic melanoma cell lines including D10 and WM115 were studied. Cell lines were phenotyped using flow cytometry and clonogenic assays were performed by limiting dilution analysis on magnetically sorted cells. Spheroidal growth was investigated in pretreated flasks. Gene expression profiles were assessed by using real-time rt-PCR and DNA microarrays. Magnetically sorted tumor cells were subcutaneously injected into the flanks of immunodeficient mice. Comparative immunohistochemistry was performed on xenografts and primary human melanoma sections. D10 cells expressed CD133 with a significantly higher clonogenic capacity as compared to CD133- cells. Na8, D10, and HBL cells formed spheroids on poly-HEMA-coated flasks. D10, Me39, RE, and WM115 cells expressed at least 2 of the 3 regulatory core transcription factors SOX2, NANOG, and OCT4 involved in the maintenance of stemness in mesenchymal stem cells. Gene expression profiling on CD133+ and CD133- D10 cells revealed 68 up- and 47 downregulated genes (+/-1.3 fold). Two genes, MGP and PROM1 (CD133), were outstandingly upregulated. CD133+ D10 cells formed tumors in NSG mice contrary to CD133- cells and CD133 expression was detected

  15. Neurovirulence of H5N1 Infection in Ferrets Is Mediated by Multifocal Replication in Distinct Permissive Neuronal Cell Regions

    PubMed Central

    Plourde, Jennifer R.; Pyles, John A.; Layton, R. Colby; Vaughan, Sarah E.; Tipper, Jennifer L.; Harrod, Kevin S.

    2012-01-01

    Highly pathogenic avian influenza A (HPAI), subtype H5N1, remains an emergent threat to the human population. While respiratory disease is a hallmark of influenza infection, H5N1 has a high incidence of neurological sequelae in many animal species and sporadically in humans. We elucidate the temporal/spatial infection of H5N1 in the brain of ferrets following a low dose, intranasal infection of two HPAI strains of varying neurovirulence and lethality. A/Vietnam/1203/2004 (VN1203) induced mortality in 100% of infected ferrets while A/Hong Kong/483/1997 (HK483) induced lethality in only 20% of ferrets, with death occurring significantly later following infection. Neurological signs were prominent in VN1203 infection, but not HK483, with seizures observed three days post challenge and torticollis or paresis at later time points. VN1203 and HK483 replication kinetics were similar in primary differentiated ferret nasal turbinate cells, and similar viral titers were measured in the nasal turbinates of infected ferrets. Pulmonary viral titers were not different between strains and pathological findings in the lungs were similar in severity. VN1203 replicated to high titers in the olfactory bulb, cerebral cortex, and brain stem; whereas HK483 was not recovered in these tissues. VN1203 was identified adjacent to and within the olfactory nerve tract, and multifocal infection was observed throughout the frontal cortex and cerebrum. VN1203 was also detected throughout the cerebellum, specifically in Purkinje cells and regions that coordinate voluntary movements. These findings suggest the increased lethality of VN1203 in ferrets is due to increased replication in brain regions important in higher order function and explains the neurological signs observed during H5N1 neurovirulence. PMID:23056366

  16. Optimization of Gene Transfection in Murine Myeloma Cell Lines using Different Transfection Reagents

    PubMed Central

    Shabani, Mahdi; Hemmati, Sheyda; Hadavi, Reza; Amirghofran, Zahra; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Shokri, Fazel

    2010-01-01

    Purification and isolation of cellular target proteins for monoclonal antibody (MAb) production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines (SP2/0, NS0, NS1, Ag8, and P3U1) were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein (EGFP), were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP2/0, 55.7%, 21.1% and 9.3% for NS0, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NS0 and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NS0 cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production. PMID:23408356

  17. Cytosolic [Ca(2+)] modulates basal GLUT1 activity and plays a permissive role in its activation by metabolic stress and insulin in rat epithelial cells.

    PubMed

    Quintanilla, R A; Porras, O H; Castro, J; Barros, L F

    2000-08-01

    The aim of this work was to investigate the role of cytosolic free calcium ([Ca(2+)]c) in the stimulation of GLUT1 by metabolic stress and insulin. Chelation of [Ca(2+)]c with bapta, introduced in rat liver epithelial Clone 9 cells in the acetoxymethyl (AM) form, decreased their basal rate of 2-deoxyglucose uptake in a dose-dependent fashion. Maximal inhibition at 75 microM bapta was by 38 +/- 8% (n = 8). The effect was partially reversed by ionomycin. Basal sugar uptake was also decreased by lowering extracellular [Ca(2+)] in ionomycin-permeabilized cells. Increasing [Ca(2+)]c over its resting level of 168 +/- 32 (n = 27) had no affect on sugar uptake. Chelation of [Ca(2+)]c did not change the abundance of surface GLUT1 and had no significant effect on the affinity of GLUT1 for sugars. In addition, calcium chelation abolished the activation of GLUT1 by azide, arsenate, 2,4-dinitrophenol and insulin. However, [Ca(2+)]c did not increase in the presence of azide. We conclude that [Ca(2+)]c, near or below its resting level, modulates GLUT1 activity over a considerable range and plays a permissive role in the activation of the carrier by metabolic stress and insulin.

  18. In vitro permissivity of bovine peripheral blood mononuclear cells to bovine viral diarrhoea virus is dependent on the animal specific immune status.

    PubMed

    Lucchini, Barbara; Ponti, Wilma; Turin, Lauretta; Bronzo, Valerio; Scaccabarozzi, Licia; Luzzago, Camilla

    2012-04-01

    The in vitro permissivity to infection with homologous and heterologous bovine viral diarrhoea virus (BVDV) strains of bovine peripheral blood mononuclear cells (PBMCs) from eight naïve and eight BVDV-1b immune animals was studied. Four reference strains (BVDV-1a NADL, BVDV-1b NY-1, BVDV-2 125 and BVDV-2 890) were selected, based on genotype, prevalence and biotype. Virus neutralizing antibody titres were determined at bleeding and the viral loads were measured in PBMCs by end point titration in cell culture and by real-time PCR. PBMCs from both naïve and immune animals became infected by all BVDV strains tested, although virus titres were lower for immune heifers than naïve ones; the differences were significant for NADL (P<0.05) and 890 (P<0.001) strains. The in vitro model used in this study showed that PBMCs from immune animals are susceptible to re-infection with both homologous and heterologous BVDV strains, albeit at a lower extent than naïve cattle.

  19. Semipermissive replication of a nuclear polyhedrosis virus of Autographa californica in a gypsy moth cell line

    SciTech Connect

    McClintock, J.T.; Dougherty, E.M.; Weiner, R.M.

    1986-01-01

    Several gypsy moth cell lines have been previously described as nonpermissive for the multiple-embedded nuclear polyhedrosis virus of Autographa californica (AcMNPV). In this report, the authors demonstrate the semipermissive infection of a gypsy moth cell line, IPLB-LD-652Y, with AcMNPV. IPLB-LD-652Y cells infected with AcMNPV produced classic cytopathic effects but failed to yield infectious progeny virus. Results of experiments employing DNA-DNA dot hybridization suggested that AcMNPV DNA synthesis was initiated from 8 to 12 h postinfection (p.i.), continued at a maximum rate from 12 to 20 h p.i., and declined from 20 to 36 h p.i. The rate of AcMNPV DNA synthesis approximated that observed in the permissive TN-368 cell line. AcMNPV-infected IPLB-LD-652Y cells, pulse-labeled with (/sup 35/S)methionine at various time intervals p.i. and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed four virus-induced proteins, one novel to the semipermissive system and three early ..cap alpha.. proteins, synthesized from 1 to 20 h p.i. Thereafter, both host and viral protein synthesis was completely suppressed. These results suggest that AcMNPV adsorbed, penetrated, and initiated limited macromolecular synthesis in the semipermissive gypsy moth cell line. However, the infection cycle was restricted during the early phase of AcMNPV replication.

  20. Pancreastatin producing cell line from human pancreatic islet cell tumor.

    PubMed

    Funakoshi, A; Tateishi, K; Tsuru, M; Jimi, A; Wakasugi, H; Ikeda, Y; Kono, A

    1990-04-30

    It has been characterized that cell line QGP-1 derived from human non-functioning pancreatic islet cell tumor produces human pancreastatin. Exponentially growing cultures produced 5.7 fmol of pancreastatin/10(6) cells/hr. Human pancreastatin immunoreactivities in plasma and tumor after xenografting with QGP-1 into nude mouse were 92.7 fmol/ml and 160.2 pmol/g wet weight, respectively. Immunocytochemical study revealed both chromogranin A and pancreastatin immunoreactive cells in the tumor. Gel filtrations of culture medium and tumor extract identified heterogenous molecular forms of PST-LI which eluted as large and smaller molecular species. These results suggest that plasma pancreastatin levels may be useful as a tumor marker of endocrine tumor of the pancreas, and the pancreastatin producing cell line may be useful for studies of the mechanism of secretions and processing of chromogranin A and pancreastatin.

  1. GPVI oligomerisation in cell lines and platelets

    PubMed Central

    2007-01-01

    Summary Background Glycoprotein VI (GPVI) is a physiological receptor for collagen expressed at the surface of platelets and megakaryocytes. Constitutive dimerisation of GPVI has been proposed as necessary for the interaction with collagen, although direct evidence of dimerisation has not been reported in cell lines or platelets. Objectives To investigate oligomerisation of GPVI in transfected cell lines and in platelets under nonstimulated conditions. Methods and Results By using a combination of molecular and biochemical techniques, we demonstrate that GPVI association occurs at the surface of transfected 293T cells under basal conditions, through an interaction at the extra-cellular domain of the receptor. Bioluminescence resonance energy transfer was used to confirm oligomerisation of GPVI under these conditions. A chemical cross-linker was used to detect constitutive oligomeric forms of GPVI at the surface of platelets, which contain the FcR γ-chain. Conclusions The present results directly demonstrate GPVI-FcR γ-chain oligomerisation at the surface of the platelet, and thereby add to the growing evidence that oligomerisation of GPVI may be a pre-requisite for binding of the receptor to collagen, and therefore for proper functioning of platelets upon vascular damage. PMID:17367493

  2. Effect of glutamate analogues on brain tumor cell lines.

    PubMed

    Campbell, G L; Bartel, R; Freidman, H S; Bigner, D D

    1985-10-01

    Glutamate analogues have been used in many different experimental approaches in neurobiology. A small number of these analogues have been classified as gliotoxic. We have examined the effect of seven glutamate analogues (five gliotoxic and two neurotoxic) on the growth and viability of four human glioma cell lines, one human medulloblastoma cell line, and one human sarcoma cell line. Aminoadipic acid and homocysteic acid predominantly affected the growth of two glioma cell lines in the presence of 4 mM glutamine. Phosphonobutyric acid predominantly affected the other two glioma cell lines and the medulloblastoma cell line in the presence of 4 mM glutamine. In medium containing no glutamine, all three analogues had marked effects on all the cell lines except the sarcoma cell line. These effects were dose dependent. We postulate that these results can in part be explained on the basis of metabolic compartmentalization.

  3. Human renal cell carcinoma induces a dendritic cell subset that uses T-cell crosstalk for tumor-permissive milieu alterations.

    PubMed

    Figel, Ainhoa-M; Brech, Dorothee; Prinz, Petra U; Lettenmeyer, Ulrike K; Eckl, Judith; Turqueti-Neves, Adriana; Mysliwietz, Josef; Anz, David; Rieth, Nicole; Muenchmeier, Niklas; Buchner, Alexander; Porubsky, Stefan; Siegert, Sabine I; Segerer, Stephan; Nelson, Peter J; Noessner, Elfriede

    2011-07-01

    Tissue dendritic cells (DCs) may influence the progression of renal cell carcinoma (RCC) by regulating the functional capacity of antitumor effector cells. DCs and their interaction with T cells were analyzed in human RCC and control kidney tissues. The frequency of CD209(+) DCs in RCCs was found to be associated with an unfavorable T(H)1 cell balance in the tissue and advanced tumor stages. The CD209(+) DCs in RCC were unusual because most of them co-expressed macrophage markers (CD14, CD163). The phenotype of these enriched-in-renal-carcinoma DCs (ercDCs) could be reiterated in vitro by carcinoma-secreted factors (CXCL8/IL-8, IL-6, and vascular endothelial growth factor). ErcDCs resembled conventional DCs in costimulatory molecule expression and antigen cross-presentation. They did not suppress cognate cytotoxic T-lymphocyte function and did not cause CD3ζ down-regulation, FOXP3 induction, or T-cell apoptosis in situ or in vitro; thus, they are different from classic myeloid-derived suppressor cells. ErcDCs secreted high levels of metalloproteinase 9 and used T-cell crosstalk to increase tumor-promoting tumor necrosis factor α and reduce chemokines relevant for T(H)1-polarized lymphocyte recruitment. This modulation of the tumor environment exerted by ercDCs suggests an immunologic mechanism by which tumor control can fail without involving cytotoxic T-lymphocyte inhibition. Pharmacologic targeting of the deviated DC differentiation could improve the efficacy of immunotherapy against RCC. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  4. High prevalence of side population in human cancer cell lines

    PubMed Central

    Boesch, Maximilian; Zeimet, Alain G.; Fiegl, Heidi; Wolf, Barbara; Huber, Julia; Klocker, Helmut; Gastl, Guenther

    2016-01-01

    Cancer cell lines are essential platforms for performing cancer research on human cells. We here demonstrate that, across tumor entities, human cancer cell lines harbor minority populations of putative stem-like cells, molecularly defined by dye extrusion resulting in the side population phenotype. These findings establish a heterogeneous nature of human cancer cell lines and argue for their stem cell origin. This should be considered when interpreting research involving these model systems. PMID:27226981

  5. A bioinformatics analysis of the cell line nomenclature.

    PubMed

    Sarntivijai, Sirarat; Ade, Alexander S; Athey, Brian D; States, David J

    2008-12-01

    Cell lines are used extensively in biomedical research, but the nomenclature describing cell lines has not been standardized. The problems are both linguistic and experimental. Many ambiguous cell line names appear in the published literature. Users of the same cell line may refer to it in different ways, and cell lines may mutate or become contaminated without the knowledge of the user. As a first step towards rationalizing this nomenclature, we created a cell line knowledgebase (CLKB) with a well-structured collection of names and descriptive data for cell lines cultured in vitro. The objectives of this work are: (i) to assist users in extracting useful information from biomedical text and (ii) to highlight the importance of standardizing cell line names in biomedical research. This CLKB contains a broad collection of cell line names compiled from ATCC, Hyper CLDB and MeSH. In addition to names, the knowledgebase specifies relationships between cell lines. We analyze the use of cell line names in biomedical text. Issues include ambiguous names, polymorphisms in the use of names and the fact that some cell line names are also common English words. Linguistic patterns associated with the occurrence of cell line names are analyzed. Applying these patterns to find additional cell line names in the literature identifies only a small number of additional names. Annotation of microarray gene expression studies is used as a test case. The CLKB facilitates data exploration and comparison of different cell lines in support of clinical and experimental research. The web ontology file for this cell line collection can be downloaded at http://www.stateslab.org/data/celllineOntology/cellline.zip.

  6. On the Ontology Based Representation of Cell Lines

    PubMed Central

    Ganzinger, Matthias; He, Shan; Breuhahn, Kai; Knaup, Petra

    2012-01-01

    Cell lines are frequently used as highly standardized and reproducible in vitro models for biomedical analyses and assays. Cell lines are distributed by cell banks that operate databases describing their products. However, the description of the cell lines' properties are not standardized across different cell banks. Existing cell line-related ontologies mostly focus on the description of the cell lines' names, but do not cover aspects like the origin or optimal growth conditions. The objective of this work is to develop an ontology that allows for a more comprehensive description of cell lines and their metadata, which should cover the data elements provided by cell banks. This will provide the basis for the standardized annotation of cell lines and corresponding assays in biomedical research. In addition, the ontology will be the foundation for automated evaluation of such assays and their respective protocols in the future. To accomplish this, a broad range of cell bank databases as well as existing ontologies were analyzed in a comprehensive manner. We identified existing ontologies capable of covering different aspects of the cell line domain. However, not all data fields derived from the cell banks' databases could be mapped to existing ontologies. As a result, we created a new ontology called cell culture ontology (CCONT) integrating existing ontologies where possible. CCONT provides classes from the areas of cell line identification, origin, cell line properties, propagation and tests performed. PMID:23144907

  7. EXAFS studies of prostate cancer cell lines

    NASA Astrophysics Data System (ADS)

    Czapla, J.; Kwiatek, W. M.; Lekki, J.; Kisiel, A.; Steininger, R.; Goettlicher, J.

    2013-04-01

    Sulphur plays a vital role in every human organism. It is known, that sulphur-bearing compounds, such as for example cysteine and glutathione, play critical roles in development and progression of many diseases. Any alteration in sulphur's biochemistry could become a precursor of serious pathological conditions. One of such condition is prostate cancer, the most frequently diagnosed malignancy in the western world and the second leading cause of cancer related death in men. The purpose of presented studies was to examine what changes occur in the nearest chemical environment of sulphur in prostate cancer cell lines in comparison to healthy cells. The Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy was used, followed by theoretical calculations. The results of preliminary analysis is presented.

  8. Establishment of hepatitis E virus infection-permissive and -non-permissive human hepatoma PLC/PRF/5 subclones.

    PubMed

    Shiota, Tomoyuki; Li, Tian-Cheng; Yoshizaki, Sayaka; Kato, Takanobu; Wakita, Takaji; Ishii, Koji

    2015-02-01

    PLC/PRF/5 cells show limited permissiveness, meaning that almost all subclones are permissive; however, some subclones do not exhibit permissiveness for hepatitis E virus (HEV) infection. In this study, the single-cell cloning of PLC/PRF/5 was performed and heterogeneous subclones characterized. Notably, the efficiency of intracellular virus replication did not correlate with the permissiveness for HEV infection. However, as well as binding permissive subclones, virus-like particles bound non-permissive subclones on various levels, suggesting that these subclones have some deficiencies in the attachment and entry steps of infection. Our data would be useful for investigating the HEV life cycle. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

  9. Generating Rho-0 Cells Using Mesenchymal Stem Cell Lines

    PubMed Central

    Fernández-Moreno, Mercedes; Hermida-Gómez, Tamara; Gallardo, M. Esther; Dalmao-Fernández, Andrea; Rego-Pérez, Ignacio; Garesse, Rafael

    2016-01-01

    Introduction The generation of Rho-0 cells requires the use of an immortalization process, or tumor cell selection, followed by culture in the presence of ethidium bromide (EtBr), incurring the drawbacks its use entails. The purpose of this work was to generate Rho-0 cells using human mesenchymal stem cells (hMSCs) with reagents having the ability to remove mitochondrial DNA (mtDNA) more safely than by using EtBr. Methodology Two immortalized hMSC lines (3a6 and KP) were used; 143B.TK-Rho-0 cells were used as reference control. For generation of Rho-0 hMSCs, cells were cultured in medium supplemented with each tested reagent. Total DNA was isolated and mtDNA content was measured by real-time polymerase chain reaction (PCR). Phenotypic characterization and gene expression assays were performed to determine whether 3a6 Rho-0 hMSCs maintain the same stem properties as untreated 3a6 hMSCs. To evaluate whether 3a6 Rho-0 hMSCs had a phenotype similar to that of 143B.TK-Rho-0 cells, in terms of reactive oxygen species (ROS) production, apoptotic levels and mitochondrial membrane potential (Δψm) were measured by flow cytometry and mitochondrial respiration was evaluated using a SeaHorse XFp Extracellular Flux Analyzer. The differentiation capacity of 3a6 and 3a6 Rho-0 hMSCs was evaluated using real-time PCR, comparing the relative expression of genes involved in osteogenesis, adipogenesis and chondrogenesis. Results The results showed the capacity of the 3a6 cell line to deplete its mtDNA and to survive in culture with uridine. Of all tested drugs, Stavudine (dt4) was the most effective in producing 3a6-Rho cells. The data indicate that hMSC Rho-0 cells continue to express the characteristic MSC cell surface receptor pattern. Phenotypic characterization showed that 3a6 Rho-0 cells resembled 143B.TK-Rho-0 cells, indicating that hMSC Rho-0 cells are Rho-0 cells. While the adipogenic capability was higher in 3a6 Rho-0 cells than in 3a6 cells, the osteogenic and chondrogenic

  10. A Neuroblastoma × Glioma Hybrid Cell Line with Morphine Receptors

    PubMed Central

    Klee, Werner A.; Nirenberg, Marshall

    1974-01-01

    A neuroblastoma × glioma hybrid cell line with well-developed neural properties was found that has high-affinity morphine receptors. The average cell contains approximately 3 × 106 receptors. In contrast, parent cells and other neuroblastoma or hybrid cell lines tested had few or no morphine receptors. PMID:4530316

  11. Quantitative analysis of cellular proteome alterations in human influenza A virus-infected mammalian cell lines.

    PubMed

    Vester, Diana; Rapp, Erdmann; Gade, Dörte; Genzel, Yvonne; Reichl, Udo

    2009-06-01

    Over the last years virus-host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2-D DIGE and nanoHPLC-nanoESI-MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2-D gels of the proteomes of uninfected and influenza-infected host cells, 16 quantitatively altered protein spots (at least +/-1.7-fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon-induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome-wide profiling of virus infection can provide insights into complexity and dynamics of virus-host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.

  12. DNA profiling and characterization of animal cell lines.

    PubMed

    Stacey, Glyn N; Byrne, Ed; Hawkins, J Ross

    2014-01-01

    The history of the culture of animal cell lines is littered with published and much unpublished experience with cell lines that have become switched, mislabelled, or cross-contaminated during laboratory handling. To deliver valid and good quality research and to avoid waste of time and resources on such rogue lines, it is vital to perform some kind of qualification for the provenance of cell lines used in research and particularly in the development of biomedical products. DNA profiling provides a valuable tool to compare different sources of the same cells and, where original material or tissue is available, to confirm the correct identity of a cell line. This chapter provides a review of some of the most useful techniques to test the identity of cells in the cell culture laboratory and gives methods which have been used in the authentication of cell lines.

  13. Neuronal cell lines as model dorsal root ganglion neurons

    PubMed Central

    Yin, Kathleen; Baillie, Gregory J

    2016-01-01

    Background Dorsal root ganglion neuron-derived immortal cell lines including ND7/23 and F-11 cells have been used extensively as in vitro model systems of native peripheral sensory neurons. However, while it is clear that some sensory neuron-specific receptors and ion channels are present in these cell lines, a systematic comparison of the molecular targets expressed by these cell lines with those expressed in intact peripheral neurons is lacking. Results In this study, we examined the expression of RNA transcripts in the human neuroblastoma-derived cell line, SH-SY5Y, and two dorsal root ganglion hybridoma cell lines, F-11 and ND7/23, using Illumina next-generation sequencing, and compared the results with native whole murine dorsal root ganglions. The gene expression profiles of these three cell lines did not resemble any specific defined dorsal root ganglion subclass. The cell lines lacked many markers for nociceptive sensory neurons, such as the Transient receptor potential V1 gene, but expressed markers for both myelinated and unmyelinated neurons. Global gene ontology analysis on whole dorsal root ganglions and cell lines showed similar enrichment of biological process terms across all samples. Conclusions This paper provides insights into the receptor repertoire expressed in common dorsal root ganglion neuron-derived cell lines compared with whole murine dorsal root ganglions, and illustrates the limits and potentials of these cell lines as tools for neuropharmacological exploration. PMID:27130590

  14. Type I and type II interferon responses in two human liver cell lines (Huh-7 and HuH6).

    PubMed

    Grünvogel, Oliver; Esser-Nobis, Katharina; Windisch, Marc P; Frese, Michael; Trippler, Martin; Bartenschlager, Ralf; Lohmann, Volker; Binder, Marco

    2016-03-01

    Most studies investigating the biology of Hepatitis C virus (HCV) have used the human hepatoma cell line Huh-7 or subclones thereof, as these are the most permissive cell lines for HCV infection and replication. Other cell lines also support replication of HCV, most notably the human hepatoblastoma cell line HuH6. HCV replication in cell culture is generally highly sensitive to interferons (IFNs) and differences in the IFN-mediated inhibition of virus replication may reflect alterations in the IFN-induced antiviral response inherent to different host cells. For example, HCV replication is highly sensitive to IFN-γ treatment in Huh-7, but not in HuH6 cells. In this study, we used microarray-based gene expression profiling to compare the response of Huh-7 and HuH6 cells to stimulation with IFN-α and IFN-γ. Furthermore, we determined whether the resistance of HCV replication in HuH6 cells can be linked to differences in the expression profile of IFN-regulated genes. Although both cells lines responded to IFNs with rapid changes in gene expression, thereby demonstrating functional type I and type II signaling pathways, differences were observed for a number of genes. Raw and normalized expression data have been deposited in GEO under accession number GSE68927.

  15. Analysis of renal cancer cell lines from two major resources enables genomics-guided cell line selection

    NASA Astrophysics Data System (ADS)

    Sinha, Rileen; Winer, Andrew G.; Chevinsky, Michael; Jakubowski, Christopher; Chen, Ying-Bei; Dong, Yiyu; Tickoo, Satish K.; Reuter, Victor E.; Russo, Paul; Coleman, Jonathan A.; Sander, Chris; Hsieh, James J.; Hakimi, A. Ari

    2017-05-01

    The utility of cancer cell lines is affected by the similarity to endogenous tumour cells. Here we compare genomic data from 65 kidney-derived cell lines from the Cancer Cell Line Encyclopedia and the COSMIC Cell Lines Project to three renal cancer subtypes from The Cancer Genome Atlas: clear cell renal cell carcinoma (ccRCC, also known as kidney renal clear cell carcinoma), papillary (pRCC, also known as kidney papillary) and chromophobe (chRCC, also known as kidney chromophobe) renal cell carcinoma. Clustering copy number alterations shows that most cell lines resemble ccRCC, a few (including some often used as models of ccRCC) resemble pRCC, and none resemble chRCC. Human ccRCC tumours clustering with cell lines display clinical and genomic features of more aggressive disease, suggesting that cell lines best represent aggressive tumours. We stratify mutations and copy number alterations for important kidney cancer genes by the consistency between databases, and classify cell lines into established gene expression-based indolent and aggressive subtypes. Our results could aid investigators in analysing appropriate renal cancer cell lines.

  16. Experimental Pathways towards Developing a Rotavirus Reverse Genetics System: Synthetic Full Length Rotavirus ssRNAs Are Neither Infectious nor Translated in Permissive Cells

    PubMed Central

    Richards, James E.; Desselberger, Ulrich; Lever, Andrew M.

    2013-01-01

    At present the ability to create rationally engineered mutant rotaviruses is limited because of the lack of a tractable helper virus-free reverse genetics system. Using the cell culture adapted bovine RV RF strain (G6P6 [1]), we have attempted to recover infectious RV by co-transfecting in vitro transcribed ssRNAs which are identical in sequence to the positive sense strand of each of the 11 dsRNA genomic segments of the RF strain. The RNAs were produced either from cDNAs cloned by a target sequence-independent procedure, or from purified double layered RV particles (DLPs). We have validated their translational function by in vitro synthesis of 35S-labelled proteins in rabbit reticulocyte lysates; all 11 proteins encoded by the RV genome were expressed. Transfection experiments with DLP- or cDNA-derived ssRNAs suggested that the RNAs do not act independently as mRNAs for protein synthesis, once delivered into various mammalian cell lines, and exhibit cytotoxicity. Transfected RNAs were not infectious since a viral cytopathic effect was not observed after infection of MA104 cells with lysates from transfected cells. By contrast, an engineered mRNA encoding eGFP was expressed when transfected under identical conditions into the same cell lines. Co-expression of plasmids encoding NSP2 and NSP5 using a fowlpox T7 polymerase recombinant virus revealed viroplasm-like structure formation, but this did not enable the translation of transfected RV ssRNAs. Attempts to recover RV from ssRNAs transcribed intracellularly from transfected cDNAs were also unsuccessful and suggested that these RNAs were also not translated, in contrast to successful translation from a transfected cDNA encoding an eGFP mRNA. PMID:24019962

  17. Immunoglobulin expression and synthesis by human haemic cell lines.

    PubMed Central

    Gordon, J; Hough, D; Karpas, A; Smith, J L

    1977-01-01

    Twenty-six human cell lines derived from a variety of lymphoid and non-lymphoid malignancies, were investigated for their immunological markers, with special reference to the class of immunoglobulin expressed. Twenty-five of the lines stained positively for surface immunoglobulin and IgD together with IgM proved to be the major immunoglobulin classes on these cells. Six of the lines were chosen for a study of their immunoglobulin synthesis patterns over an 18-h period and the immunoglobulin produced was analysed on SDS-polyacrylamide gel electrophoresis. Patterns obtained from the cell lines were similar to that from normal lymph node lymphocytes and differed markedly to plasma cells. Two of the cell lines had abnormal immunoglobulin synthesis patterns characterized as free light chains in one case. The cell lines are evaluated for their usefulness as models of immunoglobulin synthesis and analogues of normal and neoplastic states. PMID:608682

  18. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, Martha R.

    1989-01-01

    Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo[a]pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors.

  19. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, M.R.

    1985-07-01

    Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo(a)pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors. 2 tabs.

  20. Continuous human cell lines and method of making same

    SciTech Connect

    Stampfer, M.R.

    1989-02-28

    Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo[a]pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors. No Drawings

  1. Susceptibilities of 14 cell lines to bluetongue virus infection.

    PubMed Central

    Wechsler, S J; McHolland, L E

    1988-01-01

    The effect of bluetongue virus (BTV) infection was investigated in 14 cell lines. The cell lines included the following vertebrate cells: baby hamster kidney, African green monkey kidney (Vero), rabbit kidney, bovine kidney, canine kidney, bovine turbinate, bovine endothelium (CPAE), bighorn sheep tongue, equine dermis, gekko lung, rainbow trout gonad, and mouse fibroblast (L929); they also included the following invertebrate lines: mosquito and biting midge. Comparisons between the cell lines were made on the basis of time to observed cytopathic effects, titer in 50% tissue culture infectious doses, and titer in plaque-forming units. The CPAE cell line produced the highest BTV 50% tissue culture infectious dose of all cell lines tested. The Vero and L929 cells gave the most discrete plaques in plaque assays. Of the 14 cell lines tested, the CPAE cells were the most susceptible to both cell culture-adapted and animal source BTV. Bovine endothelial cells demonstrate significant potential as a cell culture system for BTV investigations. PMID:2853175

  2. Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line

    USDA-ARS?s Scientific Manuscript database

    The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...

  3. The EB66® cell line as a valuable cell substrate for MVA-based vaccines production.

    PubMed

    Léon, Arnaud; David, Anne-Laure; Madeline, Brice; Guianvarc'h, Laurence; Dureau, Elodie; Champion-Arnaud, Patrick; Hebben, Matthias; Huss, Thierry; Chatrenet, Benoît; Schwamborn, Klaus

    2016-11-21

    The selection of a cell substrate is a critical step for the development and manufacturing of a viral vaccine candidate. Several parameters such as cell susceptibility and permissiveness to the viral pathogens but also performance in terms of viral antigens quality and production yields are important considerations when identifying the ideal match between a viral vaccine and cell substrate. The modified vaccinia virus Ankara (MVA) is a replication-deficient viral vector that holds great promise as a vaccine platform, however only limited cell substrates have been tested or are available for industrialization. Here we evaluate the duck embryo-derived EB66® cell line as potential cell substrate for MVA production. To this end, we used two recombinant MVA constructs and demonstrated that EB66® cells are propagating the tested MVA viruses very efficiently, while preserving viral attenuation and transgene expression for up to 20 serial passages. Furthermore we developed upstream and downstream processes that enable industrialization of the virus production. In conclusion, we showed that EB66® cells can be used as potent cell substrate for MVA-based vaccines and represent therefore an attractive alternative for vaccine production.

  4. Re-characterization of established human retinoblastoma cell lines.

    PubMed

    Busch, Maike; Philippeit, Claudia; Weise, Andreas; Dünker, Nicole

    2015-03-01

    Retinoblastoma (RB) is the most common malignant intraocular childhood tumor. Forty years after their first description, in the present study, we re-characterized seven established retinoblastoma cell lines with regard to their RB1 mutation status, morphology, growth pattern, endogenous apoptosis levels, colony formation efficiency in soft agar and invasiveness and dissemination capacity in chick chorioallantoic membrane (CAM) assays. All RB cell lines predominantly resemble small epithelioid cells with little cytoplasm and large nucleus, which mainly grow in cell clusters, but sometimes form chain-like structures with incident loops or three-dimensional aggregates. We observed different growth rates for the different retinoblastoma cells investigated. RBL-30, RBL-13 and RBL 383 cells grew very slowly, whereas Y-79 cells grew fastest under our culture conditions. Apoptosis rates likewise differed with highest cell death levels in RB 383 and RB 355 and lowest in WERI-Rb1 and RBL-15. Contradicting former reports, six of the seven RB cell lines analyzed were able to form colonies in soft agarose after single cell seeding within 3 weeks of incubation. Upon inoculation of four out of seven RB cell lines on the dorsal CAM, GFP-positive cells were detectable in the ventral CAM and two RB cell lines caused tumor development, indicating their intravasation and dissemination potential. All RB cell lines exhibited the potential to extravasate from the capillary system after intravenous CAM injection. Our study provides valuable new details for future therapy-related retinoblastoma basic research in vitro.

  5. The pursuit of ES cell lines of domesticated ungulates

    USDA-ARS?s Scientific Manuscript database

    In contrast to differentiated cells, embryonic stem cells (ESC) maintain an undifferentiated state, have the ability to self-renew, and exhibit pluripotency, i.e., they can give rise to most if not all somatic cell types and to the germ cells, egg and sperm. These characteristics make ES cell lines...

  6. Comparative characteristics of three human embryonic stem cell lines.

    PubMed

    Lee, Jung Bok; Kim, Jin Mee; Kim, Sung Jong; Park, Jong Hyuk; Hong, Seok Ho; Roh, Sung Il; Kim, Moon Kyoo; Yoon, Hyun Soo

    2005-02-28

    Human embryonic stem (hES) cells have unique features including unlimited growth capacity, expression of specific markers, normal karyotypes and an ability to differentiate. Many investigators have tried to use hES cells for cell-based therapy, but there is little information about the properties of available hES cell lines. We compared the characteristics of three hES cell lines. The expression of SSEA-1, -3, -4, and APase, was examined by immunocytochemistry, and Oct-4 expression was analyzed by RT-PCR. Differentiation of the hES cells in vitro and in vivo led to the formation of embryoid bodies (EBs) or teratomas. We examined the expression of tissue-specific markers in the differentiated cells by semiquantitative RT-PCR, and the ability of each hES cell line to proliferate was measured by flow cytometry of DNA content and ELISA. The three hES cell lines were similar in morphology, marker expression, and teratoma formation. However there were significant differences (P < 0.05) between the differentiated cells formed by the different cell lines in levels of expression of tissue-specific markers such as renin, kallikrein, Glut-2, beta- and delta-globin, albumin, and alpha1-antitrypsin (alpha1-AT). The hES cell lines also differed in proliferative activity. Our observations should be useful in basic and clinical hES cell research.

  7. Differential Recruitment of Dendritic Cells Subsets to Lymph Nodes Correlates with a Protective or Permissive T-Cell Response during Leishmania (Viannia) Braziliensis or Leishmania (Leishmania) Amazonensis Infection.

    PubMed

    Carvalho, A K; Carvalho, K; Passero, L F D; Sousa, M G T; da Matta, V L R; Gomes, C M C; Corbett, C E P; Kallas, G E; Silveira, F T; Laurenti, M D

    2016-01-01

    Leishmania (L.) amazonensis (La) and L. (V.) braziliensis (Lb) are responsible for a large clinical and immunopathological spectrum in human disease; while La may be responsible for anergic disease, Lb infection leads to cellular hypersensitivity. To better understand the dichotomy in the immune response caused by these Leishmania species, we evaluated subsets of dendritic cells (DCs) and T lymphocyte in draining lymph nodes during the course of La and Lb infection in BALB/c mice. Our results demonstrated a high involvement of DCs in La infection, which was characterized by the greater accumulation of Langerhans cells (LCs); conversely, Lb infection led to an increase in dermal DCs (dDCs) throughout the infection. Considering the T lymphocyte response, an increase of effector, activated, and memory CD4(+) T-cells was observed in Lb infection. Interleukin- (IL-) 4- and IL-10-producing CD4(+)and CD8(+) T-cells were present in both La and Lb infection; however, interferon- (IFN-) γ-producing CD4(+)and CD8(+) T-cells were detected only in Lb infection. The results suggest that during Lb infection, the dDCs were the predominant subset of DCs that in turn was associated with the development of Th1 immune response; in contrast La infection was associated with a preferential accumulation of LCs and total blockage of the development of Th1 immune response.

  8. Trichloroethylene toxicity in a human hepatoma cell line

    SciTech Connect

    Thevenin, E.; McMillian, J.

    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.

  9. Managing clinical research permissions electronically

    PubMed Central

    Sanderson, Iain C; Obeid, Jihad S; Madathil, Kapil Chalil; Gerken, Katherine; Fryar, Katrina; Rugg, Daniel; Alstad, Colin E; Alexander, Randall; Brady, Kathleen T; Gramopadhye, Anand K; Moskowitz, Jay

    2014-01-01

    Background One mechanism to increase participation in research is to solicit potential research participants’ general willingness to be recruited into clinical trials. Such research permissions and consents typically are collected on paper upon patient registration. We describe a novel method of capturing this information electronically. Purpose The objective is to enable the collection of research permissions and informed consent data electronically to permit tracking of potential research participants’ interest in current and future research involvement and to provide a foundation for facilitating the research workflow. Methods The project involved systematic analysis focused on key areas, including existing business practices, registration processes, and permission collection workflows, and ascertaining best practices for presenting consent information to users via tablet technology and capturing permissions data. Analysis was followed by an iterative software development cycle with feedback from subject matter experts and users. Results An initial version of the software was piloted at one institution in South Carolina for a period of 1 year, during which consents and permission were collected during 2524 registrations of patients. The captured research permission data were transmitted to a clinical data warehouse. The software was later released as an open-source package that can be adopted for use by other institutions. Limitations There are significant ethical, legal, and informatics challenges that must be addressed at an institution to deploy such a system. We have not yet assessed the long-term impact of the system on recruitment of patients to clinical trials. Conclusions We propose that by improving the ability to track willing potential research participants, we can improve recruitment into clinical trials and, in the process, improve patient education by introducing multimedia to informed consent documents. PMID:23785065

  10. Electronic reserves: copyright and permissions

    PubMed Central

    Graves, Karen J.

    2000-01-01

    Electronic reserves present a new service option for libraries to provide needed materials during hours that the library is not open and to user groups located some distance from library collections. Possible changes to current copyright law and publishers permissions policies have delayed the development of electronic reserves in many libraries. This paper reviews the current state of electronic reserves materials in the publishing and library communities and presents the results of a survey of publishers to determine permissions policies for electronic materials. Issues of concern to both libraries and publishers are discussed. PMID:10658960

  11. Electronic reserves: copyright and permissions.

    PubMed

    Graves, K J

    2000-01-01

    Electronic reserves present a new service option for libraries to provide needed materials during hours that the library is not open and to user groups located some distance from library collections. Possible changes to current copyright law and publishers permissions policies have delayed the development of electronic reserves in many libraries. This paper reviews the current state of electronic reserves materials in the publishing and library communities and presents the results of a survey of publishers to determine permissions policies for electronic materials. Issues of concern to both libraries and publishers are discussed.

  12. Temperature-dependent growth rates and gene expression patterns of various medaka Oryzias latipes cell lines derived from different populations.

    PubMed

    Hirayama, Makoto; Mitani, Hiroshi; Watabe, Shugo

    2006-05-01

    Medaka Oryzias latipes has several geographically and genetically distinct populations. We examined temperature acclimation response in various medaka cell lines derived from different populations. Measurement of cell growth at various temperatures suggested that 15 degrees Celsius was the permissive growth temperature in all cell lines from the Northern Japanese and East Korean populations, but not in those from the Southern Japanese population and medaka-related species Oryzias celebensis, which inhabits a tropical zone. RT-PCR for 102 temperature-responsive genes, previously reported in other species, revealed that the accumulated mRNA level of a gene encoding HSP47 was lower at 25 degrees Celsius than at 33 degrees Celsius, and vice versa for 12 genes including IkappaBalpha and Rab-1c, in OLHNI-1 cell line from the Northern Japanese population. Further analysis by real-time PCR demonstrated that the accumulated mRNA levels of IkappaBalpha and Rab-1c in OLHNI-1 and OLSOK-e7 cell lines from the East Korean population were increased when the culture temperature was shifted from 33 to 15 degrees Celsius, but not in OLHdrR-e3 cell line from the Southern Japanese population. Since IkappaBalpha and Rab-1c are related to the NFkappaB cascade and endoplasmic reticulum-to-Golgi transport, respectively, it is inferred that immune responses and intracellular transport are possibly critical to temperature adaptation for medaka.

  13. Phenotype and Genotype of Pancreatic Cancer Cell Lines

    PubMed Central

    Deer, Emily L.; Gonzalez-Hernandez, Jessica; Coursen, Jill D.; Shea, Jill E.; Ngatia, Josephat; Scaife, Courtney L.; Firpo, Matthew A.; Mulvihill, Sean J.

    2009-01-01

    The dismal prognosis of pancreatic adenocarcinoma (PA) is due in part due to a lack of molecular information regarding disease development. Established cell lines remain a useful tool for investigating these molecular events. Here we present a review of available information on commonly used PA cell lines as a resource to help investigators select the cell lines most appropriate for their particular research needs. Information on clinical history, in vitro and in vivo growth characteristics, phenotypic characteristics, such as adhesion, invasion, migration and tumorigenesis, and genotypic status of commonly altered genes (KRAS, p53, p16, and SMAD4) was evaluated. Identification of both consensus and discrepant information in the literature suggests careful evaluation before selection of cell lines and attention be given to cell line authentication. PMID:20418756

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

    PubMed

    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.

  15. GREG cells, a dysferlin-deficient myogenic mouse cell line

    SciTech Connect

    Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S.; Morree, Antoine de; Pekkurnaz, Gulcin; Nagaraju, Kanneboyina; Zimmerberg, Joshua

    2012-01-15

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large ({approx} 200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority ({approx} 66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.

  16. Germ line, stem cells, and epigenetic reprogramming.

    PubMed

    Surani, M A; Durcova-Hills, G; Hajkova, P; Hayashi, K; Tee, W W

    2008-01-01

    The germ cell lineage has the unique attribute of generating the totipotent state. Development of blastocysts from the totipotent zygote results in the establishment of pluripotent primitive ectoderm cells in the inner cell mass of blastocysts, which subsequently develop into epiblast cells in postimplantation embryos. The germ cell lineage in mice originates from these pluripotent epiblast cells of postimplantation embryos in response to specific signals. Pluripotent stem cells and unipotent germ cells share some fundamental properties despite significant phenotypic differences between them. Additionally, early primordial germ cells can be induced to undergo dedifferentiation into pluripotent embryonic germ cells. Investigations on the relationship between germ cells and pluripotent stem cells may further elucidate the nature of the pluripotent state. Furthermore, comprehensive epigenetic reprogramming of the genome in early germ cells, including extensive erasure of epigenetic modifications, is a critical step toward establishment of totipotency. The mechanisms involved may be relevant for gaining insight into events that lead to reprogramming of somatic cells into pluripotent stem cells.

  17. Establishment of a Human Thymic Myoid Cell Line

    PubMed Central

    Wakkach, Abdel; Poea, Sandrine; Chastre, Eric; Gespach, Christian; Lecerf, Florence; De la Porte, Sabine; Tzartos, Socrates; Coulombe, Alain; Berrih-Aknin, Sonia

    1999-01-01

    The subset of myoid cells is a normal component of the thymic stroma. To characterize these cells, we immortalized stromal cells from human thymus by using a plasmid vector encoding the SV40 T oncogene. Among the eight cell lines obtained, one had myoid characteristics including desmin and troponin antigens. This new line was designated MITC (myoid immortalized thymic cells). These cells expressed both the fetal and adult forms of muscle acetylcholine receptor (AChR) at the mRNA level, as well as the myogenic transcription factor MyoD1. α-Subunit AChR protein expression was detected by flow cytometry and the AChR was functional in patch-clamp studies. In addition, AChR expression was down-modulated by myasthenia gravis sera or by monoclonal antibody anti-AChR on MITC line similarly to TE671 rhabdomyosarcoma cells, making the MITC line an interesting tool for AChR antigenic modulation experiments. Finally, the MITC line expressed LFA-3, produced several cytokines able to act on T cells, and protected total thymocytes from spontaneous apoptosis in vitro. These results are compatible with a role of thymic myoid cells in some steps of thymocyte development. Therefore MITC line appears to be a useful tool to investigate the physiological role of thymic myoid cells. PMID:10514405

  18. Deriving cell lines from zebrafish embryos and tumors.

    PubMed

    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.

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

    PubMed

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Functional calcium imaging in zebrafish lateral-line hair cells.

    PubMed

    Zhang, Q X; He, X J; Wong, H C; Kindt, K S

    2016-01-01

    Sensory hair-cell development, function, and regeneration are fundamental processes that are challenging to study in mammalian systems. Zebrafish are an excellent alternative model to study hair cells because they have an external auxiliary organ called the lateral line. The hair cells of the lateral line are easily accessible, which makes them suitable for live, function-based fluorescence imaging. In this chapter, we describe methods to perform functional calcium imaging in zebrafish lateral-line hair cells. We compare genetically encoded calcium indicators that have been used previously to measure calcium in lateral-line hair cells. We also outline equipment required for calcium imaging and compare different imaging systems. Lastly, we discuss how to set up optimal imaging parameters and how to process and visualize calcium signals. Overall, using these methods, in vivo calcium imaging is a powerful tool to examine sensory hair-cell function in an intact organism. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. When Permission Forms Work Best

    ERIC Educational Resources Information Center

    Zirkel, Perry A.

    2005-01-01

    Public schools routinely require permission or release forms for field trips and other activities of potential liability. The legal status of such forms varies, but they are generally considered neither rock-solid protection nor legally valueless in terms of immunity. This article presents a case involving a student who sustained bicycle injuries…

  2. When Permission Forms Work Best

    ERIC Educational Resources Information Center

    Zirkel, Perry A.

    2005-01-01

    Public schools routinely require permission or release forms for field trips and other activities of potential liability. The legal status of such forms varies, but they are generally considered neither rock-solid protection nor legally valueless in terms of immunity. This article presents a case involving a student who sustained bicycle injuries…

  3. The DNA methylation signature of human TCRαβ+CD4-CD8- double negative T cells reveals CG demethylation and a unique epigenetic architecture permissive to a broad stimulatory immune response.

    PubMed

    Renauer, Paul A; Coit, Patrick; Sawalha, Amr H

    2015-01-01

    T cell receptor (TCR) αβ+CD4-CD8- double negative T cells represent a rare T cell subset implicated in the pathogenesis of several autoimmune diseases. We investigated the DNA methylation signature of double negative T cells to gain insight into the epigenetic architecture of peripheral blood primary human double negative T cells compared to autologous CD4+ and CD8+ T cells. We identified 2984 CG sites across the genome with unique loss of DNA methylation in double negative T cells, and showed significant reduction in mRNA expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B. DNA methylation was increased in CD8A/CD8B and CD4 consistent with epigenetic repression of both the CD8 and CD4 genetic loci in double negative T cells. We show a consistent increase in non-CG methylation in double negative T cells, a finding suggestive of pluripotency. Network analyses indicate a strong relationship between double negative T cells and functions related to cell-cell interaction, cell adhesion, and cell activation pathways. Our data also suggest a robust pro-inflammatory epigenetic signature in double negative T cells, consistent with a transcriptional permissiveness in key inflammatory cytokines including IFNγ, IL-17F, IL-12B, IL-5, IL-18, TNFSF11 (RANKL), and TNFSF13B (BLYS or BAFF). These findings highlight an epigenetic basis for a role of double negative T cells in autoimmunity. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. The DNA methylation signature of human TCRαβ+CD4−CD8− double negative T cells reveals CG demethylation and a unique epigenetic architecture permissive to a broad stimulatory immune response

    PubMed Central

    Renauer, Paul A.; Coit, Patrick; Sawalha, Amr H.

    2014-01-01

    T cell receptor (TCR) αβ+CD4−CD8− double negative T cells represent a rare T cell subset implicated in the pathogenesis of several autoimmune diseases. We investigated the DNA methylation signature of double negative T cells to gain insight into the epigenetic architecture of peripheral blood primary human double negative T cells compared to autologous CD4+ and CD8+ T cells. We identified 2984 CG sites across the genome with unique loss of DNA methylation in double negative T cells, and showed significant reduction in mRNA expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B. DNA methylation was increased in CD8A/CD8B and CD4 consistent with epigenetic repression of both the CD8 and CD4 genetic loci in double negative T cells. We show a consistent increase in non-CG methylation in double negative T cells, a finding suggestive of pluripotency. Network analyses indicate a strong relationship between double negative T cells and functions related to cellcell interaction, cell adhesion, and cell activation pathways. Our data also suggest a robust pro-inflammatory epigenetic signature in double negative T cells, consistent with a transcriptional permissiveness in key inflammatory cytokines including IFNγ, IL-17F, IL-12B, IL-5, IL-18, TNFSF11 (RANKL), and TNFSF13B (BLYS or BAFF). These findings highlight an epigenetic basis for a role of double negative T cells in autoimmunity. PMID:25451162

  5. Generation of mesenchymal stem cell lines from murine bone marrow.

    PubMed

    Sreejit, P; Dilip, K B; Verma, R S

    2012-10-01

    Mesenchymal stem cells (MSC), because of their multipotency and ease of purification and amplification, are an ideal stem cell source for cell therapies. Bone-marrow-derived stem cells (BMSC) can be used to develop MSC-like immortalized cell lines with large proliferation and differentiation potentialities. Their immortalized status prevents the maintenance of MSC function and characters; this can be negated by modifying the isolation and maintenance protocol. Adult murine BMSC were isolated and maintained in media without additional growth factors together with passage-dependent reseeding following trypsinization. Cells maintained over 25 passages were considered as putative cell lines and characterized. The phenotypic and genotypic characteristics and multilineage differentiation potential of the cells were assessed by morphological, phenotypic, and molecular assays at various passages. The putative BMSC cell lines showed the characteristics of MSC and were able to maintain these characteristics, even after immortalization. The phenotypic data demonstrated difference among two cell lines; this was further validated by the difference in their multilineage differentiation potential following specific induction. More importantly, no changes were observed in the genotypic level in comparison with control cells, even after more than 50 passages. Our protocol thus advances the isolation and maintenance of BMSC and the development of putative BMSC cell lines that maintain characteristics of MSC, including multilineage differentiation potential, after more than 40 passages.

  6. Gastrin gene expression and regulation in rat islet cell lines.

    PubMed

    Brand, S J; Wang, T C

    1988-11-15

    Gastrin gene expression was observed in two permanent rat insulinoma (RIN) cell lines derived from a rat insulinoma. Gastrin expression was selective; highest expression was seen in a cell line which did not express other islet cell hormones. Gastrin mRNA transcription initiated from the same promoter as antral gastrin mRNA. DNA transfection studies with a gastrin chloramphenicol acetyltransferase chimeric gene showed higher expression in gastrin-expressing RIN cells than non-gastrin-expressing islet cells. This implies that gastrin-expressing RIN cells selectively express a trans-acting transcriptional activator which binds to cis-acting regulatory sequences within the 5'-flanking DNA sequence and first exon of the gastrin gene. The gastrin peptide precursor synthesized in these RIN cell lines is subject to the same repertoire of posttranslational modifications within the cell's secretory apparatus (endoproteolytic cleavage, tyrosine sulfation, and C-terminal amidation) as seen in antral G cells. Gastrin mRNA levels in these RIN cells were selectively increased by increasing the extracellular calcium concentration. Membrane depolarization also stimulated gastrin mRNA levels, probably through activation of voltage-sensitive calcium channels. Thus, these gastrin-expressing RIN cell lines provide permanent cell lines useful in analyzing the cellular regulation of gastrin gene expression.

  7. Quantitative methods to characterize morphological properties of cell lines.

    PubMed

    Mancia, Annalaura; Elliott, John T; Halter, Michael; Bhadriraju, Kiran; Tona, Alessandro; Spurlin, Tighe A; Middlebrooks, Bobby L; Baatz, John E; Warr, Gregory W; Plant, Anne L

    2012-07-01

    Descriptive terms are often used to characterize cells in culture, but the use of nonquantitative and poorly defined terms can lead to ambiguities when comparing data from different laboratories. Although recently there has been a good deal of interest in unambiguous identification of cell lines via their genetic markers, it is also critical to have definitive, quantitative metrics to describe cell phenotypic characteristics. Quantitative metrics of cell phenotype will aid the comparison of data from experiments performed at different times and in different laboratories where influences such as the age of the population and differences in culture conditions or protocols can potentially affect cellular metabolic state and gene expression in the absence of changes in the genetic profile. Here, we present examples of robust methodologies for quantitatively assessing characteristics of cell morphology and cell-cell interactions, and of growth rates of cells within the population. We performed these analyses with endothelial cell lines derived from dolphin, bovine and human, and with a mouse fibroblast cell line. These metrics quantify some characteristics of these cells lines that clearly distinguish them from one another, and provide quantitative information on phenotypic changes in one of the cell lines over large number of passages.

  8. Toll-Like Receptor 2 Ligation Enhances HIV-1 Replication in Activated CCR6+ CD4+ T Cells by Increasing Virus Entry and Establishing a More Permissive Environment to Infection

    PubMed Central

    Bolduc, Jean-François; Ouellet, Michel; Hany, Laurent

    2016-01-01

    ABSTRACT In this study, we investigated the effect of Toll-like receptor 2 (TLR2) ligation on the permissiveness of activated CD4+ T cells to HIV-1 infection by focusing our experiments on the relative susceptibility of cell subsets based on their expression of CCR6. Purified primary human CD4+ T cells were first subjected to a CD3/CD28 costimulation before treatment with the TLR2 agonist Pam3CSK4. Finally, cells were inoculated with R5-tropic HIV-1 particles that permit us to study the effect of TLR2 triggering on virus production at both population and single-cell levels. We report here that HIV-1 replication is augmented in CD3/CD28-costimulated CCR6+ CD4+ T cells upon engagement of the cell surface TLR2. Additional studies indicate that a higher virus entry and polymerization of the cortical actin are seen in this cell subset following TLR2 stimulation. A TLR2-mediated increase in the level of phosphorylated NF-κB p65 subunit was also detected in CD3/CD28-costimulated CCR6+ CD4+ T cells. We propose that, upon antigenic presentation, an engagement of TLR2 acts specifically on CCR6+ CD4+ T cells by promoting virus entry in an intracellular milieu more favorable for productive HIV-1 infection. IMPORTANCE Following primary infection, HIV-1 induces an immunological and structural disruption of the gut mucosa, leading to bacterial translocation and release of microbial components in the bloodstream. These pathogen-derived constituents include several agonists of Toll-like receptors that may affect gut-homing CD4+ T cells, such as those expressing the chemokine receptor CCR6, which are highly permissive to HIV-1 infection. We demonstrate that TLR2 ligation in CD3/CD28-costimulated CCR6+ CD4+ T cells leads to enhanced virus production. Our results highlight the potential impact of bacterial translocation on the overall permissiveness of CCR6+ CD4+ T cells to productive HIV-1 infection. PMID:27928019

  9. Toll-Like Receptor 2 Ligation Enhances HIV-1 Replication in Activated CCR6+ CD4+ T Cells by Increasing Virus Entry and Establishing a More Permissive Environment to Infection.

    PubMed

    Bolduc, Jean-François; Ouellet, Michel; Hany, Laurent; Tremblay, Michel J

    2017-02-15

    In this study, we investigated the effect of Toll-like receptor 2 (TLR2) ligation on the permissiveness of activated CD4(+) T cells to HIV-1 infection by focusing our experiments on the relative susceptibility of cell subsets based on their expression of CCR6. Purified primary human CD4(+) T cells were first subjected to a CD3/CD28 costimulation before treatment with the TLR2 agonist Pam3CSK4. Finally, cells were inoculated with R5-tropic HIV-1 particles that permit us to study the effect of TLR2 triggering on virus production at both population and single-cell levels. We report here that HIV-1 replication is augmented in CD3/CD28-costimulated CCR6(+) CD4(+) T cells upon engagement of the cell surface TLR2. Additional studies indicate that a higher virus entry and polymerization of the cortical actin are seen in this cell subset following TLR2 stimulation. A TLR2-mediated increase in the level of phosphorylated NF-κB p65 subunit was also detected in CD3/CD28-costimulated CCR6(+) CD4(+) T cells. We propose that, upon antigenic presentation, an engagement of TLR2 acts specifically on CCR6(+) CD4(+) T cells by promoting virus entry in an intracellular milieu more favorable for productive HIV-1 infection. Following primary infection, HIV-1 induces an immunological and structural disruption of the gut mucosa, leading to bacterial translocation and release of microbial components in the bloodstream. These pathogen-derived constituents include several agonists of Toll-like receptors that may affect gut-homing CD4(+) T cells, such as those expressing the chemokine receptor CCR6, which are highly permissive to HIV-1 infection. We demonstrate that TLR2 ligation in CD3/CD28-costimulated CCR6(+) CD4(+) T cells leads to enhanced virus production. Our results highlight the potential impact of bacterial translocation on the overall permissiveness of CCR6(+) CD4(+) T cells to productive HIV-1 infection. Copyright © 2017 American Society for Microbiology.

  10. Cell line models for differentiation: preadipocytes and adipocytes.

    PubMed

    Poulos, Sylvia P; Dodson, Michael V; Hausman, Gary J

    2010-10-01

    In vitro models have been invaluable in determining the mechanisms involved in adipocyte proliferation, differentiation, adipokine secretion and gene/protein expression. The cells presently available for research purposes all have unique advantages and disadvantages that one should be aware of when selecting cells. Established cell lines, such as 3T3-L1 cells, are easier and less costly to use than freshly isolated cells, even though freshly isolated cells allow for various comparisons such as the in vitro evaluation of different in vivo conditions that may not be possible using cell lines. Moreover, stem cells, transdifferentiated cells or dedifferentiated cells are relatively new cell models being evaluated for the study of adipocyte regulation and physiology. The focus of this brief review is to highlight similarities and differences in adipocyte models to aid in appropriate model selection and data interpretation for successful advancement of our understanding of adipocyte biology.

  11. Chikungunya Virus Infection of Cell Lines: Analysis of the East, Central and South African Lineage

    PubMed Central

    Wikan, Nitwara; Sakoonwatanyoo, Prirayapak; Ubol, Sukathida; Yoksan, Sutee; Smith, Duncan R.

    2012-01-01

    Chikungunya virus (CHIKV) is a re-emerging mosquito borne alphavirus that has caused large scale epidemics in the countries around the Indian Ocean, as well as leading to autochthonous transmission in some European countries. The transmission of the disease has been driven by the emergence of an African lineage of CHIKV with enhanced transmission and dissemination in Aedes mosquito hosts. Two main genotypes of this lineage have been circulating, characterized by the presence of a substitution of a valine for an alanine at position 226 of the E1 protein. The outbreak, numbering in millions of cases in the infected areas, has been associated with increasing numbers of cases with non-classical presentation including encephalitis and meningitis. This study sought to compare the original Ross strain with two isolates from the recent outbreak of chikungunya fever in respect of infectivity and the induction of apoptosis in eight mammalian cell lines and two insect cell lines, in addition to generating a comprehensive virus production profile for one of the newer isolates. Results showed that in mammalian cells there were few differences in either tropism or pathogenicity as assessed by induction of apoptosis with the exception of Hela cells were the recent valine isolate showed less infectivity. The Aedes albopictus C6/36 cell line was however significantly more permissive for both of the more recent isolates than the Ross strain. The results suggest that the increased infectivity seen in insect cells derives from an evolution of the CHIKV genome not solely associated with the E1:226 substitution. PMID:22299053

  12. Failure in activation of the canonical NF-κB pathway by human T-cell leukemia virus type 1 Tax in non-hematopoietic cell lines

    SciTech Connect

    Mizukoshi, Terumi; Komori, Hideyuki; Mizuguchi, Mariko; Abdelaziz, Hussein; Hara, Toshifumi; Higuchi, Masaya; Tanaka, Yuetsu; Ohara, Yoshiro; Funato, Noriko; Fujii, Masahiro; Nakamura, Masataka

    2013-09-01

    Human T-cell leukemia virus type 1 (HTLV-1) Tax (Tax1) plays crucial roles in leukemogenesis in part through activation of NF-κB. In this study, we demonstrated that Tax1 activated an NF-κB binding (gpκB) site of the gp34/OX40 ligand gene in a cell type-dependent manner. Our examination showed that the gpκΒ site and authentic NF-κB (IgκB) site were activated by Tax1 in hematopoietic cell lines. Non-hematopoietic cell lines including hepatoma and fibroblast cell lines were not permissive to Tax1-mediated activation of the gpκB site, while the IgκB site was activated in those cells in association with binding of RelB. However RelA binding was not observed in the gpκB and IgκB sites. Our results suggest that HTLV-1 Tax1 fails to activate the canonical pathway of NF-κB in non-hematopoietic cell lines. Cell type-dependent activation of NF-κB by Tax1 could be associated with pathogenesis by HTLV-1 infection. - Highlights: • HTLV-1 Tax1 does not activate RelA of NF-κB in non-hematopoietic cell lines. • Tax1 activates the NF-κB non-canonical pathway in non-hematopoietic cell lines. • Tax1 does not induce RelA nuclear translocation in those cell lines, unlike TNFα. • The OX40L promoter κB site is activated by ectopic, but not endogenous, RelA.

  13. Antineoplastic activity of rinvanil and phenylacetylrinvanil in leukaemia cell lines

    PubMed Central

    LUVIANO, AXEL; AGUIÑIGA-SÁNCHEZ, ITZEN; DEMARE, PATRICIA; TIBURCIO, REYNALDO; LEDESMA-MARTÍNEZ, EDGAR; SANTIAGO-OSORIO, EDELMIRO; REGLA, IGNACIO

    2014-01-01

    In the search for novel chemotherapeutic agents for cancer treatment, capsaicin has been shown to inhibit proliferation and induce apoptosis in various types of cancer cell line, including leukaemia cell lines. The capsaicin analogues, rinvanil and phenylacetylrinvanil (PhAR), share a binding affinity for vanilloid receptors and may have biological activities similar to capsaicin; however, their anticancer potential has not yet been reported. This study analyses the antineoplastic activities of rinvanil and PhAR in leukaemia versus normal cells. P388, J774 and WEHI-3 leukaemia cell lines, as well as mouse bone marrow mononuclear cells, were cultured with varying concentrations of rinvanil and PhAR. Following this, proliferation and apoptosis were determined by the sulforhodamine B (SRB) assay and DNA ladder. Cultured leukaemia cell lines and mouse bone marrow mononuclear cells demonstrated a dose-dependent inhibition of proliferation, while non-diseased cells were less sensitive to the cytotoxic effect of capsaicin, rinvanil and PhAR. Rinvanil and PhAR also induced apoptosis in leukaemia cell lines but not in bone marrow. Given the lower IC50 values for apoptosis induction in leukaemia cells compared with that of normal cells, PhAR is a promising selective anticancer agent. PMID:24765194

  14. Generation and sequencing of pulmonary carcinoid tumor cell lines.

    PubMed

    Asiedu, Michael K; Thomas, Charles F; Tomaszek, Sandra C; Peikert, Tobias; Sanyal, Bharati; Sutor, Shari L; Aubry, Marie-Christine; Li, Peter; Wigle, Dennis A

    2014-12-01

    Pulmonary carcinoid tumors account for approximately 5% of all lung malignancies in adults, and comprise 30% of all carcinoid tumors. There are limited reagents available to study these rare tumors, and consequently no major advances have been made for patient treatment. We report the generation and characterization of human pulmonary carcinoid tumor cell lines to study underlying biology, and to provide models for testing novel chemotherapeutic agents. Tissue was harvested from three patients with primary pulmonary typical carcinoid tumors undergoing surgical resection. The tumor was dissociated and plated onto dishes in culture media. The established cell lines were characterized by immunohistochemistry, Western blotting, and cell proliferation assays. Tumorigenicity was confirmed by soft agar growth and the ability to form tumors in a mouse xenograft model. Exome and RNA sequencing of patient tumor samples and cell lines was performed using standard protocols. Three typical carcinoid tumor lines grew as adherent monolayers in vitro, expressed neuroendocrine markers consistent with the primary tumor, and formed colonies in soft agar. A single cell line produced lung tumors in nude mice after intravenous injection. Exome and RNA sequencing of this cell line showed lineage relationship with the primary tumor, and demonstrated mutations in a number of genes related to neuronal differentiation. Three human pulmonary typical carcinoid tumor cell lines have been generated and characterized as a tool for studying the biology and novel treatment approaches for these rare tumors.

  15. Growth of Murine Cytomegalovirus in Various Cell Lines

    PubMed Central

    Kim, Kwang Soo; Carp, Richard I.

    1971-01-01

    Murine cytomegalovirus (MCMV) was capable of infecting and replicating in both primary and continuous cell lines obtained from various species. In African green monkey kidney (BSC-1) cells, primary rabbit kidney cells, and baby hamster kidney (BHK-21) cells, there were cytopathic effects (CPE) and virus replication upon initial exposure of cells to virus. In primary fetal sheep brain (FSB) cells, L cells, and rabbit kidney (RK-13) cells, it was necessary to subculture the infected cells one or more times before appearance of CPE and replication of virus. In the case of the infected FSB cultures, it was found that the virus effect could be induced if subculturing were accomplished by trypsinization but did not occur if cells were subcultured by scraping. FSB-grown virus replicated better in FSB than in mouse embryo fibroblast (MEF) cells. The CPE produced in all of the above cell lines was similar to that observed in MEF infected with MCMV. The virus grown in different cell lines was completely neutralized when mixed with several reference sera prepared in rabbits or mice. The populations of virions released from infected MEF and FSB cells were compared by isopycnic centrifugation in potassium tartrate, and no differences were revealed in the buoyant densities of the populations. Human embryonic brain cells, human embryonic kidney cells, a human lung fibroblast cell strain (WI-38), HeLa, and Hep-2 were not susceptible to MCMV. PMID:4327583

  16. Investigation of Radiosensitivity Gene Signatures in Cancer Cell Lines

    PubMed Central

    Hall, John S.; Iype, Rohan; Senra, Joana; Taylor, Janet; Armenoult, Lucile; Oguejiofor, Kenneth; Li, Yaoyong; Stratford, Ian; Stern, Peter L.; O’Connor, Mark J.; Miller, Crispin J.; West, Catharine M. L.

    2014-01-01

    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. PMID:24466029

  17. Cytotoxicity of gemcitabine enhanced by polyphenolics from Aronia melanocarpa in pancreatic cancer cell line AsPC-1.

    PubMed

    Thani, Noor Azela Abdullah; Keshavarz, Sholeh; Lwaleed, Bashir A; Cooper, Alan J; Rooprai, Harcharan K

    2014-11-01

    Extending work with brain tumours, the hypothesis that micronutrients may usefully augment anticancer regimens, chokeberry (Aronia melanocarpa) extract was tested to establish whether it has pro-apoptotic effects in AsPC-1, an established human pancreatic cell line, and whether it potentiates cytotoxicity in combination with gemcitabine. Pancreatic cancer was chosen as a target, as its prognosis remains dismal despite advances in therapy. An MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was used to assess the growth of the single pancreatic cancer cell line AsPC-1, alone and in comparison or combination with gemcitabine. This was backed up by flow cytometric DRAQ7 cell viability analysis. TUNEL assays were also carried out to investigate pro-apoptotic properties as responsible for the effects of chokeberry extract. Chokeberry extract alone and its IC75 value (1 µg/mL) in combination with gemcitabine were used to assess the growth of the AsPC-1 cell line. Gemcitabine in combination with chokeberry extract was more effective than gemcitabine alone. TUNEL assays showed apoptosis to be a mechanism occurring at 1 µg/mL concentration of chokeberry, with apoptotic bodies detected by both colourimetric and fluorometric methods. The implication of this study, using single cancer cell line, is that chemotherapy (at least with gemcitabine) might be usefully augmented with the use of micronutrients such as chokeberry extract. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  18. Regulation of germ line stem cell homeostasis

    PubMed Central

    Garcia, T.X.; Hofmann, M.C.

    2015-01-01

    Mammalian spermatogenesis is a complex process in which spermatogonial stem cells of the testis (SSCs) develop to ultimately form spermatozoa. In the seminiferous epithelium, SSCs self-renew to maintain the pool of stem cells throughout life, or they differentiate to generate a large number of germ cells. A balance between SSC self-renewal and differentiation is therefore essential to maintain normal spermatogenesis and fertility. Stem cell homeostasis is tightly regulated by signals from the surrounding microenvironment, or SSC niche. By physically supporting the SSCs and providing them with these extrinsic molecules, the Sertoli cell is the main component of the niche. Earlier studies have demonstrated that GDNF and CYP26B1, produced by Sertoli cells, are crucial for self-renewal of the SSC pool and maintenance of the undifferentiated state. Down-regulating the production of these molecules is therefore equally important to allow germ cell differentiation. We propose that NOTCH signaling in Sertoli cells is a crucial regulator of germ cell fate by counteracting these stimulatory factors to maintain stem cell homeostasis. Dysregulation of this essential niche component can lead by itself to sterility or facilitate testicular cancer development.

  19. Radiosensitivity of hepatoma cell lines and human normal liver cell lines exposed to 12C6+ ions

    NASA Astrophysics Data System (ADS)

    Jing, X.; Yang, J.; Li, W.; Guo, C.; Dang, B.; Wang, J.; Zhou, L.; Wei, W.; Gao, Q.

    AIM To investigate the radiosensitivity of hepatoma cell lines and human normal liver cell lines METHODS Accelerated carbon ions by heavy ion research facility in Lanzhou HIRFL have high LET We employed it to study the radiosensitivity of hepatoma cell lines SMMC-7721 and human normal liver cell lines L02 using premature chromosome condensation technique PCC Cell survive was documented by a colony assay Chromatid breaks were measured by counting the number of chromatid breaks and isochromatid breaks immediately after prematurely chromosome condensed by Calyculin-A RESULTS The survival curve of the two cell lines presented a good linear relationship and the survival fraction of L02 is higher than that of SMMC-7721 Additionally the two types of G 2 phase chromosome breaks chromatid breaks and isochromatid breaks of L02 are lower than that of SMMC-7721 CONCLUSION Human normal liver cell line have high radioresistance than that of hepatoma cell line It imply that it is less damage to normal organs when radiotherapy to hepatoma

  20. Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure.

    PubMed

    Yang, Ying; Adachi, Katsuyuki; Sheridan, Megan A; Alexenko, Andrei P; Schust, Danny J; Schulz, Laura C; Ezashi, Toshihiko; Roberts, R Michael

    2015-05-05

    Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling. Here, we report the acquisition of a unique stem cell phenotype by both human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) in response to transient (24-36 h) exposure to bone morphogenetic protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and FGF2 (PD173074), followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. The self-renewing cell lines stain weakly for CDX2 and strongly for NANOG, can be propagated clonally on either Matrigel or gelatin, and are morphologically distinct from human PSC progenitors on either substratum but still meet standard in vitro criteria for pluripotency. They form well-differentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG, LEFTY1, and LEFTY2). In nonconditioned medium lacking FGF2, the colonies spontaneously differentiated along multiple lineages, including trophoblast. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast, and especially syncytiotrophoblast, whereas an A83-01/PD173074 combination favored increased expression of HLA-G, a marker of extravillous trophoblast. Together, these data suggest that the cell lines exhibit totipotent potential and that BMP4 can prime human PSCs to a self-renewing alternative state permissive for trophoblast development. The results may have implications for regulation of lineage decisions in the early embryo.

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

    PubMed

    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.

  2. Development and characterization of a largemouth bass cell line.

    PubMed

    Getchell, Rodman G; Groocock, Geoffrey H; Cornwell, Emily R; Schumacher, Vanessa L; Glasner, Lindsay I; Baker, Barry J; Frattini, Stephen A; Wooster, Gregory A; Bowser, Paul R

    2014-09-01

    Abstract The development and characterization of a new cell line, derived from the ovary of Largemouth Bass Micropterus salmoides, is described. Gonad tissue was collected from Largemouth Bass that were electrofished from Oneida Lake, New York. The tissue was processed and grown in culture flasks at approximately 22°C for more than 118 passages during an 8-year period from 2004 to 2011. The identity of these cells as Largemouth Bass origin was confirmed by sequencing a portion of the cytochrome b gene. Growth rate at three different temperatures was documented. The cell line was susceptible to Largemouth Bass virus (LMBV) and its replication was compared with that of Bluegill Lepomis macrochirus fry (BF-2), one of the cell lines recommended for LMBV isolation by the American Fisheries Society Fish Health Section Blue Book. Quantitative PCR results from the replication trial showed the BF-2 cell line produced approximately 10-fold more LMBV copies per cell than the new Largemouth Bass cell line after 6 d, while the titration assay showed similar quantities in each cell line after 1 week. Received February 18, 2014; accepted April 16, 2014.

  3. [Decontamination of continual cell lines spontaneously infected with mycoplasmas].

    PubMed

    Machatková, M; Jurmanová, K; Snejdar, V

    1986-07-01

    The continual cell lines of bovine kidneys MDBK and AUBEK, and porcine kidneys RPD and IBRS, spontaneously infected with Mycoplasma arginini and Acholeplasma laidlawii, were decontaminated by the method of selective elimination. Two elimination procedures were modified to be used for the decontamination: one based on the reduction of infection by the light treatment of the cultures, the other based on the selection of mycoplasma-free cell population through cell clonation. On the basis of a long-continued control of the cell clones a methodical procedure of the preparation of mycoplasma-free cell lines was worked out.

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

    PubMed

    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.

  5. Sensitivity of Hodgkin's lymphoma cell lines to the cell cycle inhibitor roscovitine.

    PubMed

    Foell, Juergen L; Max, Daniela; Giersberg, Corinna; Korholz, Dieter; Staege, Martin S

    2008-01-01

    The prognosis of patients with Hodgkin's lymphoma (HL) has improved in recent decades. However, not all patients can be cured and the development of alternative treatment strategies is necessary. Gene expression in HL cell lines was analyzed using DNA microarrays and both conventional and quantitative reverse transcriptase-polymerase chain reaction. Sensitivity of HL cell lines to the cell cycle inhibitor roscovitine was assessed in vitro. All HL cell lines express high levels of cyclin D2. Treatment of HL cells with roscovitine induced cell death in some cell lines whereas other cell lines were resistant to roscovitine. Roscovitine-sensitive cell lines were characterized by expression of T-cell markers and expressed high levels of the unusual cytokine interleukin-26. Roscovitine is a cytotoxic drug for a subpopulation of HL cells and might be an interesting agent for the treatment of patients with HL.

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

    PubMed

    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

  7. Programmed Genetic Instability: A Tumor-Permissive Mechanism for Maintaining the Evolvability of Higher Species through Methylation-Dependent Mutation of DNA Repair Genes in the Male Germ Line

    PubMed Central

    Zhao, Yongzhong

    2008-01-01

    Tumor suppressor genes are classified by their somatic behavior either as caretakers (CTs) that maintain DNA integrity or as gatekeepers (GKs) that regulate cell survival, but the germ line role of these disease-related gene subgroups may differ. To test this hypothesis, we have used genomic data mining to compare the features of human CTs (n = 38), GKs (n = 36), DNA repair genes (n = 165), apoptosis genes (n = 622), and their orthologs. This analysis reveals that repair genes are numerically less common than apoptosis genes in the genomes of multicellular organisms (P < 0.01), whereas CT orthologs are commoner than GK orthologs in unicellular organisms (P < 0.05). Gene targeting data show that CTs are less essential than GKs for survival of multicellular organisms (P < 0.0005) and that CT knockouts often permit offspring viability at the cost of male sterility. Patterns of human familial oncogenic mutations confirm that isolated CT loss is commoner than is isolated GK loss (P < 0.00001). In sexually reproducing species, CTs appear subject to less efficient purifying selection (i.e., higher Ka/Ks) than GKs (P = 0.000003); the faster evolution of CTs seems likely to be mediated by gene methylation and reduced transcription-coupled repair, based on differences in dinucleotide patterns (P = 0.001). These data suggest that germ line CT/repair gene function is relatively dispensable for survival, and imply that milder (e.g., epimutational) male prezygotic repair defects could enhance sperm variation—and hence environmental adaptation and speciation—while sparing fertility. We submit that CTs and repair genes are general targets for epigenetically initiated adaptive evolution, and propose a model in which human cancers arise in part as an evolutionarily programmed side effect of age- and damage-inducible genetic instability affecting both somatic and germ line lineages. PMID:18535014

  8. Establishment and characterization of a chicken mononuclear cell line.

    PubMed

    Qureshi, M A; Miller, L; Lillehoj, H S; Ficken, M D

    1990-11-01

    A new chicken mononuclear cell line (MQ-NCSU) has been established. The starting material used to initiate this cell line was a transformed spleen from a female Dekalb XL chicken which had been experimentally challenged with the JM/102W strain of the Marek's disease virus. After homogenization, a single cell suspension of splenic cells was cultured using L.M. Hahn medium supplemented with 10 microM 2-mercaptoethanol. Under these culture conditions, a rapidly proliferating cell was observed and then expanded after performing limiting dilution cultures. These cells were moderately adherent and phagocytic for sheep red blood cells and Salmonella typhimurium. When tested against a panel of monoclonal antibodies (mAb) using the flow cytometry, MQ-NCSU cells stained readily with anti-chicken monocyte specific (K-1) mAb but did not stain with mAb detecting T-helper, T-cytotoxic/suppressor, and NK cells. MQ-NCSU cells expressed very high levels of Ia antigens and transferrin receptors. In addition, cell-free supernatant obtained from MQ-NCSU culture contained a factor which exhibited cytolytic activity against tumor cell targets. Based on their cultural, morphological, and functional characteristics and mAb reactivity profile, we conclude that MQ-NCSU cell line represents a malignantly-transformed cell which shares features characteristic of cells of the mononuclear phagocyte lineage.

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

    PubMed

    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.

  10. Establishment of shrimp cell lines: perception and orientation.

    PubMed

    Jayesh, P; Seena, Jose; Singh, I S Bright

    2012-09-01

    Development of continuous shrimp cell lines for effective investigation on shrimp viruses remains elusive with an arduous history of over 25 years. Despite presenting challenges to researchers in developing a cell line, the billion dollar aquaculture industry is under viral threat. Advances in molecular biology and various gene transfer technologies for immortalization of cells have resulted in the development of hundreds of cell lines from insects and mammals, but yet not a single cell line has been developed from shrimp and other marine invertebrates. Though improved growth and longevity of shrimp cells in vitro could be achieved by using modified growth media this did not make any leap to spontaneous transformation; probably due to the fact that shrimp cells inhibited neoplastic transformations. Oncogenic induction and immortalization are considered as the possible ways, and an exclusive medium for shrimp cell culture and an appropriate mode of transformation are crucial. In this review status of shrimp cell line development and its future orientation are discussed.

  11. Induction of apoptosis by opium in some tumor cell lines.

    PubMed

    Khaleghi, M; Farsinejad, A; Dabiri, S; Asadikaram, G

    2016-09-30

    The current study is aimed at investigation of the opium effects on the apoptosis of different cell lines in culture medium and compares such effects with one another. The study is carried out on over 8 cell lines (AA8, AGS, Hela, HepG2, MCF7, N2a, PC12, WEHI). A 2.86 x 10-4 g/ml opium concentration was prepared and added to the culture medium of the cell lines for 48 hours. Cytotoxicity was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic effect of opium on the cell lines was analyzed by Annexin-PI test. Opium with concentration of 2.86 x 10-4 g/ml in 48 hours significantly induces apoptosis in certain cell lines (i.e. AA8, N2a, WEHI), apoptosis and necrosis in some others (i.e. Hela, HepG2, MCF7, and PC12), and also solely necrosis in the AGS cell line. One could infer that the usage of opium with different levels in different tissues leads to certain disorders in some tissues and may have therapeutic effects under distinctive conditions (i.e. unchecked growth of cells) as confirmed by the results.

  12. Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: improving the efficiency of cell line generation.

    PubMed

    Porter, Alison J; Racher, Andrew J; Preziosi, Richard; Dickson, Alan J

    2010-01-01

    Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide-ranging transfectant population. This identification process is on the critical path for first-in-human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One-hundred and seventy-five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines.

  13. Natural Killer Cells for Immunotherapy - Advantages of the NK-92 Cell Line over Blood NK Cells.

    PubMed

    Klingemann, Hans; Boissel, Laurent; Toneguzzo, Frances

    2016-01-01

    Natural killer (NK) cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells from a patient's blood since they represent only 10% of the lymphocytes and are often dysfunctional. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T cells to prevent graft-versus-host reactions. Cytotoxic cell lines have been established from patients with clonal NK-cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. Except for the NK-92 cell line, though, none of the other six known NK cell lines has consistently and reproducibly shown high antitumor cytotoxicity. Only NK-92 cells can easily be genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through antibody-dependent cellular cytotoxicity. NK-92 is also the only cell line product that has been infused into patients with advanced cancer with clinical benefit and minimal side effects.

  14. Guanylate-Binding Protein-1 protects ovarian cancer cell lines but not breast cancer cell lines from killing by paclitaxel.

    PubMed

    Tipton, Aaron R; Nyabuto, Geoffrey O; Trendel, Jill A; Mazur, Travis M; Wilson, John P; Wadi, Suzan; Justinger, Jacob S; Moore, Garret L; Nguyen, Peter T; Vestal, Deborah J

    2016-09-30

    Forced expression of the cytokine-induced large GTPase, human Guanylate-Binding Protein-1 (hGBP-1), in ovarian cancer cell lines increases resistance to paclitaxel. Elevated hGBP-1 RNA in ovarian tumors correlates with shorter recurrence-free survival. In contract, hGBP-1 is part of a gene signature predicting improved prognosis in all subtypes of breast cancers. hGBP-1 does not confer paclitaxel resistance on MCF-7 and TMX2-28 breast cancer cells. Expression of the isotype of the hGBP-1-interacting protein, PIM1, which may contribute to paclitaxel resistance when associated with hGBP-1, is different in breast and ovarian cancer cell lines. Breast cancer cell lines express the 44 kDa isoform of PIM-1, and ovarian cancer cell lines express the 33 kDa isoform. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. MORPHOMETRIC SUBTYPING FOR A PANEL OF BREAST CANCER CELL LINES

    SciTech Connect

    Han, Ju; Chang, Hang; Fontenay, Gerald; Wang, Nicholas J.; Gray, Joe W.; Parvin, Bahram

    2009-05-08

    A panel of cell lines of diverse molecular background offers an improved model system for high-content screening, comparative analysis, and cell systems biology. A computational pipeline has been developed to collect images from cell-based assays, segment individual cells and colonies, represent segmented objects in a multidimensional space, and cluster them for identifying distinct subpopulations. While each segmentation strategy can vary for different imaging assays, representation and subpopulation analysis share a common thread. Application of this pipeline to a library of 41 breast cancer cell lines is demonstrated. These cell lines are grown in 2D and imaged through immunofluorescence microscopy. Subpopulations in this panel are identified and shown to correlate with previous subtyping literature that was derived from transcript data.

  16. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

    Nano, J.L.; Cefai, D.; Rampal, P. )

    1990-02-01

    The effect of exposure of an intestinal epithelial cell line to various concentrations of ethanol (217 mM (1%) to 652 mM (3%)) during 24, 48, and 72 hr was investigated in vitro using a rat intestinal epithelial cell line (IRD 98). Incubation of these cells in the presence of ethanol significantly decreased cell growth. This inhibition was accompanied by a strong increase in cellular protein. Stimulation of specific disaccharidases, gamma-glutamyl transferase, and aminopeptidase activities by ethanol was dose- and time-dependent. Ethanol induces a change in the relative proportions of the different lipid classes synthesized; triglycerides, fatty acids, and cholesterol esters were preferentially synthethysed. Our findings show that cell lines are good models for investigation of the effects of ethanol, and that alcohol considerably modifies the functions of intestinal epithelial cells.

  17. Growth hormone is permissive for neoplastic colon growth

    PubMed Central

    Chesnokova, Vera; Zonis, Svetlana; Zhou, Cuiqi; Recouvreux, Maria Victoria; Ben-Shlomo, Anat; Araki, Takako; Barrett, Robert; Workman, Michael; Wawrowsky, Kolja; Ljubimov, Vladimir A.; Uhart, Magdalena; Melmed, Shlomo

    2016-01-01

    Growth hormone (GH) excess in acromegaly is associated with increased precancerous colon polyps and soft tissue adenomas, whereas short-stature humans harboring an inactivating GH receptor mutation do not develop cancer. We show that locally expressed colon GH is abundant in conditions predisposing to colon cancer and in colon adenocarcinoma-associated stromal fibroblasts. Administration of a GH receptor (GHR) blocker in acromegaly patients induced colon p53 and adenomatous polyposis coli (APC), reversing progrowth GH signals. p53 was also induced in skin fibroblasts derived from short-statured humans with mutant GHR. GH-deficient prophet of pituitary-specific positive transcription factor 1 (Prop1)−/− mice exhibited induced colon p53 levels, and cross-breeding them with Apcmin+/− mice that normally develop intestinal and colon tumors resulted in GH-deficient double mutants with markedly decreased tumor number and size. We also demonstrate that GH suppresses p53 and reduces apoptosis in human colon cell lines as well as in induced human pluripotent stem cell-derived intestinal organoids, and confirm in vivo that GH suppresses colon mucosal p53/p21. GH excess leads to decreased colon cell phosphatase and tensin homolog deleted on chromosome 10 (PTEN), increased cell survival with down-regulated APC, nuclear β-catenin accumulation, and increased epithelial–mesenchymal transition factors and colon cell motility. We propose that GH is a molecular component of the “field change” milieu permissive for neoplastic colon growth. PMID:27226307

  18. Growth hormone is permissive for neoplastic colon growth.

    PubMed

    Chesnokova, Vera; Zonis, Svetlana; Zhou, Cuiqi; Recouvreux, Maria Victoria; Ben-Shlomo, Anat; Araki, Takako; Barrett, Robert; Workman, Michael; Wawrowsky, Kolja; Ljubimov, Vladimir A; Uhart, Magdalena; Melmed, Shlomo

    2016-06-07

    Growth hormone (GH) excess in acromegaly is associated with increased precancerous colon polyps and soft tissue adenomas, whereas short-stature humans harboring an inactivating GH receptor mutation do not develop cancer. We show that locally expressed colon GH is abundant in conditions predisposing to colon cancer and in colon adenocarcinoma-associated stromal fibroblasts. Administration of a GH receptor (GHR) blocker in acromegaly patients induced colon p53 and adenomatous polyposis coli (APC), reversing progrowth GH signals. p53 was also induced in skin fibroblasts derived from short-statured humans with mutant GHR. GH-deficient prophet of pituitary-specific positive transcription factor 1 (Prop1)(-/-) mice exhibited induced colon p53 levels, and cross-breeding them with Apc(min+/-) mice that normally develop intestinal and colon tumors resulted in GH-deficient double mutants with markedly decreased tumor number and size. We also demonstrate that GH suppresses p53 and reduces apoptosis in human colon cell lines as well as in induced human pluripotent stem cell-derived intestinal organoids, and confirm in vivo that GH suppresses colon mucosal p53/p21. GH excess leads to decreased colon cell phosphatase and tensin homolog deleted on chromosome 10 (PTEN), increased cell survival with down-regulated APC, nuclear β-catenin accumulation, and increased epithelial-mesenchymal transition factors and colon cell motility. We propose that GH is a molecular component of the "field change" milieu permissive for neoplastic colon growth.

  19. Reliable in vitro studies require appropriate ovarian cancer cell lines

    PubMed Central

    2014-01-01

    Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past, in vitro studies using cell line models have been an invaluable tool for basic, discovery-driven cancer research. However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future. PMID:24936210

  20. Permissible performance limits of regression analyses in method comparisons.

    PubMed

    Haeckel, Rainer; Wosniok, Werner; Al Shareef, Nadera

    2011-11-01

    Method comparisons are indispensable tools for the extensive validation of analytic procedures. Laboratories often only want to know whether an established procedure (x-method) can be replaced by another one (y-method) without interfering with diagnostic purposes. Then split patients' samples are analyzed more or less simultaneously with both procedures designed to measure the same quantity. The measured values are usually presented graphically as a scatter or difference plots. The two methods are considered to be equivalent (comparable) if the data pairs scatter around the line of equality (x=y line) within permissible equivalence lines. It is proposed to derive these limits of permissible imprecision limits which are based on false-positive error rates. If all data pairs are within the limits, both methods lead to comparable false error rates. If one or more data pairs are outside the permissible equivalence limits, the x-method cannot simply be replaced by the y-method and further studies are required. The discordance may be caused either by aberrant values (outliers), non-linearity, bias or a higher variation of e.g., the y-values. The spread around the line of best fit can detect possible interferences if more than 1% of the data pairs are outside permissible spread lines in a scatter plot. Because bias between methods and imprecision can be inter-related, both require specific examinations for their identification.

  1. Mammalian cell line developments in speed and efficiency.

    PubMed

    Estes, Scott; Melville, Mark

    2014-01-01

    Mammalian cell expression systems are the dominant tool today for producing complex biotherapeutic proteins. In this chapter, we discuss the basis for this dominance, and further explore why the Chinese hamster ovary (CHO) cell line has become the prevalent choice of hosts to produce most recombinant biologics. Furthermore, we explore some of the innovations that are currently in development to improve the CHO cell platform, from cell line specific technologies to overarching technologies that are designed to improve the overall workflow of bioprocess development.

  2. Antiproliferative Effect of Solanum nigrum on Human Leukemic Cell Lines

    PubMed Central

    Gabrani, Reema; Jain, Ramya; Sharma, Anjali; Sarethy, Indira P.; Dang, Shweta; Gupta, S.

    2012-01-01

    Solanum nigrum is used in various traditional medical systems for antiproliferative, antiinflammatory, antiseizure and hepatoprotective activities. We have evaluated organic solvent and aqueous extracts obtained from berries of Solanum nigrum for antiproliferative activity on leukemic cell lines, Jurkat and HL-60 (Human promyelocytic leukemia cells). The cell viability after the treatment with Solanum nigrum extract was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Results indicated increased cytotoxicity with increasing extract concentrations. Comparative analysis indicated that 50% inhibitory concentration value of methanol extract is the lowest on both cell lines. PMID:23716874

  3. Transcriptomic profiling of 39 commonly-used neuroblastoma cell lines.

    PubMed

    Harenza, Jo Lynne; Diamond, Maura A; Adams, Rebecca N; Song, Michael M; Davidson, Heather L; Hart, Lori S; Dent, Maiah H; Fortina, Paolo; Reynolds, C Patrick; Maris, John M

    2017-03-28

    Neuroblastoma cell lines are an important and cost-effective model used to study oncogenic drivers of the disease. While many of these cell lines have been previously characterized with SNP, methylation, and/or mRNA expression microarrays, there has not been an effort to comprehensively sequence these cell lines. Here, we present raw whole transcriptome data generated by RNA sequencing of 39 commonly-used neuroblastoma cell lines. These data can be used to perform differential expression analysis based on a genetic aberration or phenotype in neuroblastoma (e.g., MYCN amplification status, ALK mutation status, chromosome arm 1p, 11q and/or 17q status, sensitivity to pharmacologic perturbation). Additionally, we designed this experiment to enable structural variant and/or long-noncoding RNA analysis across these cell lines. Finally, as more DNase/ATAC and histone/transcription factor ChIP sequencing is performed in these cell lines, our RNA-Seq data will be an important complement to inform transcriptional targets as well as regulatory (enhancer or repressor) elements in neuroblastoma.

  4. Baculovirus studies in new, indigenous lepidopteran cell lines.

    PubMed

    Pant, U; Sudeep, A B; Athawale, S S; Vipat, V C

    2002-01-01

    Eight lepidopteran cell lines were established recently and their susceptibility to different insect viruses was studied. Two Spodoptera litura cell lines from the larval and pupal ovaries, were found highly susceptible to S. litura nuclear polyhedrosis virus (SLNPV, 5-6 x 10(6) NPV/ml). The Helicoverpa armigera cell line from the embryonic tissue was highly susceptible to H. armigera NPV (HaNPV, 6.3 x 10(6) NPV/ml). These in vitro grown SLNPV and HaNPV caused 100% mortality to respective 2nd instar larvae. The susceptibility of the cryo-preserved cell lines to respective baculoviruses (SLNPV/HaNPV) was studied and no significant difference in their susceptibility status was observed. The cultures could grow as suspension culture on shakers and may find application for in vitro production of wild type/recombinant baculoviruses as bio-insecticides. S. litura and Bombyx mori cell lines from larval ovaries, were highly susceptible to Autographa californica NPV (5.5 x 10(6) NPV/ml) and Bombyx mori NPV (BmNPV, 6.1 x 10(6) NPV/ml) respectively. These cell lines may find application in baculovirus expression vector studies for the production of recombinant proteins, useful in the development of diagnostic kits or as vaccines.

  5. Transcriptomic profiling of 39 commonly-used neuroblastoma cell lines

    PubMed Central

    Harenza, Jo Lynne; Diamond, Maura A.; Adams, Rebecca N.; Song, Michael M.; Davidson, Heather L.; Hart, Lori S.; Dent, Maiah H.; Fortina, Paolo; Reynolds, C. Patrick; Maris, John M.

    2017-01-01

    Neuroblastoma cell lines are an important and cost-effective model used to study oncogenic drivers of the disease. While many of these cell lines have been previously characterized with SNP, methylation, and/or mRNA expression microarrays, there has not been an effort to comprehensively sequence these cell lines. Here, we present raw whole transcriptome data generated by RNA sequencing of 39 commonly-used neuroblastoma cell lines. These data can be used to perform differential expression analysis based on a genetic aberration or phenotype in neuroblastoma (e.g., MYCN amplification status, ALK mutation status, chromosome arm 1p, 11q and/or 17q status, sensitivity to pharmacologic perturbation). Additionally, we designed this experiment to enable structural variant and/or long-noncoding RNA analysis across these cell lines. Finally, as more DNase/ATAC and histone/transcription factor ChIP sequencing is performed in these cell lines, our RNA-Seq data will be an important complement to inform transcriptional targets as well as regulatory (enhancer or repressor) elements in neuroblastoma. PMID:28350380

  6. Establishment of the DU.528 human lymphohemopoietic stem cell line

    PubMed Central

    1985-01-01

    We have established the DU.528 cell line from the pretreatment leukemia cells of a patient who underwent a T lymphoblastic-to-promyelocytic phenotype conversion during treatment with the adenosine deaminase inhibitor, deoxycoformycin. The cell line and clones obtained from it by limiting dilution have the same karyotype previously found in the patient's pretreatment T lymphoblasts and post-deoxycoformycin treatment promyelocytes. DU.528 cells in continuous culture for greater than 2 yr display a predominant undifferentiated T lymphoblastoid phenotype. These cells spontaneously generate progeny of at least three lineages, T lymphoid, granulocytic/monocytic, and erythroid. The surface marker most consistently expressed by DU.528 cells in the undifferentiated state is the 3A1 antigen, which has been found on prothymocytes in the embryonic thymus. Some undifferentiated DU.528 cells also expressed the IL-2 receptor, but no other T cell differentiation antigens. Exposure of DU.528 cells to a variety of agents induced myeloid maturation; adenosine and deoxyadenosine, in the presence of deoxycoformycin, induced expression of myeloid differentiation antigens. Our results suggest that DU.528 is a lymphohematopoietic stem cell line and support the hypothesis that differentiation of pluripotent stem cells may be altered by genetic deficiency of adenosine deaminase. DU.528 cells may provide a useful model for examining factors that regulate stem cell proliferation and differentiation. PMID:4056659

  7. Picking Cell Lines for High-Throughput Transcriptomic Toxicity ...

    EPA Pesticide Factsheets

    High throughput, whole genome transcriptomic profiling is a promising approach to comprehensively evaluate chemicals for potential biological effects. To be useful for in vitro toxicity screening, gene expression must be quantified in a set of representative cell types that captures the diversity of potential responses across chemicals. The ideal dataset to select these cell types would consist of hundreds of cell types treated with thousands of chemicals, but does not yet exist. However, basal gene expression data may be useful as a surrogate for representing the relevant biological space necessary for cell type selection. The goal of this study was to identify a small (< 20) number of cell types that capture a large, quantifiable fraction of basal gene expression diversity. Three publicly available collections of Affymetrix U133+2.0 cellular gene expression data were used: 1) 59 cell lines from the NCI60 set; 2) 303 primary cell types from the Mabbott et al (2013) expression atlas; and 3) 1036 cell lines from the Cancer Cell Line Encyclopedia. The data were RMA normalized, log-transformed, and the probe sets mapped to HUGO gene identifiers. The results showed that <20 cell lines capture only a small fraction of the total diversity in basal gene expression when evaluated using either the entire set of 20960 HUGO genes or a subset of druggable genes likely to be chemical targets. The fraction of the total gene expression variation explained was consistent when

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

    PubMed

    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.

  9. Inducible human immunodeficiency virus type 1 packaging cell lines.

    PubMed Central

    Yu, H; Rabson, A B; Kaul, M; Ron, Y; Dougherty, J P

    1996-01-01

    Packaging cell lines are important tools for transferring genes into eukaryotic cells. Human immunodeficiency virus type 1 (HIV-1)-based packaging cell lines are difficult to obtain, in part owing to the problem that some HIV-1 proteins are cytotoxic in a variety of cells. To overcome this, we have developed an HIV-1-based packaging cell line which has an inducible expression system. The tetracycline-inducible expression system was utilized to control the expression of the Rev regulatory protein, which in turn controls the expression of the late proteins including Gag, Pol, and Env. Western blotting (immunoblotting) demonstrated that the expression of p24gag and gp120env from the packaging cells peaked on days 6 and 7 postinduction. Reverse transcriptase activity could be detected by day 4 after induction and also peaked on days 6 and 7. Defective vector virus could be propagated, yielding titers as high as 7 x 10(3) CFU/ml, while replication-competent virus was not detectable at any time. Thus, the cell line should enable the transfer of specific genes into CD4+ cells and should be a useful tool for studying the biology of HIV-1. We have also established an inducible HIV-1 Env-expressing cell line which could be used to propagate HIV-1 vectors that require only Env in trans. The env-minus vector virus titer produced from the Env-expressing cells reached 2 x 10(4) CFU/ml. The inducible HIV-1 Env-expressing cell line should be a useful tool for the study of HIV-1 Env as well. PMID:8676479

  10. Establishment and characterization of a novel osteosarcoma cell line: CHOS.

    PubMed

    Liu, Yunlu; Feng, Xiaobo; Zhang, Yukun; Jiang, Hongyan; Cai, Xianyi; Yan, Xinxin; Huang, Zengfa; Mo, Fengbo; Yang, Wen; Yang, Cao; Yang, Shuhua; Liu, Xianzhe

    2016-12-01

    Osteosarcoma has a well-recognized bimodal distribution, with the first peak in adolescence and another in the elderly age-group. The elderly patients have different clinical features and a poorer prognosis as compared to adolescents. To better understand the biological features of osteosarcoma in the elderly population, we established a new human osteosarcoma cell line from a 58-year-old man with primary chondroblastic osteosarcoma. After 6 months of continuous culture in vitro for over 50 passages, an immortalized cell line CHOS was established. The cell line was well-characterized by cytogenetic, biomarker, functional, and histological analyses. The CHOS cells exhibited a spindle-shaped morphology and a doubling time of 36 h. Cytogenetic analysis of CHOS cells revealed the loss of chromosome Y and the gain of chromosome 12. Quantitative real-time polymerase chain reaction (RT-PCR), Western blotting and/or immunofluorescence revealed the expression of chondroblastic, mesenchymal and tumor metastasis markers in the CHOS cells. Compared with the osteosarcoma cell line, the CHOS cells were found to be more sensitive to cisplatin and doxorubicin, but were resistant to methotrexate. The cell line was highly tumorigenic and maintained the histological characteristics and invasive nature of the original tumor. Furthermore, on immunohistochemical analysis, the xenografts and metastases were found to co-express collagen II, aggrecan, vimentin and S100A4 that resembled the original tumor cells. Our results indicate, the potential of CHOS cell line to serve as a useful tool for further studies on the molecular biology of osteosarcoma, especially in the elderly patients. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2116-2125, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  11. The Type 1 Alveolar Lining Cells of the Mammalian Lung

    PubMed Central

    Rosenbaum, Robert M.; Picciano, Paul

    1978-01-01

    Using a newly described dissociation and isolation technique, Type 1 alveolar lining cells were obtained from adult rabbit lung within a heterogeneous population. Identification of many lung cell types in this mixed population was by a) comparison of isolated cells with in situ lung cells in lung sections using identical parallel staining, b) stepwise ultrastructural examination of cells during all stages of lung dissociation so that intercellular associations were monitored throughout, and c) Type 1 cell surface changes following collagenase treatment. This phenomenon was studied with both electron and light microscopy, the latter employing tetrachrome staining of basophilic blebs as well as characteristic staining of nucleus and cytoplasm. Following their isolation, most Type 1 cells lost their surface blebs and assumed a “relaxed” state. In this condition, Type 1 cells were exposed to cytochalasin D (CD) for various times and at several concentrations. Surface knobs, having all the characteristics of zeiotic knobs produced in a number of cultured cell lines by exposure to CD, were produced in isolated Type 1 epithelial cells within 45 minutes. The reaction to CD was temperature-dependent, proceeding maximally at 37 C with inhibition at lower temperatures and was inhibited by antimetabolites such as dinitrophenol and 2-deoxyglucose in the presence of CD. As with established cell lines, formation of zeiotic knobs at the isolated Type 1 cell surface appeared closely related to microfilamentous nets located beneath the plasmalemma. The density of this net appeared to vary as isolated Type 1 cells underwent expansion and contraction in response to CD. Zeiotic knobs were formed as the result of herniation of endoplasm through the cell cortex. The significance of such a labile cortical zone is considered in relation to the deformation changes Type 1 cells undergo during inflation-deflation of alveoli and the folding-unfolding of alveolar lining cells as a result of

  12. Human embryonic stem cell lines model experimental human cytomegalovirus latency.

    PubMed

    Penkert, Rhiannon R; Kalejta, Robert F

    2013-05-28

    Herpesviruses are highly successful pathogens that persist for the lifetime of their hosts primarily because of their ability to establish and maintain latent infections from which the virus is capable of productively reactivating. Human cytomegalovirus (HCMV), a betaherpesvirus, establishes latency in CD34(+) hematopoietic progenitor cells during natural infections in the body. Experimental infection of CD34(+) cells ex vivo has demonstrated that expression of the viral gene products that drive productive infection is silenced by an intrinsic immune defense mediated by Daxx and histone deacetylases through heterochromatinization of the viral genome during the establishment of latency. Additional mechanistic details about the establishment, let alone maintenance and reactivation, of HCMV latency remain scarce. This is partly due to the technical challenges of CD34(+) cell culture, most notably, the difficulty in preventing spontaneous differentiation that drives reactivation and renders them permissive for productive infection. Here we demonstrate that HCMV can establish, maintain, and reactivate in vitro from experimental latency in cultures of human embryonic stem cells (ESCs), for which spurious differentiation can be prevented or controlled. Furthermore, we show that known molecular aspects of HCMV latency are faithfully recapitulated in these cells. In total, we present ESCs as a novel, tractable model for studies of HCMV latency.

  13. On-line characterization of a hybridoma cell culture process.

    PubMed

    Zhou, W; Hu, W S

    1994-06-20

    The on-line determination of the physiological state of a cell culture process requires reliable on-line measurements of various parameters and calculations of specific rates from these measurements. The cell concentration of a hybridoma culture was estimated on-line by measuring optical density (OD) with a laser turbidity probe. The oxygen uptake rate (OUR) was determined by monitoring dynamically dissolved oxygen concentration profiles and closing oxygen balances in the culture. The base addition for neutralizing lactate produced by cells was also monitored on-line via a balance. Using OD and OUR measurements, the specific growth and specific oxygen consumption rates were determined on-line. By combining predetermined stoichiometric relationships among oxygen and glucose consumption and lactate production, the specific glucose consumption and lactate production rates were also calculated on-line. Using these on-line measurements and calculations, the hybridoma culture process was characterized on-line by identifying the physiological states. They will also facilitate the implementation of nutrient feeding strategies for fed-batch and perfusion cultures. (c) 1994 John Wiley & Sons, Inc.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26293811

  15. Experimental Adaptation of Rotaviruses to Tumor Cell Lines

    PubMed Central

    Guerrero, Carlos A.; Guerrero, Rafael A.; Silva, Elver; Acosta, Orlando; Barreto, Emiliano

    2016-01-01

    A number of viruses show a naturally extended tropism for tumor cells whereas other viruses have been genetically modified or adapted to infect tumor cells. Oncolytic viruses have become a promising tool for treating some cancers by inducing cell lysis or immune response to tumor cells. In the present work, rotavirus strains TRF-41 (G5) (porcine), RRV (G3) (simian), UK (G6-P5) (bovine), Ym (G11-P9) (porcine), ECwt (murine), Wa (G1-P8), Wi61 (G9) and M69 (G8) (human), and five wild-type human rotavirus isolates were passaged multiple times in different human tumor cell lines and then combined in five different ways before additional multiple passages in tumor cell lines. Cell death caused by the tumor cell-adapted isolates was characterized using Hoechst, propidium iodide, 7-AAD, Annexin V, TUNEL, and anti-poly-(ADP ribose) polymerase (PARP) and -phospho-histone H2A.X antibodies. Multiple passages of the combined rotaviruses in tumor cell lines led to a successful infection of these cells, suggesting a gain-of-function by the acquisition of greater infectious capacity as compared with that of the parental rotaviruses. The electropherotype profiles suggest that unique tumor cell-adapted isolates were derived from reassortment of parental rotaviruses. Infection produced by such rotavirus isolates induced chromatin modifications compatible with apoptotic cell death. PMID:26828934

  16. Myelination in coculture of established neuronal and Schwann cell lines.

    PubMed

    Sango, Kazunori; Kawakami, Emiko; Yanagisawa, Hiroko; Takaku, Shizuka; Tsukamoto, Masami; Utsunomiya, Kazunori; Watabe, Kazuhiko

    2012-06-01

    Establishing stable coculture systems with neuronal and Schwann cell lines has been considered difficult, presumably because of their high proliferative activity and phenotypic differences from primary cultured cells. The present study is aimed at developing methods for myelin formation under coculture of the neural crest-derived pheochromocytoma cell line PC12 and the immortalized adult rat Schwann cell line IFRS1. Prior to coculture, PC12 cells were seeded at low density (3 × 10(2)/cm(2)) and maintained in serum-free medium with N2 supplement, ascorbic acid (50 μg/ml), and nerve growth factor (NGF) (50 ng/ml) for a week. Exposure to such a NGF-rich environment with minimum nutrients accelerated differentiation and neurite extension, but not proliferation, of PC12 cells. When IFRS1 cells were added to NGF-primed PC12 cells, the cell density ratio of PC12 cells to IFRS1 cells was adjusted from 1:50 to 1:100. The cocultured cells were then maintained in serum-free medium with B27 supplement, ascorbic acid (50 μg/ml), NGF (10 ng/ml), and recombinant soluble neuregulin-1 type III (25 ng/ml). Myelin formation was illustrated by light and electron microscopy performed at day 28 of coculture. The stable PC12-IFRS1 coculture system is free of technical and ethical problems arising from the primary culture and can be a valuable tool to study peripheral nerve degeneration and regeneration.

  17. Protein Expression Profiles of Permissive, Semi-Permissive and Non-Permissive Cells Infected by Baculovirus

    USDA-ARS?s Scientific Manuscript database

    Amassing information on the in vitro protein expression of an insect host challenged by an entomopathogenic agent, such as a baculovirus, is paramount to an enhanced understanding of how host-pathogen interactions determine the success or failure of a pathogen. In this study, 2D-gel electrophoresis...

  18. Sclerostin Antibody Administration Converts Bone Lining Cells Into Active Osteoblasts.

    PubMed

    Kim, Sang Wan; Lu, Yanhui; Williams, Elizabeth A; Lai, Forest; Lee, Ji Yeon; Enishi, Tetsuya; Balani, Deepak H; Ominsky, Michael S; Ke, Hua Zhu; Kronenberg, Henry M; Wein, Marc N

    2016-11-14

    Sclerostin antibody (Scl-Ab) increases osteoblast activity, in part through increasing modeling-based bone formation on previously quiescent surfaces. Histomorphometric studies have suggested that this might occur through conversion of bone lining cells into active osteoblasts. However, direct data demonstrating Scl-Ab-induced conversion of lining cells into active osteoblasts are lacking. Here, we used in vivo lineage tracing to determine if Scl-Ab promotes the conversion of lining cells into osteoblasts on periosteal and endocortical bone surfaces in mice. Two independent, tamoxifen-inducible lineage-tracing strategies were used to label mature osteoblasts and their progeny using the DMP1 and osteocalcin promoters. After a prolonged "chase" period, the majority of labeled cells on bone surfaces assumed a thin, quiescent morphology. Then, mice were treated with either vehicle or Scl-Ab (25 mg/kg) twice over the course of the subsequent week. After euthanization, marked cells were enumerated, their thickness quantified, and proliferation and apoptosis examined. Scl-Ab led to a significant increase in the average thickness of labeled cells on periosteal and endocortical bone surfaces, consistent with osteoblast activation. Scl-Ab did not induce proliferation of labeled cells, and Scl-Ab did not regulate apoptosis of labeled cells. Therefore, direct reactivation of quiescent bone lining cells contributes to the acute increase in osteoblast numbers after Scl-Ab treatment in mice. © 2017 American Society for Bone and Mineral Research.

  19. Establishment and characterization of 10 cell lines derived from patients with adult T-cell leukemia.

    PubMed Central

    Hoshino, H; Esumi, H; Miwa, M; Shimoyama, M; Minato, K; Tobinai, K; Hirose, M; Watanabe, S; Inada, N; Kinoshita, K; Kamihira, S; Ichimaru, M; Sugimura, T

    1983-01-01

    By using human T-cell growth factor (TCGF), 10 cell lines were established from tissue samples of 10 patients with adult T-cell leukemia (ATL). Three cell lines were adapted to growth in medium lacking TCGF. The surface markers of all cell lines were characteristic of inducer/helper T cells, i.e., OKT3+, OKT4+, OKT6-, OKT8-, OKIa1+, and human Lyt2+ and Lyt3+, except that one cell line was OKT3-. The expression of the viral antigen was examined during establishment of 8 of the 10 cell lines. The viral antigen was not expressed in leukemic cells before cultivation. In 5 lines, the viral antigen was detected by immunofluorescent staining after a short period of cultivation. However, 3 cell lines, ATL-6A, ATL-9Y, and ATL-1K did not express the viral antigen during short-term culture: the ATL-6A and ATL-9Y cell lines became positive for the viral antigen after 5 and 2 months of cultivation, respectively; the ATL-1K cell line remained antigen-negative throughout a culture period of 13 months. Southern blot hybridization assay showed that all of the cell lines, including the viral antigen-negative ATL-1K cell line, contained the viral genome. Thus, the retrovirus was associated with all 10 cell lines established from ATL patients, but there was a heterogeneity in the expression time of the retroviral antigen in leukemic cells maintained in vitro. Our findings suggested that the expression of the viral antigen was not required for maintenance of the leukemic state in vivo and for growth of leukemic cells in vitro. Images PMID:6193528

  20. Three-dimensional cultured glioma cell lines

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R. (Inventor); Marley, Garry M. (Inventor)

    1991-01-01

    Three-dimensional glioma spheroids were produced in vitro with size and histological differentiation previously unattained. The spheroids were grown in liquid media suspension in a Johnson Space Center (JSC) Rotating Wall Bioreactor without using support matrices such as microcarrier beads. Spheroid volumes of greater than 3.5 cu mm and diameters of 2.5 mm were achieved with a viable external layer or rim of proliferating cells, a transitional layer beneath the external layer with histological differentiation, and a degenerative central region with a hypoxic necrotic core. Cell debris was evident in the degenerative central region. The necrotics centers of some of the spheroids had hyaline droplets. Granular bodies were detected predominantly in the necrotic center.

  1. Solid Oxide Fuel Cell Systems PVL Line

    SciTech Connect

    Susan Shearer - Stark State College; Gregory Rush - Rolls-Royce Fuel Cell Systems

    2012-05-01

    In July 2010, Stark State College (SSC), received Grant DE-EE0003229 from the U.S. Department of Energy (DOE), Golden Field Office, for the development of the electrical and control systems, and mechanical commissioning of a unique 20kW scale high-pressure, high temperature, natural gas fueled Stack Block Test System (SBTS). SSC worked closely with subcontractor, Rolls-Royce Fuel Cell Systems (US) Inc. (RRFCS) over a 13 month period to successfully complete the project activities. This system will be utilized by RRFCS for pre-commercial technology development and training of SSC student interns. In the longer term, when RRFCS is producing commercial products, SSC will utilize the equipment for workforce training. In addition to DOE Hydrogen, Fuel Cells, and Infrastructure Technologies program funding, RRFCS internal funds, funds from the state of Ohio, and funding from the DOE Solid State Energy Conversion Alliance (SECA) program have been utilized to design, develop and commission this equipment. Construction of the SBTS (mechanical components) was performed under a Grant from the State of Ohio through Ohio's Third Frontier program (Grant TECH 08-053). This Ohio program supported development of a system that uses natural gas as a fuel. Funding was provided under the Department of Energy (DOE) Solid-state Energy Conversion Alliance (SECA) program for modifications required to test on coal synthesis gas. The subject DOE program provided funding for the electrical build, control system development and mechanical commissioning. Performance testing, which includes electrical commissioning, was subsequently performed under the DOE SECA program. Rolls-Royce Fuel Cell Systems is developing a megawatt-scale solid oxide fuel cell (SOFC) stationary power generation system. This system, based on RRFCS proprietary technology, is fueled with natural gas, and operates at elevated pressure. A critical success factor for development of the full scale system is the capability to

  2. Hedgehog signaling pathway is inactive in colorectal cancer cell lines.

    PubMed

    Chatel, Guillaume; Ganeff, Corine; Boussif, Naima; Delacroix, Laurence; Briquet, Alexandra; Nolens, Gregory; Winkler, Rosita

    2007-12-15

    The Hedgehog (Hh) signaling pathway plays an important role in human development. Abnormal activation of this pathway has been observed in several types of human cancers, such as the upper gastro-intestinal tract cancers. However, activation of the Hh pathway in colorectal cancers is controversial. We analyzed the expression of the main key members of the Hh pathway in 7 colon cancer cell lines in order to discover whether the pathway is constitutively active in these cells. We estimated the expression of SHH, IHH, PTCH, SMO, GLI1, GLI2, GLI3, SUFU and HHIP genes by RT-PCR. Moreover, Hh ligand, Gli3 and Sufu protein levels were quantified by western blotting. None of the cell lines expressed the complete set of Hh pathway members. The ligands were absent from Colo320 and HCT116 cells, Smo from Colo205, HT29 and WiDr. GLI1 gene was not expressed in SW480 cells nor were GLI2/GLI3 in Colo205 or Caco-2 cells. Furthermore the repressive form of Gli3, characteristic of an inactive pathway, was detected in SW480 and Colo320 cells. Finally treatment of colon cancer cells with cyclopamine, a specific inhibitor of the Hh pathway, did not downregulate PTCH and GLI1 genes expression in the colorectal cells, whereas it did so in PANC1 control cells. Taken together, these results indicate that the aberrant activation of the Hh signaling pathway is not common in colorectal cancer cell lines.

  3. Permissive hypofiltration: an alternative view

    PubMed Central

    2012-01-01

    If permissive hypercapnia is used in the context of protective ventilation for patients with acute respiratory distress syndrome, it must be highlighted that the alveoli in these patients are still exposed to significant stress. Similarly, early renal replacement therapy is not necessarily a protective therapy for acute kidney injury and loop diuretics are not necessarily harmful. It is conceivable that early initiation of 'protective' low-dose (10 ml/kg/h) continuous renal replacement therapy with zero balanced ultrafiltration in association with administration of (high dose) diuretics may help to rest the kidneys while ensuring preservation of urine output. PMID:23136903

  4. Permissive hypofiltration: an alternative view.

    PubMed

    Ricci, Zaccaria; Romagnoli, Stefano; Emma, Francesco

    2012-11-08

    If permissive hypercapnia is used in the context of protective ventilation for patients with acute respiratory distress syndrome, it must be highlighted that the alveoli in these patients are still exposed to significant stress. Similarly, early renal replacement therapy is not necessarily a protective therapy for acute kidney injury and loop diuretics are not necessarily harmful. It is conceivable that early initiation of 'protective' low-dose (10 ml/kg/h) continuous renal replacement therapy with zero balanced ultrafiltration in association with administration of (high dose) diuretics may help to rest the kidneys while ensuring preservation of urine output.

  5. Comparison of seven cell lines derived from human gastric carcinomas.

    PubMed

    Motoyama, T; Hojo, H; Watanabe, H

    1986-01-01

    In an attempt to elucidate various histological features of gastric cancers, seven human gastric adenocarcinomas were studied in vitro and in nude mice. Growth pattern of each cultured cell line in vitro corresponded well to the histological type of parent tumor. The cell lines, MKN7, MKN74, and MKN28 derived from differentiated carcinomas showed morphological characteristics of intestinal differentiation in cell polarity and microvilli with core-filaments in vitro as well as in nude mice. However, they gradually diminished the characteristics in course of time. The cell lines, MKN 45 and OKAJIMA, derived from undifferentiated carcinomas, had natures of not only ordinary gastric mucosa but also intestinal metaplastic mucosa. They seem to have multipotentiality for differentiation, and preserved well the natures for long periods of culture. The KWS-I cell line composed of undifferentiated cells in vitro displayed the potential for differentiation in nude mice. However, the differentiation of KATO-III cells derived from a signet-ring cell carcinoma was suppressed in nude mice. The common abnormality of chromosome was not found, and the growth rate in vitro was not dependent on the histological type of parent tumor.

  6. Rabeprazole exhibits antiproliferative effects on human gastric cancer cell lines

    PubMed Central

    GU, MENGLI; ZHANG, YAN; ZHOU, XINXIN; MA, HAN; YAO, HANGPING; JI, FENG

    2014-01-01

    Intracellular proton extrusion in gastric cancer cells has been reported to promote cancer cell survival under acidic conditions via hydrogen/potassium adenosine triphosphatase (H+/K+-ATPase). Rabeprazole is a frequently used second-generation proton pump inhibitor (PPI) that irreversibly inactivates gastric H+/K+-ATPase. Therefore, we hypothesized that rabeprazole could reduce the viability of gastric cancer cells. In the present study, four human gastric cancer cell lines and one non-cancer gastric cell line were cultured. Cell viability, the α- and β-subunits of H+/K+-ATPase and cellular apoptosis were analyzed by dye exclusion assay, reverse transcription-polymerase chain reaction and annexin V-fluorescein isothiocyanate/propidium iodide staining, respectively. The expression level of total extracellular signal-regulated protein kinase 1/2 (ERK 1/2) and phosphorylated-ERK protein was detected by western blot analysis. Gastric cancer cell lines were more tolerant of the acidic culture media than non-cancer cells. Administration of rabeprazole led to a marked decrease in the viability of MKN-28 cells. Exposure to rabeprazole induced significant apoptosis in AGS cells. Rabeprazole completely inhibited the phosphorylation of ERK 1/2 in the MKN-28 cells, whereas the same effect was not observed in either the KATO III or MKN-45 cells. The ERK 1/2 inhibitor, PD98059, attenuated the viability of the AGS cells. A similar antiproliferative effect was observed in the rabeprazole treatment group. In addition, PD98059 and rabeprazole were able to efficaciously inhibit the phosphorylation of ERK 1/2 in the gastric cancer cells. Therefore, it was concluded that rabeprazole can attenuate the cell viability of human gastric cancer cells through inactivation of the ERK1/2 signaling pathway. The results of the present study demonstrate that rabeprazole inhibits the viability of gastric cancer cells in vitro and may serve as a novel antineoplastic agent. PMID:25202402

  7. Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors

    SciTech Connect

    Watanabe, S.; Temin, H.M.

    1983-12-01

    The authors wished to construct cell lines that supply the gene products of gag, pol, and env for the growth of replication-defective reticuloendotheliosis retrovirus vectors without production of the helper virus. To do this, first they located by S1 mapping the donor and acceptor splice sites of reticuloendotheliosis virus strain A. The donor splice site is ca. 850 base pairs from the 5' end of proviral DNA. It is close to or overlaps the encapsidation sequences for viral RNA. The splice acceptor site is ca. 5.6 kilobase pairs from the 5' end of proviral DNA. Therefore, the encapsidation sequences and the donor splice site were removed from viral DNA to give expression of the gag and pol genes without virus production. The promoter in the long terminal repeat was fused to a site near the first ATG codon of the env gene, thereby deleting the encapsidation sequences and the gag and pol genes to give expression of the env gene without virus production. The permissive canine cell line D17 was transfected with the two modified viral DNAs. Two cell clones that contain both modified viral DNAs support the production of replication-defective spleen necrosis virus-thymidine kinase recombinant retrovirus vectors without the production of helper virus. To prevent recombination, the vector contains deletions that overlap with deletions in the integrated helper virus DNAs. This helper cell-vector system will be useful to derive infectious recombinant virus stocks of high titer (over 10/sup 5/ thymidine kinase transforming units per ml) which are able to infect avian, rat, and dog cells without the aid of helper virus.

  8. Canine mammary tumour cell lines established in vitro.

    PubMed

    Hellmén, E

    1993-01-01

    Mammary tumours are the most common tumours in the female dog. The tumours have a complex histology and exist in epithelial, mixed and mesenchymal forms. To study the biology of canine mammary tumours, five cell lines have been established and characterized. The results indicate that canine mammary tumours might be derived from mammary stem cells and that the tumour growth is independent of oestrogens. The established canine mammary tumour cell lines will be valuable tools in further studies of the histogenesis and pathogenesis of these tumours.

  9. Definitive Molecular Cytogenetic Characterization of 15 Colorectal Cancer Cell Lines

    PubMed Central

    Knutsen, Turid; Padilla-Nash, Hesed M.; Wangsa, Danny; Barenboim-Stapleton, Linda; Camps, Jordi; McNeil, Nicole; Difilippantonio, Michael J.; Ried, Thomas

    2009-01-01

    In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. We here present the results of a comprehensive investigation of 15 established colorectal cancer cell lines utilizing spectral karyotyping (SKY), fluorescence in situ hybridization, and comparative genomic hybridization (CGH). Detailed karyotypic analysis by SKY on five of the lines (P53HCT116, T84, NCI-H508, NCI-H716, and SK-CO-1) are described here for the first time. The five lines with karyotypes in the diploid range and that are characterized by defects in DNA mismatch repair had a mean of 4.8 chromosomal abnormalities per line, whereas the 10 aneuploid lines exhibited complex karyotypes and a mean of 30 chromosomal abnormalities. Of the 150 clonal translocations, only eight were balanced and none were recurrent among the lines. We also reviewed the karyotypes of 345 cases of adenocarcinoma of the large intestine listed in the Mitelman Database of Chromosome Aberrations in Cancer. The types of abnormalities observed in the cell lines reflected those seen in primary tumors: there were no recurrent translocations in either tumors or cell lines, isochromosomes were the most common recurrent abnormalities, and breakpoints occurred most frequently at the centromeric/pericentromeric and telomere regions. Of the genomic imbalances detected by array CGH, 87% correlated with chromosome aberrations observed in the SKY studies. The fact that chromosome abnormalities result predominantly in copy number changes rather than specific chromosome or gene fusions, suggests this may be the major mechanism leading to carcinogenesis in colorectal cancer. PMID:19927377

  10. Definitive molecular cytogenetic characterization of 15 colorectal cancer cell lines.

    PubMed

    Knutsen, Turid; Padilla-Nash, Hesed M; Wangsa, Danny; Barenboim-Stapleton, Linda; Camps, Jordi; McNeil, Nicole; Difilippantonio, Michael J; Ried, Thomas

    2010-03-01

    In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. Here, we present the results of a comprehensive investigation of 15 established colorectal cancer cell lines using spectral karyotyping (SKY), fluorescence in situ hybridization, and comparative genomic hybridization (CGH). Detailed karyotypic analysis by SKY on five of the lines (P53HCT116, T84, NCI-H508, NCI-H716, and SK-CO-1) is described here for the first time. The five lines with karyotypes in the diploid range and that are characterized by defects in DNA mismatch repair had a mean of 4.8 chromosomal abnormalities per line, whereas the 10 aneuploid lines exhibited complex karyotypes and a mean of 30 chromosomal abnormalities. Of the 150 clonal translocations, only eight were balanced and none were recurrent among the lines. We also reviewed the karyotypes of 345 cases of adenocarcinoma of the large intestine listed in the Mitelman Database of Chromosome Aberrations in Cancer. The types of abnormalities observed in the cell lines reflected those seen in primary tumors: there were no recurrent translocations in either tumors or cell lines; isochromosomes were the most common recurrent abnormalities; and breakpoints occurred most frequently at the centromeric/pericentromeric and telomere regions. Of the genomic imbalances detected by array CGH, 87% correlated with chromosome aberrations observed in the SKY studies. The fact that chromosome abnormalities predominantly result in copy number changes rather than specific chromosome or gene fusions suggests that this may be the major mechanism leading to carcinogenesis in colorectal cancer.

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

    PubMed

    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.

  12. Design of tunable microwave transmission lines using metamaterial cells

    NASA Astrophysics Data System (ADS)

    Bensafieddine, D.; Djerfaf, F.; Chouireb, F.; Vincent, D.

    2017-04-01

    In this paper, frequency tunable transmission lines are designed using metasurface split ring resonator unit cell. We prove that the tuning principle in metasurface transmission lines is based on the variation of the resonance frequency of the permeability. The frequency-tuning arises by changing the values of two gaps in the inner and outer rings of unit cell ( g1 and g2). The branches of a disconnected gaps type conductor of each unit cell can be joined by switches (PIN diodes, MEMs, etc.). According to switch states ON or OFF, the unit cell has four different commutable behaviors which are 00, 01, 11, and 10. The results show that the resonance frequency of our metasurface transmission line is strongly shifted by about 2.5 GHz between the cases (01) and (11).

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

    PubMed Central

    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

    2014-01-01

    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. PMID:25117809

  14. Susceptibility of nonprimate cell lines to hepatitis A virus infection.

    PubMed Central

    Dotzauer, A; Feinstone, S M; Kaplan, G

    1994-01-01

    Hepatitis A virus (HAV) has been adapted to grow in primate cell cultures. We investigated replication of HAV in nonprimate cells by inoculating 20 cell lines from different species with the tissue culture-adapted HM175 strain. Slot blot hybridization and immunofluorescence analysis revealed that HAV replicated in GPE, SP 1K, and IB-RS-2 D10 cells of guinea pig, dolphin, and pig origin, respectively. Studies in IB-RS-2 D10 cells were discontinued because cultures were contaminated with classical swine fever virus. A growth curve showed that HAV grew poorly in GPE cells and intermediately in SP 1K cells compared with growth in FRhK-4 cells. Therefore, the cell surface receptor(s) and other host factor(s) required for HAV replication are present in nonprimate as well as primate cells. Images PMID:8057483

  15. Non-targeted radiation effects in vertebrate cell lines

    NASA Astrophysics Data System (ADS)

    Ryan, Lorna

    Radiation effects, such as bystander effects, hyper radiosensitivity/induced radioresistance (HRS/IRR) and adaptive response that are not related to direct DNA damage are now accepted. However the inter-relationship between them and the possible impact on the scientific basis for radiation protection are highly controversial. This thesis attempts to elucidate the mechanisms of some of these well known but little understood effects. Each paper examines some aspect of bystander effects, adaptive responses and HRS/IRR in an effort to understand how they vary with cell type, dose and time of exposure to single or multiple doses. All the effects involve non-linear dose effect curves and are mainly evident following low doses. Overall findings of the thesis include (1) A clear difference was observed between radioresistant, tumorigenic cell lines with mutant p53 gene expression, and radiosensitive, more normal, cell lines with wild type p53. In general death inducing bystander responses are induced in normal cell populations exposed to low doses of radiation while survival inducing IRR and adaptive responses are seen in the radioresistant tumorigenic cell lines. (2) A cohort of fish cell lines which demonstrated survival promoting bystander effects, also did not show a protective adaptive responses. (3) Adaptive responses traditionally occur when a large challenge dose is given 4--6hrs following low (10--100mGy) priming doses but this thesis shows that for the epithelial cell lines tested, the size of the priming dose (range 0.1--2Gy) does not appear to alter the size of the recovery response. Additionally increased survival could be detected in some cases when the challenge dose was given within one hour of the priming dose. The overall conclusion is that cell lines induce either a bystander response or a protective/adaptive response depending on genetic background and other factors. Care is needed in the interpretation of data generated from only one or two cell lines

  16. Induction of experimental autoimmune uveoretinitis by T-cell lines.

    PubMed Central

    Rozenszajn, L A; Muellenberg-Coulombre, C; Gery, I; el-Saied, M; Kuwabara, T; Mochizuki, M; Lando, Z; Nussenblatt, R B

    1986-01-01

    Experimental autoimmune uveoretinitis was induced in genetically susceptible Lewis rats by passive transfer of T-lymphocyte cell lines from long-term cultures primed against soluble retinal antigen (S-Ag). A continuous T-cell line was established from non-adherent lymph node cells of S-Ag-immunized Lewis rats. The lymphoid cells were propagated in vitro by serially restimulating them with S-Ag in the presence of irradiated syngeneic spleen cells and expanding them in IL-2-containing media. The cell lines exhibited markers specific for T lymphocytes and the majority had the helper phenotype. When naïve rats were inoculated intravenously with anti S-Ag T-cell lines re-exposed to the antigen prior to injection, they developed uveoretinitis with both clinical and histological characteristics in half the time required by S-Ag to induce the disease by active immunization. The rats exhibited a delayed hypersensitivity skin reaction towards S-Ag. Images Figure 2 Figure 3 PMID:3485569

  17. Cytogenetic characteristics of cell lines from Ixodes scapularis (Acari: Ixodidae).

    PubMed

    Chen, C; Munderloh, U G; Kurtti, T J

    1994-05-01

    Three new cell lines, IDE8 and IDE12 from embryos of northern specimens of Ixodes scapularis Say and ISE18 from southern specimens of I. scapularis, were compared cytogenetically via conventional karyotyping, C- and G-banding, and nucleolar organizing regions (NORs). The karyotypes were very similar. The standard karyotype in the three cell lines consisted of 28 chromosomes with 26 autosomes and XX (female) or XY (male) sex chromosomes. The X chromosome was the largest, and the Y chromosome the smallest chromosome of the karyotype. Constitutive heterochromatin (C-bands) was almost entirely restricted to the centromeric region. An additional interstitial C-band in chromosome 7 was an important notable characteristic of the three cell lines. In sets showing a similar degree of condensation, individual chromosomes of the three lines had identical G-banding patterns. In addition, there was no difference among the cells in number and position of NORs. There were approximately 100 G-bands per haploid set in chromosomes from cells in metaphase, with three to 18 G-bands in each chromosome arm. After staining with silver nitrate, interstitial NORs were identified in chromosomes 7, 10, and the X chromosome. Male cells had five and female cells had six NORs. These findings support the notion that I. scapularis and I. dammini Spielman et al. are conspecific.

  18. SENSORY HAIR CELL REGENERATION IN THE ZEBRAFISH LATERAL LINE

    PubMed Central

    Lush, Mark E.; Piotrowski, Tatjana

    2014-01-01

    Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. PMID:25045019

  19. Detection of Dichlorvos Adducts in a Hepatocyte Cell Line

    DTIC Science & Technology

    2014-06-30

    modified targets in lysed human hepatocyte- like cells (HepaRG) using a direct liquid chromatography−mass spectrometry (LC−MS) assay of cell lysates...parasympathetic autonomic nervous system5 and the neuromuscular systems.3 Recent studies also suggest that DDVP affects non-neuronal targets in human ... human hepatocyte-like cell line (HepaRG) with DDVP. Then, we identified DDVP-modified targets in these lysates either with shotgun proteomics or with a

  20. Butyrate-Induced Apoptosis in Prostate Cancer Cell Lines

    DTIC Science & Technology

    2001-09-01

    butyrate-induced apoptosis was independent of cell cycle phase. 14. SUBJECT TERMS 15. NUMBER OF PAGES prostate cancer, histone deacetylase inhibitors, bone...of cells plated) HDI histone deacetylase inhibitor SBHA suberoylbishydroxamate PKC protein kinase C activator SDS-PAGE SDS polyacrylamide gel...cancer cell lines 1. Summary of goals and findings Histone deacetylase inhibitors (HDI) such as butyrate and suberoylbishydroxamate (SBHA) have

  1. Oxidant generation, DNA damage and cytotoxicity by a panel of engineered nanomaterials in three different human epithelial cell lines.

    PubMed

    Thongkam, Waluree; Gerloff, Kirsten; van Berlo, Damien; Albrecht, Catrin; Schins, Roel P F

    2017-01-01

    Due to the steeply increased use of nanomaterials (NMs) for commercial and industrial applications, toxicological assessment of their potential harmful effects is urgently needed. In this study, we compared the DNA-damaging properties and concurrent cytotoxicity of a panel of 10 engineered NMs in three different cell lines in relation to their intrinsic oxidant generating properties. The human epithelial cell lines A549, HK-2 and HepG2 were chosen to represent relevant target organs for NMs in the lung, kidney and liver. Cytotoxicity, evaluated by WST-1 assay in the treatment concentration range of 0.3-80 µg/cm(2), was shown for Ag and ZnO NM in all three cell lines. Cytotoxicity was absent for all other NMs, i.e. five types of TiO2 and two types of multiwalled carbon nanotubes. DNA damage, evaluated by the alkaline comet assay, was observed with Ag and ZnO, albeit only at cytotoxic concentrations. DNA damage varied considerably with the cell line. The oxidant generating properties of the NMs, evaluated by electron spin resonance spectroscopy in cell free conditions, did not correlate with their cytotoxic or DNA-damaging properties. DNA damage by the nanosilver could be partly attributed to its surfactant-containing dispersant. The coating of a TiO2 sample with the commercial surfactant Curosurf augmented its DNA-damaging properties in A549 cells, while surface modification with serum tended to reduce damage. Our findings indicate that measurement of the intrinsic oxidant-generating capacity of NMs is a poor predictor of DNA damage and that the cytotoxic and DNA-damaging properties of NMs can vary substantially with experimental conditions. Our study also underlines the critical importance of selecting appropriate cell systems and aligned testing protocols. Selection of a cell line on the mere basis of its origin may provide only poor insight on organ-specific hazards of NMs. © The Author 2016. Published by Oxford University Press on behalf of the UK

  2. Biological behaviors and proteomics analysis of hybrid cell line EAhy926 and its parent cell line A549.

    PubMed

    Lu, Ze Jun; Ren, Ya Qiong; Wang, Guo Ping; Song, Qi; Li, Mei; Jiang, Sa Sa; Ning, Tao; Guan, Yong Song; Yang, Jin Liang; Luo, Feng

    2009-02-13

    It is well established that cancer cells can fuse with endothelial cells to form hybrid cells spontaneously, which facilitates cancer cells traversing the endothelial barrier to form metastases. However, up to now, little is known about the biologic characteristics of hybrid cells. Therefore, we investigate the malignant biologic behaviors and proteins expression of the hybrid cell line EAhy926 with its parent cell line A549. Cell counting and flow cytometry assay were carried out to assess cell proliferation. The number of cells attached to the extracellular matrix (Matrigel) was measured by MTT assay for the adhesion ability of cells. Transwell chambers were established for detecting the ability of cell migration and invasion. Tumor xenograft test was carried out to observe tumorigenesis of the cell lines. In addition, two-dimensional electrophoresis (2-DE) and mass spectrometry were utilized to identify differentially expressed proteins between in Eahy926 cells and in A549 cells. The doubling time of EAhy926 cell and A549 cell proliferation was 25.32 h and 27.29 h, respectively (P > 0.1). Comparing the phase distribution of cell cycle of EAhy926 cells with that of A549 cells, the percentage of cells in G0/G1 phase, in S phase and in G2/M phase was (63.7% +/- 2.65%) VS (60.0% +/- 3.17%), (15.4% +/- 1.52%) VS (13.8% +/- 1.32%), and (20.9% +/- 3.40%) VS (26.3% +/- 3.17%), respectively (P > 0.05). For the ability of cell adhesion of EAhy926 cells and A549 cells, the value of OD in Eahy926 cells was significantly higher than that in A549 cells (0.3236 +/- 0.0514 VS 0.2434 +/- 0.0390, P < 0.004). We also found that the migration ability of Eahy926 cells was stronger than that of A549 cells (28.00 +/- 2.65 VS 18.00 +/- 1.00, P < 0.01), and that the invasion ability of Eahy926 cells was significantly weak than that of A549 cells (15.33 +/- 0.58 VS 26.67 +/- 2.52, P < 0.01). In the xenograft tumor model, expansive masses of classic tumor were found in the A549 cells

  3. Seven Murine Cell Lines with Properties of Macrophages,

    DTIC Science & Technology

    1981-02-01

    of this study; BALB-G-F, a fibroblast-like line derived from the same culture as BALB-G-M by cloning; L929 cells, a gift from Dr. Rolf Zinkernagel...less than 3% of cells ingested E under the same conditions. BW-J-T, NZW-D-T, BALB-G-T, BALB-G-F, L929 and TE-1 control cells were all nonphagocytic under...induced spreading. Exp. Cell Res. 79, 423, 1973. 30. Rabinovitch, M. and DeStefano, M. J. Use of the local anesthetic lidocaine for cell harvesting

  4. Comparison of Rift Valley fever virus replication in North American livestock and wildlife cell lines.

    PubMed

    Gaudreault, Natasha N; Indran, Sabarish V; Bryant, P K; Richt, Juergen A; Wilson, William C

    2015-01-01

    Rift Valley fever virus (RVFV) causes disease outbreaks across Africa and the Arabian Peninsula, resulting in high morbidity and mortality among young domestic livestock, frequent abortions in pregnant animals, and potentially severe or fatal disease in humans. The possibility of RVFV spreading to the United States or other countries worldwide is of significant concern to animal and public health, livestock production, and trade. The mechanism for persistence of RVFV during inter-epidemic periods may be through mosquito transovarial transmission and/or by means of a wildlife reservoir. Field investigations in endemic areas and previous in vivo studies have demonstrated that RVFV can infect a wide range of animals, including indigenous wild ruminants of Africa. Yet no predominant wildlife reservoir has been identified, and gaps in our knowledge of RVFV permissive hosts still remain. In North America, domestic goats, sheep, and cattle are susceptible hosts for RVFV and several competent vectors exist. Wild ruminants such as deer might serve as a virus reservoir and given their abundance, wide distribution, and overlap with livestock farms and human populated areas could represent an important risk factor. The objective of this study was to assess a variety of cell lines derived from North American livestock and wildlife for susceptibility and permissiveness to RVFV. Results of this study suggest that RVFV could potentially replicate in native deer species such as white-tailed deer, and possibly a wide range of non-ruminant animals. This work serves to guide and support future animal model studies and risk model assessment regarding this high-consequence zoonotic pathogen.

  5. Comparison of Rift Valley fever virus replication in North American livestock and wildlife cell lines

    PubMed Central

    Gaudreault, Natasha N.; Indran, Sabarish V.; Bryant, P. K.; Richt, Juergen A.; Wilson, William C.

    2015-01-01

    Rift Valley fever virus (RVFV) causes disease outbreaks across Africa and the Arabian Peninsula, resulting in high morbidity and mortality among young domestic livestock, frequent abortions in pregnant animals, and potentially severe or fatal disease in humans. The possibility of RVFV spreading to the United States or other countries worldwide is of significant concern to animal and public health, livestock production, and trade. The mechanism for persistence of RVFV during inter-epidemic periods may be through mosquito transovarial transmission and/or by means of a wildlife reservoir. Field investigations in endemic areas and previous in vivo studies have demonstrated that RVFV can infect a wide range of animals, including indigenous wild ruminants of Africa. Yet no predominant wildlife reservoir has been identified, and gaps in our knowledge of RVFV permissive hosts still remain. In North America, domestic goats, sheep, and cattle are susceptible hosts for RVFV and several competent vectors exist. Wild ruminants such as deer might serve as a virus reservoir and given their abundance, wide distribution, and overlap with livestock farms and human populated areas could represent an important risk factor. The objective of this study was to assess a variety of cell lines derived from North American livestock and wildlife for susceptibility and permissiveness to RVFV. Results of this study suggest that RVFV could potentially replicate in native deer species such as white-tailed deer, and possibly a wide range of non-ruminant animals. This work serves to guide and support future animal model studies and risk model assessment regarding this high-consequence zoonotic pathogen. PMID:26175725

  6. Phase transitions in tumor growth: II prostate cancer cell lines

    NASA Astrophysics Data System (ADS)

    Llanos-Pérez, J. A.; Betancourt-Mar, A.; De Miguel, M. P.; Izquierdo-Kulich, E.; Royuela-García, M.; Tejera, E.; Nieto-Villar, J. M.

    2015-05-01

    We propose a mechanism for prostate cancer cell lines growth, LNCaP and PC3 based on a Gompertz dynamics. This growth exhibits a multifractal behavior and a "second order" phase transition. Finally, it was found that the cellular line PC3 exhibits a higher value of entropy production rate compared to LNCaP, which is indicative of the robustness of PC3, over to LNCaP and may be a quantitative index of metastatic potential tumors.

  7. Electrophysiological characterization of Nsc-34 cell line using Microelectrode Array.

    PubMed

    Sabitha, K R; Sanjay, D; Savita, B; Raju, T R; Laxmi, T R

    2016-11-15

    Neurons communicate with each other through intricate network to evolve higher brain functions. The electrical activity of the neurons plays a crucial role in shaping the connectivity. With motor neurons being vulnerable to neurodegenerative diseases, understanding the electrophysiological properties of motor neurons is the need of the hour, in order to comprehend the impairment of connectivity in these diseases. NSC-34 cell line serves as an excellent model to study the properties of motor neurons as they express Choline acetyltransferase (ChAT). Although NSC-34 cell lines have been used to study the effect of various toxicological, neurotrophic and neuroprotective agents, the electrical activity of these cells has not been elucidated. In the current study, we have characterized the electrophysiological properties of NSC-34 cell lines using Micro-Electrode Array (MEA) as a tool. Based on the spike waveform, firing frequency, auto- and cross-correlogram analysis, we demonstrate that NSC-34 cell culture has >2 distinct types of neuronal population: principal excitatory neurons, putative interneurons and unclassified neurons. The presence of interneurons in the NSC-34 culture was characterized by increased expression of GAD-67 markers. Thus, finding an understanding of the electrophysiological properties of different population of neurons in NSC-34 cell line, will have multiple applications in the treatment of neurological disorders.

  8. Cytokine profile of breast cell lines after different radiation doses.

    PubMed

    Bravatà, Valentina; Minafra, Luigi; Forte, Giusi Irma; Cammarata, Francesco Paolo; Russo, Giorgio; Di Maggio, Federica Maria; Augello, Giuseppa; Lio, Domenico; Gilardi, Maria Carla

    2017-09-01

    Ionizing radiation (IR) treatment activates inflammatory processes causing the release of a great amount of molecules able to affect the cell survival. The aim of this study was to analyze the cytokine signature of conditioned medium produced by non-tumorigenic mammary epithelial cell line MCF10A, as well as MCF7 and MDA-MB-231 breast cancer cell lines, after single high doses of IR in order to understand their role in high radiation response. We performed a cytokine profile of irradiated conditioned media of MCF10A, MCF7 and MDA-MB-231 cell lines treated with 9 or 23 Gy, by Luminex and ELISA analyses. Overall, our results show that both 9 Gy and 23 Gy of IR induce the release within the first 72 h of cytokines and growth factors potentially able to influence the tumor outcome, with a dose-independent and cell-line dependent signature. Moreover, our results show that the cell-senescence phenomenon does not correlate with the amount of 'senescence-associated secretory phenotype' (SASP) molecules released in media. Thus, additional mechanisms are probably involved in this process. These data open the possibility to evaluate cytokine profile as useful marker in modulating the personalized radiotherapy in breast cancer care.

  9. Caffeine augments Alprazolam induced cytotoxicity in human cell lines.

    PubMed

    Saha, Biswarup; Mukherjee, Ananda; Samanta, Saheli; Saha, Piyali; Ghosh, Anup Kumar; Santra, Chitta Ranjan; Karmakar, Parimal

    2009-09-01

    Combined effects of alprazolam (Alp), a member of benzodiazepine group of drugs and caffeine on human cell lines, HeLa and THP1 were investigated in this study. Alp mediated cytotoxicity was enhanced while caffeine was present. The cell death was confirmed by observing morphological changes, LDH assay and membrane anisotropic study. Also such combined effects induced elevated level of ROS and depletion of GSH. The mechanism of cell death induced by simultaneous treatment of Alp and caffeine was associated with the calcium-mediated activation of mu-calpain, release of lysosomal protease cathepsin B, activation of PARP and cleavage of caspase 3. Our results indicate that, Alp alone induces apoptosis in human cells but in the presence of caffeine it augments necrosis in a well-regulated pathway. Thus our observations strongly suggest that, alprazolam and caffeine together produce severe cytotoxicity in human cell lines.

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

    PubMed Central

    Mari, Emanuela; Mardente, Stefania; Morgante, Emanuela; Tafani, Marco; Lococo, Emanuela; Fico, Flavia; Valentini, Federica; Zicari, Alessandra

    2016-01-01

    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. PMID:27916824

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

    PubMed

    Mari, Emanuela; Mardente, Stefania; Morgante, Emanuela; Tafani, Marco; Lococo, Emanuela; Fico, Flavia; Valentini, Federica; Zicari, Alessandra

    2016-11-29

    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.

  12. Effects of curcumin on stem-like cells in human esophageal squamous carcinoma cell lines

    PubMed Central

    2012-01-01

    Background Many cancers contain cell subpopulations that display characteristics of stem cells. Because these cancer stem cells (CSCs) appear to provide resistance to chemo-radiation therapy, development of therapeutic agents that target CSCs is essential. Curcumin is a phytochemical agent that is currently used in clinical trials to test its effectiveness against cancer. However, the effect of curcumin on CSCs is not well established. The current study evaluated curcumin-induced cell death in six cancer cell lines derived from human esophageal squamous cell carcinomas. Moreover, these cell lines and the ones established from cells that survived curcumin treatments were characterized. Methods Cell loss was assayed after TE-1, TE-8, KY-5, KY-10, YES-1, and YES-2 cells were exposed to 20–80 μM curcumin for 30 hrs. Cell lines surviving 40 or 60 μM curcumin were established from these six original lines. The stem cell markers aldehyde dehydrogenase-1A1 (ALDH1A1) and CD44 as well as NF-κB were used to compare CSC-like subpopulations within and among the original lines as well as the curcumin-surviving lines. YES-2 was tested for tumorsphere-forming capabilities. Finally, the surviving lines were treated with 40 and 60 μM curcumin to determine whether their sensitivity was different from the original lines. Results The cell loss after curcumin treatment increased in a dose-dependent manner in all cell lines. The percentage of cells remaining after 60 μM curcumin treatment varied from 10.9% to 36.3% across the six lines. The cell lines were heterogeneous with respect to ALDH1A1, NF-κB and CD44 expression. KY-5 and YES-1 were the least sensitive and had the highest number of stem-like cells whereas TE-1 had the lowest. The curcumin-surviving lines showed a significant loss in the high staining ALDH1A1 and CD44 cell populations. Tumorspheres formed from YES-2 but were small and rare in the YES-2 surviving line. The curcumin-surviving lines showed a small but

  13. Generation of cell lines for monoclonal antibody production.

    PubMed

    Alvin, Krista; Ye, Jianxin

    2014-01-01

    Monoclonal antibodies (mAbs) represent the largest group of therapeutic proteins with 30 products approved in the USA and hundreds of therapies currently undergoing clinical trials. The complex nature of mAbs makes their development as therapeutic agents constrained by numerous criteria such as quality, safety, regulation, and quantity. Identification of a clonal cell line expressing high levels of mAb with adequate quality attributes and generated in compliance with regulatory standards is a necessary step prior to a program moving to large-scale production for clinical material. This chapter outlines the stable transfection technology that generates clonal cell lines for commercial manufacturing processes.

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

    PubMed

    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.

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

    PubMed

    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.

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

    SciTech Connect

    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).

  17. Thyroid hormone transport in a human glioma cell line.

    PubMed

    Goncalves, E; Lakshmanan, M; Pontecorvi, A; Robbins, J

    1990-03-05

    The uptake of 3,5,3'-triiodothyronine (T3) and thyroxine (T4) was studied in human glioma cells (Hs 683) and compared with that in several other neural cell lines. At 25 degrees C or 37 degrees C, total cell uptake rose rapidly and reached equilibrium within 60 min. The glioma cells had the highest uptake: 47.6 fmol of L-T3 and 43.4 fmol of L-T4 per 10(6) cells at 37 degrees C. These were inhibited 77% and 72%, respectively, by excess unlabeled hormone. Uptake in the nuclei reached equilibrium between 90 and 120 min and was also highest in glioma cells: 1.46 fmol of L-T3 and 0.49 fmol of L-T4 per 10(6) cells. When expressed as percent of total cell uptake, however, glioma cells had the lowest values (3.1% for L-T3 and 1.1% for L-T4). Also in contrast to other cell lines, glioma cells transported L-T4 almost as effectively as L-T3. D-T3 and D-T4 total cell uptake was 86% and 96% lower than that of the respective L-isomers, and the nuclear uptake as a fraction of the cell uptake was similar. Kinetic analysis of the initial rate of cell uptake gave Vmax values for D-T3 and D-T4 that were 97% and 98% lower than for the L-isomers. Antimycin and monodansylcadaverine decreased the Vmax as well as the equilibrium cell and nuclear uptake of the L-isomers. The apparent nuclear affinity constant for L-T4 in intact cells was inhibited 90% in the presence of antimycin, whereas no effect was observed in isolated nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Increased EGF receptors on human squamous carcinoma cell lines.

    PubMed Central

    Cowley, G. P.; Smith, J. A.; Gusterson, B. A.

    1986-01-01

    Characterisation and quantitation of epidermal growth factor receptors (EGFR) have been carried out on eight human squamous carcinoma cell lines and the results compared with those from simian virus transformed keratinocytes and normal keratinocytes grown under similar conditions. All cells tested possess both high and low affinity receptors with dissociation constants ranging from 2.4 X 10(-10) M to 5.4 X 10(-9) M. When epidermal growth factor (EGF) binds to its receptor it is internalised and degraded and the receptor is down regulated. Malignant cells and virally transformed cells possess 5-50 times more EGF receptors than normal keratinocytes and one cell line LICR-LON-HN-5 possesses up to 1.4 X 10(7) receptors per cell, which is the highest number yet reported for a cell line. These results are discussed in the context of recent data that suggest that the increased expression of EGF receptors in epidermoid malignancies may be an important component of the malignant phenotype in these tumours. PMID:2420349

  19. Mouse DRG Cell Line with Properties of Nociceptors.

    PubMed

    Doran, Ciara; Chetrit, Jonathan; Holley, Matthew C; Grundy, David; Nassar, Mohammed A

    2015-01-01

    In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons.

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

    PubMed Central

    Krampe, Britta

    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 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. PMID:20502964

  1. Transthyretin expression in medulloblastomas and medulloblastoma cell lines.

    PubMed

    Albrecht, S; Bayer, T A; Kraus, J A; Pietsch, T

    1995-10-01

    Transthyretin is a protein crucial to the transport of lipophilic molecules such as thyroid hormones and retinoids. In the central nervous system, large amounts of transthyretin are synthesized by the choroid plexus and are secreted into the cerebrospinal fluid. The choroid plexus is the only site of transthyretin synthesis in the brain. Transthyretin is expressed by most benign and malignant choroid plexus tumours while gliomas and meningiomas do not express transthyretin. Other major sites of transthyretin synthesis are the retinal pigment epithelium and hepatocytes. Medulloblastoma is the prototypical primitive neuroectodermal tumour of the cerebellum and can show multiple lines of differentiation, including the expression of retinal markers. In this study, we examined transthyretin expression both at the RNA and protein level in four medulloblastomas and six medulloblastoma cell lines using Northern and Western blot analysis, reverse transcription polymerase chain reaction (PCR), RNA in situ hybridization, and immunohistochemistry. All four medulloblastomas and five of the six medulloblastoma cell lines expressed transthyretin-mRNA as demonstrated by reverse PCR and in situ hybridization while three medulloblastomas and one cell line were positive on Northern blot. The medulloblastoma with the most abundant RNA expression was transthyretin-immunoreactive on cryosections and the medulloblastoma cell line that was positive on Northern blot also expressed transthyretin at levels detectable by Western blot. No transthyretin-immunoreactivity was seen in 16 additional medulloblastomas studied on paraffin sections. These findings indicate that low-level expression of transthyretin-mRNA is common in medulloblastomas and medulloblastoma cell lines. Expression of transthyretin protein occurs rarely but can reach significant levels. Transthyretin expression in medulloblastoma is consistent with retinal pigment epithelium differentiation in medulloblastomas and reflects

  2. Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines

    PubMed Central

    Coram, Marc A.; Reddy, Anupama; Deng, Glenn; Telli, Melinda L.; Advani, Ranjana H.; Carlson, Robert W.; Mollick, Joseph A.; Sheth, Shruti; Kurian, Allison W.; Ford, James M.; Stockdale, Frank E.; Quake, Stephen R.; Pease, R. Fabian; Mindrinos, Michael N.; Bhanot, Gyan; Dairkee, Shanaz H.; Davis, Ronald W.; Jeffrey, Stefanie S.

    2012-01-01

    Background To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery. Methodology/Principal Findings We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy. Conclusions/Significance For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs

  3. Engineering Retina from Human Retinal Progenitors (Cell Lines)

    PubMed Central

    Cao, Yang

    2009-01-01

    Retinal degeneration resulting in the loss of photoreceptors is the leading cause of blindness. Several therapeutic protocols are under consideration for treatment of this disease. Tissue replacement is one such strategy currently being explored. However, availability of tissues for transplant poses a major obstacle. Another strategy with great potential is the use of adult stem cells, which could be expanded in culture and then utilized to engineer retinal tissue. In this study, we have explored a spontaneously immortalized human retinal progenitor cell line for its potential in retinal engineering using rotary cultures to generate three-dimensional (3D) structures. Retinal progenitors cultured alone or cocultured with retinal pigment epithelial cells form aggregates. The aggregate size increases between days 1 and 10. The cells grown as a 3D culture rotary system, which promotes cell–cell interaction, retain a spectrum of differentiation capability. Photoreceptor differentiation in these cultures is confirmed by significant upregulation of rhodopsin and AaNat, an enzyme implicated in melatonin synthesis (immunohistochemistry and Western blot analysis). Photoreceptor induction and differentiation is further attested to by the upregulation of rod transcription factor Nrl, Nr2e3, expression of interstitial retinal binding protein, and rhodopsin kinase by reverse transcription–polymerase chain reaction. Differentiation toward other cell lineages is confirmed by the expression of tyrosine hydroxylase in amacrine cells, thy 1.1 expression in ganglion cells and calbindin, and GNB3 expression in cone cells. The capability of retinal progenitors to give rise to several retinal cell types when grown as aggregated cells in rotary culture offers hope that progenitor stem cells under appropriate culture conditions will be valuable to engineer retinal constructs, which could be further tested for their transplant potential. The fidelity with which this multipotential cell

  4. The silver lining of induced pluripotent stem cell variation

    PubMed Central

    Jain, Tanya; Sevilla, Ana

    2016-01-01

    Induced pluripotent stem cells (iPSCs) are being generated using various reprogramming methods and from different cell sources. Hence, a lot of effort has been devoted to evaluating the differences among iPSC lines, in particular with respect to their differentiation capacity. While line-to-line variability should mainly reflect the genetic diversity within the human population, here we review some studies that have brought attention to additional variation caused by genomic and epigenomic alterations. We discuss strategies to evaluate aberrant changes and to minimize technical and culture-induced noise, in order to generate safe cells for clinical applications. We focus on the findings from a recent study, which compared the differentiation capacity of several iPSC lines committed to the hematopoietic lineage and correlated the differential maturation capacity with aberrant DNA methylations. Although iPSC variation represents a challenge for the field, we embrace the authors’ perspective that iPSC variations should be used to our advantage for predicting and selecting the best performing iPSC lines, depending on the desired application. PMID:28066788

  5. Maslinic Acid Inhibits Proliferation of Renal Cell Carcinoma Cell Lines and Suppresses Angiogenesis of Endothelial Cells

    PubMed Central

    Thakor, Parth; Song, Wenzhe; Subramanian, Ramalingam B.; Thakkar, Vasudev R.; Vesey, David A.

    2017-01-01

    Despite the introduction of many novel therapeutics in clinical practice, metastatic renal cell carcinoma (RCC) remains a treatment-resistant cancer. As red and processed meat are considered risk factors for RCC, and a vegetable-rich diet is thought to reduce this risk, research into plant-based therapeutics may provide valuable complementary or alternative therapeutics for the management of RCC. Herein, we present the antiproliferative and antiangiogenic effects of maslinic acid, which occurs naturally in edible plants, particularly in olive fruits, and also in a variety of medicinal plants. Human RCC cell lines (ACHN, Caki-1, and SN12K1), endothelial cells (human umbilical vein endothelial cell line [HUVEC]), and primary cultures of kidney proximal tubular epithelial cells (PTEC) were treated with maslinic acid. Maslinic acid was relatively less toxic to PTEC when compared with RCC under similar experimental conditions. In RCC cell lines, maslinic acid induced a significant reduction in proliferation, proliferating cell nuclear antigen, and colony formation. In HUVEC, maslinic acid induced a significant reduction in capillary tube formation in vitro and vascular endothelial growth factor. This study provides a rationale for incorporating a maslinic acid–rich diet either to reduce the risk of developing kidney cancer or as an adjunct to existing antiangiogenic therapy to improve efficacy. PMID:28405545

  6. Volatile metabolomic signature of human breast cancer cell lines.

    PubMed

    Silva, Catarina L; Perestrelo, Rosa; Silva, Pedro; Tomás, Helena; Câmara, José S

    2017-03-03

    Breast cancer (BC) remains the most prevalent oncologic pathology in women, causing huge psychological, economic and social impacts on our society. Currently, the available diagnostic tools have limited sensitivity and specificity. Metabolome analysis has emerged as a powerful tool for obtaining information about the biological processes that occur in organisms, and is a useful platform for discovering new biomarkers or make disease diagnosis using different biofluids. Volatile organic compounds (VOCs) from the headspace of cultured BC cells and normal human mammary epithelial cells, were collected by headspace solid-phase microextraction (HS-SPME) and analyzed by gas chromatography combined with mass spectrometry (GC-MS), thus defining a volatile metabolomic signature. 2-Pentanone, 2-heptanone, 3-methyl-3-buten-1-ol, ethyl acetate, ethyl propanoate and 2-methyl butanoate were detected only in cultured BC cell lines. Multivariate statistical methods were used to verify the volatomic differences between BC cell lines and normal cells in order to find a set of specific VOCs that could be associated with BC, providing comprehensive insight into VOCs as potential cancer biomarkers. The establishment of the volatile fingerprint of BC cell lines presents a powerful approach to find endogenous VOCs that could be used to improve the BC diagnostic tools and explore the associated metabolomic pathways.

  7. Volatile metabolomic signature of human breast cancer cell lines

    PubMed Central

    Silva, Catarina L.; Perestrelo, Rosa; Silva, Pedro; Tomás, Helena; Câmara, José S.

    2017-01-01

    Breast cancer (BC) remains the most prevalent oncologic pathology in women, causing huge psychological, economic and social impacts on our society. Currently, the available diagnostic tools have limited sensitivity and specificity. Metabolome analysis has emerged as a powerful tool for obtaining information about the biological processes that occur in organisms, and is a useful platform for discovering new biomarkers or make disease diagnosis using different biofluids. Volatile organic compounds (VOCs) from the headspace of cultured BC cells and normal human mammary epithelial cells, were collected by headspace solid-phase microextraction (HS-SPME) and analyzed by gas chromatography combined with mass spectrometry (GC–MS), thus defining a volatile metabolomic signature. 2-Pentanone, 2-heptanone, 3-methyl-3-buten-1-ol, ethyl acetate, ethyl propanoate and 2-methyl butanoate were detected only in cultured BC cell lines. Multivariate statistical methods were used to verify the volatomic differences between BC cell lines and normal cells in order to find a set of specific VOCs that could be associated with BC, providing comprehensive insight into VOCs as potential cancer biomarkers. The establishment of the volatile fingerprint of BC cell lines presents a powerful approach to find endogenous VOCs that could be used to improve the BC diagnostic tools and explore the associated metabolomic pathways. PMID:28256598

  8. DIVERSITY OF ARSENIC METABOLISM IN CULTURED HUMAN CANCER CELL LINES

    EPA Science Inventory

    Diversity of arsenic metabolism in cultured human cancer cell lines.

    Arsenic has been known to cause a variety of malignancies in human. Pentavalent As (As 5+) is reduced to trivalent As (As3+) which is further methylated by arsenic methyltransferase(s) to monomethylarson...

  9. Antiproliferative Properties of Clausine-B against Cancer Cell Lines

    PubMed Central

    Wan Mohd Zain, Wan Nor I’zzah; Rahmat, Asmah; Othman, Fauziah; Yap, Taufiq Yun Hin

    2009-01-01

    Background: Clausine B, a carbazole alkaloid isolated from the stem bark of Clausena excavata, was investigated for its antiproliferative activities against five human cancer cell lines: HepG2 (hepatic cancer), MCF-7 (hormone-dependent breast cancer), MDA-MB-231 (non-hormone-dependent breast cancer), HeLa (cervical cancer), and CAOV3 (ovarian cancer). Methods: Chang liver (normal cells) was used as a control. The effect of clausine-B was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Results: Clausine-B was found to be active (IC50<30 μg/mL) against four of the cancer cell lines tested. The IC50 values for these four lines were: 21.50 μg/mL (MDA-MB-231), 22.90 g/ml (HeLa), 27.00 μg/mL (CAOV3) and 28.94 μg/mL (HepG2). Clausine-B inhibited the MCF-7 cancer cell line at 52.90 μg/mL, and no IC50 value was obtained against Chang liver. Conclusion: It is possible that the phenolic group in clausine-B responsible for the antiproliferative activities found in this study. PMID:22589662

  10. DIVERSITY OF ARSENIC METABOLISM IN CULTURED HUMAN CANCER CELL LINES

    EPA Science Inventory

    Diversity of arsenic metabolism in cultured human cancer cell lines.

    Arsenic has been known to cause a variety of malignancies in human. Pentavalent As (As 5+) is reduced to trivalent As (As3+) which is further methylated by arsenic methyltransferase(s) to monomethylarson...

  11. METHYLATION OF ARSENITE BY SOME MAMMALIAN CELL LINES

    EPA Science Inventory

    THIS ABSTRACT WAS SUBMITTED ELECTRONICALLY;. SPACE CONSTRAINTS WERE SEVERE)

    Methylation of Arsenite by Some Mammalian Cell Lines.

    Methylation of arsenite is thought to play an important role in the carcinogenicity of arsenic.
    Aim 1: Determine if there is diffe...

  12. Use of Cell Lines in the Investigation of Pharmacogenetic Loci

    PubMed Central

    Zhang, Wei; Dolan, M. Eileen

    2009-01-01

    Drug response and toxicity, complex traits that are often highly varied among individuals, likely involve multiple genetic and non-genetic factors. Pharmacogenomic research aims to individualize therapy in an effort to maximize efficacy and minimize toxicity for each patient. Cell lines can be used as a model system for cellular pharmacologic effects, which include, but are not limited to, drug-induced cytotoxicity or apoptosis, biochemical effects and enzymatic reactions. Because severe toxicities may be associated with drugs such as chemotherapeutics, cell lines derived from healthy individuals or patients provide a convenient model to study how human genetic variation alters response to these drugs that would be unsafe or unethical to administer to human volunteers. In addition to the traditional candidate gene approaches that focus on well-understood candidate genes and pathways, the availability of extensive genotypic and phenotypic data on some cell line models has begun to allow genome-wide association (GWA) studies to simultaneously test the entire human genome for associations with drug response and toxicity. Though with some important limitations, the use of these cell lines in pharmacogenomic discovery demonstrates the promise of constructing a more comprehensive model that may ultimately integrate both genetic and non-genetic factors to predict individual response and toxicity to anticancer drugs. PMID:19925429

  13. 77 FR 5489 - Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ... identification as part of this project will undergo STR profiling, a DNA profiling method that examines/screens for STRs (DNA elements 2-6 bps long repeated in tandem) in the human chromosomes, that has been shown... are expected between cell line DNA samples originating from unrelated individuals. Each unique...

  14. METHYLATION OF ARSENITE BY SOME MAMMALIAN CELL LINES

    EPA Science Inventory

    THIS ABSTRACT WAS SUBMITTED ELECTRONICALLY;. SPACE CONSTRAINTS WERE SEVERE)

    Methylation of Arsenite by Some Mammalian Cell Lines.

    Methylation of arsenite is thought to play an important role in the carcinogenicity of arsenic.
    Aim 1: Determine if there is diffe...

  15. Cryopreservation of specialized chicken lines using cultured primordial germ cells.

    PubMed

    Nandi, S; Whyte, J; Taylor, L; Sherman, A; Nair, V; Kaiser, P; McGrew, M J

    2016-08-01

    Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells ( PGCS: ). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- ( MHC-: ) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs. © The Author 2016. Published by Oxford University Press on behalf of Poultry Science Association.

  16. Curbing rampant cross-contamination and misidentification of cell lines.

    PubMed

    Nardone, Roland M

    2008-09-01

    A son's challenge started an emeritus professor of biology on a three-year odyssey to get biological researchers to correct a decades-long problem with cross-contaminated and misidentified cell lines. These errors may account for more than 15% of mammalian cultures, wasting resources and undermining the integrity of research.

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

    Cancer.gov

    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.

  18. Drug transporter gene expression in human colorectal tissue and cell lines: modulation with antiretrovirals for microbicide optimization.

    PubMed

    Mukhopadhya, Indrani; Murray, Graeme I; Berry, Susan; Thomson, John; Frank, Bruce; Gwozdz, Garry; Ekeruche-Makinde, Julia; Shattock, Robin; Kelly, Charles; Iannelli, Francesco; Pozzi, Gianni; El-Omar, Emad M; Hold, Georgina L; Hijazi, Karolin

    2016-02-01

    The objectives of this study were to comprehensively assess mRNA expression of 84 drug transporters in human colorectal biopsies and six representative cell lines, and to investigate the alteration of drug transporter gene expression after exposure to three candidate microbicidal antiretroviral (ARV) drugs (tenofovir, darunavir and dapivirine) in the colorectal epithelium. The outcome of the objectives informs development of optimal ARV-based microbicidal formulations for prevention of HIV-1 infection. Drug transporter mRNA expression was quantified from colorectal biopsies and cell lines by quantitative real-time PCR. Relative mRNA expression was quantified in Caco-2 cells and colorectal explants after induction with ARVs. Data were analysed using Pearson's product moment correlation (r), hierarchical clustering and principal component analysis (PCA). Expression of 58 of the 84 transporters was documented in colorectal biopsies, with genes for CNT2, P-glycoprotein (P-gp) and MRP3 showing the highest expression. No difference was noted between individual subjects when analysed by age, gender or anatomical site (rectum or recto-sigmoid) (r = 0.95-0.99). High expression of P-gp and CNT2 proteins was confirmed by immunohistochemical staining. Similarity between colorectal tissue and cell-line drug transporter gene expression was variable (r = 0.64-0.84). PCA showed distinct clustering of human colorectal biopsy samples, with the Caco-2 cells defined as the best surrogate system. Induction of Caco-2 cell lines with ARV drugs suggests that darunavir-based microbicides incorporating tenofovir may result in drug-drug interactions likely to affect distribution of individual drugs to sub-epithelial target cells. These findings will help optimize complex formulations of rectal microbicides to realize their full potential as an effective approach for pre-exposure prophylaxis against HIV-1 infection. © The Author 2015. Published by Oxford University Press on behalf of the

  19. Radiation-induced delayed cell death in a hypomorphic Artemis cell line.

    PubMed

    Evans, Paul M; Woodbine, Lisa; Riballo, Enriquetta; Gennery, Andrew R; Hubank, Michael; Jeggo, Penny A

    2006-04-15

    Null mutations in Artemis confer a condition described as RS-SCID, in which patients display radiosensitivity combined with severe combined immunodeficiency. Here, we characterize the defect in Artemis in a patient who displayed progressive combined immunodeficiency (CID) and elevated lymphocyte apoptosis. The patient is a compound heterozygote with novel mutations in both alleles, resulting in Artemis proteins with either L70 deletion or G126D substitution. Both mutational changes impact upon Artemis function and a fibroblast cell line derived from the patient (F96-224) has greatly reduced Artemis protein. In contrast to Artemis null cell lines, which fail to repair a subset of DNA double strand breaks (DSBs) induced by ionizing radiation, F96-224 cells show slow but residual DSB rejoining. Despite showing intermediate cellular and clinical features, F96-224 cells are as radiosensitive as Artemis null cell lines. We developed a FACS-based assay to examine cell division and cellular characteristics for 10 days following exposure to ionizing radiation (2 and 4 Gy). This analysis demonstrated that F96-224 cells show delayed cell death when compared with rapid growth arrest of an Artemis null cell line, and the emergence of a cycling population shown by a control line. F96-224 cells also display elevated chromosome aberrations when compared with control cells. F96-224 therefore represents a novel phenotype for a hypomorphic cell line. We suggest that delayed cell death contributes to the progressive CID phenotype of the Artemis patient.

  20. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    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 described...

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

    PubMed Central

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

    2017-01-01

    Summary 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 a comprehensive system of mouse melanoma cell lines that are syngeneic to C57Bl/6J, have well-defined human-relevant driver mutations, and are genomically stable. These will be a useful tool for the study of tumor immunology and genotype-specific cancer biology. PMID:27287723

  2. Establishment a CHO Cell Line Expressing Human CD52 Molecule

    PubMed Central

    Kadijeh, Tati; Mahsa, Yazdanpanah-Samani; Amin, Ramezani; Elham, Mahmoudi Maymand; Abbas, Ghaderi

    2016-01-01

    Background: CD52 is a small glycoprotein with a GPI anchor at its C-terminus. CD52 is expressed by Normal and malignant T and B lymphocytes and monocytes. There are detectable amounts of soluble CD52 in plasma of patients with CLL and could be used as a tumor marker. Although the biological function of CD52 is unknown but it seems that CD52 may be involved in migration and activation of T-cells .The aim of this study was to clone and express human CD52 gene in CHO cell line and studying its function in more details Methods: Based on GenBank databases two specific primers were designed for amplification of cd52 gene. Total RNA was extracted from Raji cell line and cDNA synthesized. Amplified fragment was cloned in pBudCE4.1 vector. The new construct was transfected to CHO-K1 cell line using electroporation method. Expression of recombinant CD52 protein was evaluated by Real time PCR and flow cytometry methods. Results: Amplification of CD52 gene using specific primers on Raji cDNA showed a 209 bp band. New construct was confirmed by PCR and restriction pattern and sequence analysis. The new construct was designated as pBudKT1. RT-PCR analysis detected cd52 mRNAs in transfected cells and Flow cytometry Results showed that 78.4 % of cells represented CD52 in their surfaces. Conclusion: In conclusion, we established a human CD52 positive cell line, CHO-CD52, and the protein was expressed on the membrane. Cloning of the CD52 gene could be the first step for the production of therapeutic monoclonal antibodies and detection systems for soluble CD52 in biological fluids PMID:28070536

  3. Immortality of cell lines: challenges and advantages of establishment.

    PubMed

    Maqsood, Muhammad Irfan; Matin, Maryam M; Bahrami, Ahmad Reza; Ghasroldasht, Mohammad M

    2013-10-01

    Cellular immortality happens upon impairment of cell-cycle checkpoint pathways (p53/p16/pRb), reactivation or up-regulation of telomerase enzyme, or upregulation of some oncogenes or oncoproteins leading to a higher rate of cell division.There are also some other factors and mechanisms involved in immortalisation, which need to be discovered. Immortalisation of cells derived from different sources and establishment of immortal cell lines has proven useful in understanding the molecular pathways governing cell developmental cascades in eukaryotic, especially human, cells. After the breakthrough of achieving the immortal cells and understanding their critical importance in the field of molecular biology, intense efforts have been dedicated to establish cell lines useful for elucidating the functions of telomerase, developmental lineage of progenitors, self-renewal potency, cellular transformation, differentiation patterns and some bioprocesses, like odontogenesis. Meanwhile, discovering the exact mechanisms of immortality, a major challenge for science yet, is believed to open new gateways toward understanding and treatment of cancer in the long term. This review summarises the methods involved in establishing immortality, its advantages and the challenges still being faced in this field.

  4. The effect of sclareol on growth and cell cycle progression of human leukemic cell lines.

    PubMed

    Dimas, K; Kokkinopoulos, D; Demetzos, C; Vaos, B; Marselos, M; Malamas, M; Tzavaras, T

    1999-03-01

    Sclareol, a labdane-type diterpene, was tested for cytotoxic effect against a panel of established human leukemic cell lines. The compound showed an IC50 lower than 20 microg/ml in most cell lines tested, while it was higher for resting peripheral blood mononuclear leukocytes (PBML). Furthermore, the compound was tested for cytostatic activity against four of the leukemic cell lines used. At a concentration of 20 microg/ml the compound showed a significant cytostatic effect as soon as 4 h after continuous incubation against two from B and two from T lineage cell lines. The morphology and the kind of death induced from sclareol in three cell lines, was also investigated. The effect of sclareol on the cell cycle progression of two cell lines, using flow cytometry, was examined. The results show that sclareol kills cell lines, through the process of apoptosis. The appearance of the apoptotic signs is time and dose dependent. From the flow cytometry experiments, a delay of the cell population on G0/1 seems to take place. This is the first report, that a labdane type diterpene kills tumor cells via a phase specific mechanism which induces apoptosis.

  5. Biological characteristics of side population cells in a self-established human ovarian cancer cell line

    PubMed Central

    WEI, ZHENTONG; LV, SHUANG; WANG, YISHU; SUN, MEIYU; CHI, GUANGFAN; GUO, JUN; SONG, PEIYE; FU, XIAOYU; ZHANG, SONGLING; LI, YULIN

    2016-01-01

    The aim of the present study was to establish an ovarian cancer (OC) cell line from ascites of an ovarian serous cystadenocarcinoma patient and investigate the biological characteristics of its side population (SP) cells. The OC cell line was established by isolating, purifying and subculturing primary cells from ascites of an ovarian serous cystadenocarcinoma patient (stage IIIc; grade 3). SP and non-SP (NSP) cells were isolated by fluorescence-activated cell sorting and cultured in serum-free medium and soft agar to compare the tumorsphere and colony formation capacities. Furthermore, SP and NSP cell tumorigenesis was examined by subcutaneous and intraperitoneal injection of the cells to non-obese diabetic/severe combined immune deficiency (NOD/SCID) mice. Drug resistance to cisplatin was examined by cell counting kit-8. The OC cell line was successfully established from ascites of an ovarian serous cystadenocarcinoma patient, which exhibited properties similar to primary tumors subsequent to >50 passages and >2 years of culture. The SP cell ratio was 0.38% in the OC cell line, and a similar SP cell ratio (0.39%) was observed when sorted SP cells were cultured for 3 weeks. Compared with NSP cells, SP cells exhibited increased abilities in differentiation and tumorsphere and colony formation, in addition to the formation of xenografted tumors and ascites and metastasis of the tumors in NOD/SCID mice, even at low cell numbers (3.0×103 cells). The xenografted tumors demonstrated histological features similar to primary tumors and expressed the ovarian serous cystadenocarcinoma marker CA125. In addition, SP cells demonstrated a significantly stronger drug resistance to cisplatin compared with NSP and unsorted cells, while treatment with verapamil, an inhibitor of ATP-binding cassette transporters, potently abrogated SP cell drug resistance. In conclusion, the present study verified SP cells from an established OC cell line and characterized the cells with self

  6. Cytotoxic effects of Euterpe oleracea Mart. in malignant cell lines

    PubMed Central

    2014-01-01

    Background Euterpe oleracea Mart., a plant from the Amazon region, is commonly known as açaí or juçara; it has high nutritional value and elevated levels of lipids, proteins, and minerals. Açaí is an abundant and much consumed fruit by the Amazon local population, and studies have demonstrated that it is rich in phytochemicals with antioxidant, anti-inflammatory, and anticancer activities. Therefore, the aim of this study was to test this plant for anticancer activity in different human malignant cell lines. Methods Cell lines derived from breast and colorectal adenocarcinomas were treated with 10, 20, and 40 μg/mL of bark, seed, and total açaí fruit hydroalcoholic extracts for 24 and 48 h. After treatment, cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and cell morphological features were observed by light and transmission electron microscopy. The type of cell death was also evaluated. The data were analyzed statistically by one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s post hoc tests, as appropriate. Results We observed that of all the cell lines tested, MCF-7 was the only line that responded to açaí treatment. The extracts caused significant reduction (p < 0.01) in cell viability and altered cell morphological features by inducing the appearance of autophagic vacuoles, as observed by transmission electron microscopy. Furthermore, increased expression of LC3BII, a protein marker of autophagosome formation, was observed by western blotting. Caspase Glo™ assays and morphologic observations by DAPI nuclear staining and transmission electron microscopy did not indicate any apoptotic events. Conclusions The present study demonstrated that açaí possesses antitumorigenic potential in the MCF-7 cell line. Further studies are needed to identify the compound (s) responsible for this cytotoxic activity and the molecular target in the cell. This discovery of the

  7. Diffuse Large B Cell Lymphoma Cell Line U-2946: Model for MCL1 Inhibitor Testing.

    PubMed

    Quentmeier, Hilmar; Drexler, Hans G; Hauer, Vivien; MacLeod, Roderick A F; Pommerenke, Claudia; Uphoff, Cord C; Zaborski, Margarete; Berglund, Mattias; Enblad, Gunilla; Amini, Rose-Marie

    2016-01-01

    Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma worldwide. We describe the establishment and molecular characteristics of the DLBCL cell line U-2946. This cell line was derived from a 52-year-old male with DLBCL. U-2946 cells carried the chromosomal translocation t(8;14) and strongly expressed MYC, but not the mature B-cell lymphoma associated oncogenes BCL2 and BCL6. Instead, U-2946 cells expressed the antiapoptotic BCL2 family member MCL1 which was highly amplified genomically (14n). MCL1 amplification is recurrent in DLBCL, especially in the activated B cell (ABC) variant. Results of microarray expression cluster analysis placed U-2946 together with ABC-, but apart from germinal center (GC)-type DLBCL cell lines. The 1q21.3 region including MCL1 was focally coamplified with a short region of 17p11.2 (also present at 14n). The MCL1 inhibitor A-1210477 triggered apoptosis in U-2946 (MCL1pos/BCL2neg) cells. In contrast to BCL2pos DLBCL cell lines, U-2946 did not respond to the BCL2 inhibitor ABT-263. In conclusion, the novel characteristics of cell line U-2946 renders it a unique model system to test the function of small molecule inhibitors, especially when constructing a panel of DLBCL cell lines expressing broad combinations of antiapoptotic BCL2-family members.

  8. Diffuse Large B Cell Lymphoma Cell Line U-2946: Model for MCL1 Inhibitor Testing

    PubMed Central

    Quentmeier, Hilmar; Drexler, Hans G.; Hauer, Vivien; MacLeod, Roderick A. F.; Pommerenke, Claudia; Uphoff, Cord C.; Zaborski, Margarete; Berglund, Mattias

    2016-01-01

    Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma worldwide. We describe the establishment and molecular characteristics of the DLBCL cell line U-2946. This cell line was derived from a 52-year-old male with DLBCL. U-2946 cells carried the chromosomal translocation t(8;14) and strongly expressed MYC, but not the mature B-cell lymphoma associated oncogenes BCL2 and BCL6. Instead, U-2946 cells expressed the antiapoptotic BCL2 family member MCL1 which was highly amplified genomically (14n). MCL1 amplification is recurrent in DLBCL, especially in the activated B cell (ABC) variant. Results of microarray expression cluster analysis placed U-2946 together with ABC-, but apart from germinal center (GC)-type DLBCL cell lines. The 1q21.3 region including MCL1 was focally coamplified with a short region of 17p11.2 (also present at 14n). The MCL1 inhibitor A-1210477 triggered apoptosis in U-2946 (MCL1pos/BCL2neg) cells. In contrast to BCL2pos DLBCL cell lines, U-2946 did not respond to the BCL2 inhibitor ABT-263. In conclusion, the novel characteristics of cell line U-2946 renders it a unique model system to test the function of small molecule inhibitors, especially when constructing a panel of DLBCL cell lines expressing broad combinations of antiapoptotic BCL2-family members. PMID:27907212

  9. Leukemia L1210 cell lines resistant to ribonucleotide reductase inhibitors.

    PubMed

    Cory, J G; Carter, G L

    1988-02-15

    Leukemia L1210 cell lines, ED1 and ED2, were generated which were resistant to the cytotoxic effects of deoxyadenosine/erythro-9-(2-hydroxyl-3-nonyl)adenine and deoxyadenosine/erythro-9-(2-hydroxyl-3-nonyl)adenine plus 2,3-dihydro-1H-pyrazole[2,3a]imidazole/Desferal, respectively. The ED1 and ED2 were characterized to show that these cell lines had increased levels of ribonucleotide reductase as measured by CDP reduction. The reductase activity in crude cell-free extracts from the ED1 and ED2 cells was not inhibited by dATP. For CDP reductase, the activation by adenylylimido diphosphate and inhibition by dGTP and dTTP in these extracts from the ED1 and ED2 cells were the same as for the wild-type L1210 cells. The ED1 and ED2 cells were highly cross-resistant, as measured by growth inhibition, to deoxyguanosine/8-aminoguanosine, 2-fluorodeoxyadenosine, and 2-fluoroadenine arabinoside. While the ED2 cells showed resistance to 2,3-dihydro-1H-pyrazole-[2,3a]-imidazole/Desferal (6-fold), the ED1 and ED2 cell lines showed less resistance to hydroxyurea, 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone, and the dialdehyde of inosine. These data indicate that the mechanisms of resistance to the ribonucleotide reductase inhibitors are related to the increased level of ribonucleotide reductase activity and to the decreased sensitivity of the effector-binding subunit to dATP.

  10. Zebrafish kidney stromal cell lines support multilineage hematopoiesis

    PubMed Central

    Stachura, David L.; Reyes, Jason R.; Bartunek, Petr; Paw, Barry H.; Zon, Leonard I.

    2009-01-01

    Studies of zebrafish hematopoiesis have been largely performed using mutagenesis approaches and retrospective analyses based upon gene expression patterns in whole embryos. We previously developed transplantation assays to test the repopulation potentials of candidate hematopoietic progenitor cells. We have been impaired, however, in determining cellular differentiation potentials by a lack of short-term functional assays. To enable more precise analyses of hematopoietic progenitor cells, we have created zebrafish kidney stromal (ZKS) cell lines. Culture of adult whole kidney marrow with ZKS cells results in the maintenance and expansion of hematopoietic precursor cells. Hematopoietic growth is dependent upon ZKS cells, and we show that ZKS cells express many growth factors and ligands previously demonstrated to be important in maintaining mammalian hematopoietic cells. In the absence of exogenous growth factors, ZKS cells maintain early hematopoietic precursors and support differentiation of lymphoid and myeloid cells. With the addition of zebrafish erythropoietin, ZKS cells also support the differentiation of erythroid precursors. These conditions have enabled the ability to ascertain more precisely the points at which hematopoietic mutants are defective. The development of robust in vitro assays now provide the means to track defined, functional outcomes for prospectively isolated blood cell subsets in the zebrafish. PMID:19433857

  11. Susceptibility of the PER.C6 cell line for infection with clinical human respiratory syncytial virus isolates.

    PubMed

    Houspie, Lieselot; Keyaerts, Els; Maes, Piet; Van Ranst, Marc

    2012-04-01

    Human respiratory syncytial virus (HRSV) is one of the most important agents causing upper respiratory infection in infants. The susceptibility of the PER.C6 cell line for infection with clinical HRSV strains was investigated. During the HRSV season of 2006/2007 in Belgium, 90 isolates were inoculated into the PER.C6 and HEp-2 cells and viral growth was evaluated by quantification of the extracellular viral RNA concentration. Initially, viral growth of HRSV strains was observed after 7 days of inoculation in the PER.C6 cells, demonstrated by an increase in HRSV RNA levels. However, HRSV strains that were selected for continued propagation in the PER.C6 cells no longer seemed to replicate, which was reflected by consecutive decreasing viral RNA levels. Several approaches that were investigated to improve the efficiency of infection of clinical HRSV isolates in the PER.C6 cells, such as the influence of rotation and centrifugation of the virus inoculum into the cells were unsuccessful. In contrast, when clinical HRSV strains were inoculated in the HEp-2 cells substantial virus stocks could be produced and the virus could be propagated up to infectious titers of 5 logPFU per ml. In conclusion, the PER.C6 cell line was not permissive for persistent HRSV infection in our experiments and did not support long term production of large virus stocks at high titers. This possibly limits the use of this cell line for many in vitro applications using HRSV, e.g. plaque or virus yield reduction assays or the preparation of virus stocks for in vivo challenge experiments or in vitro vaccine production.

  12. 9-{beta}-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

    SciTech Connect

    Heaton, D.; Mustafi, R.; Schwartz, J.L. |

    1992-06-01

    The effect of 9-{beta}-arabinofuranosyladenine (ara-A) on sensitivity to the deleterious effects of x-rays was studied in six squamous cell carcinoma cell lines. Three lines were relatively radioresistant, having D{sub 0} values of 2.31 to 2.89 Gy, and the other three lines were relatively radiosensitive, having D{sub 0} values of between 1.07 and 1.45 Gy. Ara-A (50 or 500 {mu}M) was added to cultures 30 min prior to irradiation and removed 30 min after irradiation, and sensitivity was measured in terms of cell survival. The radiosensitizing effect of ara-A was very dependent on the inherent radiosensitivity of the tumor cell line. Fifty micromolar concentrations of ara-A sensitized only the two most radioresistant lines, SCC-12B.2 and JSQ-3. Five hundred micromolar concentrations of ara-A sensitized the more sensitive cell lines, SQ-20B and SQ-9G, but failed to have any effect on the radiation response of the two most sensitive cell lines, SQ-38 and SCC-61. Concentrations of ara-A as low as 10 {mu}M were equally efficient in inhibiting DNA synthesis in all six cell lines. These results suggest that the target for the radiosensitizing effect of ara-A is probably related to the factor controlling the inherent radiosensitivity of human tumor cells. Therefore, ara-A might be useful in overcoming radiation resistance in vivo.

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

    PubMed Central

    2011-01-01

    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. PMID:21669008

  14. Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines.

    PubMed

    Kowarz, Eric; Löscher, Denise; Marschalek, Rolf

    2015-04-01

    Stable gene expression in mammalian cells is a prerequisite for many in vitro and in vivo experiments. However, either the integration of plasmids into mammalian genomes or the use of retro-/lentiviral systems have intrinsic limitations. The use of transposable elements, e.g. the Sleeping Beauty system (SB), circumvents most of these drawbacks (integration sites, size limitations) and allows the quick generation of stable cell lines. The integration process of SB is catalyzed by a transposase and the handling of this gene transfer system is easy, fast and safe. Here, we report our improvements made to the existing SB vector system and present two new vector types for robust constitutive or inducible expression of any gene of interest. Both types are available in 16 variants with different selection marker (puromycin, hygromycin, blasticidin, neomycin) and fluorescent protein expression (GFP, RFP, BFP) to fit most experimental requirements. With this system it is possible to generate cell lines from stable transfected cells quickly and reliably in a medium-throughput setting (three to five days). Cell lines robustly express any gene-of-interest, either constitutively or tightly regulated by doxycycline. This allows many laboratory experiments to speed up generation of data in a rapid and robust manner. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Fucose-targeted glycoengineering of pharmaceutical cell lines.

    PubMed

    Ogorek, Christiane; Jordan, Ingo; Sandig, Volker; von Horsten, Hans Henning

    2012-01-01

    Glycosylation is known to have an impact on pharmacokinetics and pharmacodynamics of therapeutic proteins. While the production of pharmaceutically desirable glycosylation forms of a therapeutic protein can in certain cases be influenced by the upstream process parameters, certain specialized glycan structures can only be produced in large quantities from cell lines that have been genetically engineered.One particular case where a specialized glycostructure has a major impact on pharmacodynamic mode of action is the enhanced ADCC-effector function of afucosylated IgG1-type monoclonal antibodies. Here we describe the methodological details of a powerful yet simple glycoengineering approach targeted at the fucosylation machinery within eukaryotic cells. As an example we demonstrate the modification of the permanent avian cell line AGE1.CR.pIX which is characterized by a unique glycosylation machinery.

  16. Androglobin knockdown inhibits growth of glioma cell lines

    PubMed Central

    Huang, Bo; Lu, Yi-Sheng; Li, Xia; Zhu, Zhi-Chuan; Li, Kui; Liu, Ji-Wei; Zheng, Jing; Hu, Ze-Lan

    2014-01-01

    Globin family was famous for oxygen supply function of its members such as hemoglobin and myoglobin. With the progress of research, several members of this protein family have been proven to play roles in tumors including glioma. Androglobin (ADGB) is a recently identified member of globin family with very few studies about its function. In the present study, we show that ADGB plays an oncogene role in glioma. Lentiviral vector mediated ADGB knockdown inhibited the proliferation of glioma cell lines determined by MTT assay and colony formation assay. ADGB knockdown also increased the apoptosis of glioma cell line U251 assessed by flow cytometry. In addition, western blot showed that ADGB knockdown altered levels of several proteins related to proliferation, survival or apoptosis in U251 cells. These findings suggest ADGB is involved in the progression of glioma in vitro. PMID:24966926

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

    DOEpatents

    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.

  18. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  19. [Sorting of side population cells from multiple myeloma cell lines and analysis of their biological characteristics].

    PubMed

    Zhang, Xiao-Li; Zhang, Li-Na; Huang, Hong-Ming; Ding, Run-Sheng; Shi, Wei; Xu, Rui-Rong; Yu, Xiao-Tang; Jiang, Sheng-Hua

    2014-06-01

    This study was aimed to sort the side population (SP) cells from human multiple myeloma cell lines, then detect the biological characteristics of those SP cells. After Hoechst33342 staining, intracellular Hoechst33342 fluorescence staining differences of myeloma cell lines observed by the fluorescence microscopy. The fluorescence-activated cell sorting (FACS) technology was used to isolate SP cells and main population (MP) cells; proliferative capacity in vitro was determined by cell growth curve; the cell colony forming ability was compared by colony forming test. The CD138 expression was detected by flow cytometry. The expression of ABCG2 mRNA was detected by reverse transcription PCR; CCK-8 assay and colony forming test were used to evaluate the effect of bortezomib on the cell proliferation, vitality and colony forming ability of the two populations. The results showed that the myeloma cell lines had a small proportion of SP cells, especially, RPMI 8226 cells accounted for the highest proportion of SP cells (7.10 ± 2.69)%, which have also been confirmed under the fluorescence microscope; the proliferative activity and cell colony forming ability of SP cells were significantly higher than those of MP cells (P < 0.05). The expression levels of CD138 in SP and MP cells were not significantly different (P > 0.05). RT-PCR results showed that SP cells expressed the drug-resistance gene ABCG2, but MP cells hardly express these genes. The inhibition rate of bortezomib on SP cells was significantly lower than that on MP cells (P < 0.05), however, the difference was not significant (P > 0.05) at bortezomib 40 nmol/L. Bortezomib could reduce colony formation in the both two cell populations, but more severe reduction appeared in the MP cells. It is concluded that the myeloma cell line contain a small amount of SP cells with the cancer stem cell characteristics.

  20. Zika virus infects cells lining the blood-retinal barrier and causes chorioretinal atrophy in mouse eyes

    PubMed Central

    Singh, Pawan Kumar; Guest, John-Michael; Kanwar, Mamta; Gao, Nan; Juzych, Mark S.; Abrams, Gary W.; Yu, Fu-Shin

    2017-01-01

    Zika virus (ZIKV) is an important pathogen that causes not only neurologic, but also ocular, abnormalities. Thus, it is imperative that models to study ZIKV pathogenesis in the eye are developed to identify potential targets for interventions. Here, we studied ZIKV interactions with human retinal cells and evaluated ZIKV’s pathobiology in mouse eyes. We showed that cells lining the blood-retinal barrier (BRB), the retinal endothelium, and retinal pigment epithelium (RPE) were highly permissive and susceptible to ZIKV-induced cell death. Direct inoculation of ZIKV in eyes of adult C57BL/6 and IFN-stimulated gene 15 (ISG15) KO mice caused chorioretinal atrophy with RPE mottling, a common ocular manifestation of congenital ZIKV infection in humans. This response was associated with induced expression of multiple inflammatory and antiviral (IFNs) response genes in the infected mouse retina. Interestingly, ISG15 KO eyes exhibited severe chorioretinitis, which coincided with increased retinal cell death and higher ZIKV replication. Collectively, our study provides the first evidence to our knowledge that ZIKV causes retinal lesions and infects the cells lining the BRB and that ISG15 plays a role in retinal innate defense against ZIKV infection. Our mouse model can be used to study mechanisms underlying ZIKV-induced chorioretinitis and to gauge ocular antiviral therapies. PMID:28239662

  1. Zika virus infects cells lining the blood-retinal barrier and causes chorioretinal atrophy in mouse eyes.

    PubMed

    Singh, Pawan Kumar; Guest, John-Michael; Kanwar, Mamta; Boss, Joseph; Gao, Nan; Juzych, Mark S; Abrams, Gary W; Yu, Fu-Shin; Kumar, Ashok

    2017-02-23

    Zika virus (ZIKV) is an important pathogen that causes not only neurologic, but also ocular, abnormalities. Thus, it is imperative that models to study ZIKV pathogenesis in the eye are developed to identify potential targets for interventions. Here, we studied ZIKV interactions with human retinal cells and evaluated ZIKV's pathobiology in mouse eyes. We showed that cells lining the blood-retinal barrier (BRB), the retinal endothelium, and retinal pigment epithelium (RPE) were highly permissive and susceptible to ZIKV-induced cell death. Direct inoculation of ZIKV in eyes of adult C57BL/6 and IFN-stimulated gene 15 (ISG15) KO mice caused chorioretinal atrophy with RPE mottling, a common ocular manifestation of congenital ZIKV infection in humans. This response was associated with induced expression of multiple inflammatory and antiviral (IFNs) response genes in the infected mouse retina. Interestingly, ISG15 KO eyes exhibited severe chorioretinitis, which coincided with increased retinal cell death and higher ZIKV replication. Collectively, our study provides the first evidence to our knowledge that ZIKV causes retinal lesions and infects the cells lining the BRB and that ISG15 plays a role in retinal innate defense against ZIKV infection. Our mouse model can be used to study mechanisms underlying ZIKV-induced chorioretinitis and to gauge ocular antiviral therapies.

  2. Establishment and characterization of human non-small cell lung cancer cell lines.

    PubMed

    Li, Jiangchao; Yang, Hong; Chen, Leilei; Li, Yan; Zhu, Yinghui; Dai, Yongdong; Chen, Kai; Ai, Jiaoyu; Zeng, Tingting; Mao, Xueying; Liu, Lulu; Li, Xiaodong; Guan, Xin-Yuan

    2012-01-01

    Non-small cell lung cancer (NSCLC), a highly malignant tumor, is common in China and is associated with a very poor 5-year survival rate. To better understand the cancer biology of this disease, we report here the establishment of three new NSCLC cell lines, SCC210011, SCC211441 and ACC212102, from the tumor tissue of three NSCLC patients. By histological analysis, we found that all three cell lines displayed the typical features of endothelial cancer cells. The population doubling times of SCC210011, SCC211441 and ACC212102 cells were 42, 38 and 25 h, respectively. Our cytogenetic studies indicated that these cell lines exhibit structural and numerical chromosomal abnormalities. Furthermore, the tumorigenicity in nude mice was confirmed, and H&E staining results revealed that they resembled the primary tissue. These newly established cell lines may serve as useful models for studying the molecular pathogenesis of NSCLC.

  3. Characterization of Murine Thymic Stromal-Cell Lines Immortalized by Temperature-Sensitive Simian Virus 40 Large T or Adenovirus 5 E1a

    PubMed Central

    Larsson, Lena; Timms, Emma; Blight, Kenneth; Restall, Deborah E.; Jat, Parmjit S.; Fisher, Amanda G.

    1991-01-01

    The heterogeneity of thymic stromal cells is probably related to their role in providing different microenvironments where T cells can develop. We have immortalized thymic stromal elements using recombinant retroviral constructs containing a temperature-sensitive simian virus 40 (SV40tsA58) large-T antigen gene or the adenovirus 5 E1a region linked to the gene coding for resistance to G418. Cell lines containing the thermolabile large T antigen encoded by SV40 proliferate at the permissive temperature of 33°C and arrest growth when transferred to the nonpermissive temperature of 39°C. At the nonpermissive temperature, ts-derived cell lines are shown to alter their phenotype but remain metabolically active, as indicated by the inducible expression of class I and class II MHC antigens. Here we describe the generation of a total of 84 thymic stromal-cell lines, many of which show distinct morphologic, phenotypic, and functional properties consistent with fibroblastoid, epithelial, or monocytoid origins. Several E1a and SV40tsA58-derived cell lines generated exhibit the epithelial characteristic of desmosome formation and, in addition, two of these lines (15.5 and 15.18) form multicellular complexes (rosettes) when incubated with unfractionated thymocytes from syngeneic mice. A single line (14.5) displays very strong nonspecific esterase activity, suggesting it may represent a macrophagelike cell type. We describe the generation of stromal cell lines with different properties, which is consistent with the heterogeneity found in the thymic microenvironment. In addition to documenting this diversity, these cell lines may be useful tools for studying T-cell development in vitro and give access to model systems in which stromal-thymocyte interactions can be examined. PMID:1668372

  4. Heterogeneity of a human T-lymphoblastoid cell line

    SciTech Connect

    Snow, K.; Judd, W.

    1987-08-01

    A human T-lymphoblastoid cell line (Jurkat) was cloned, and four resulting sublines were characterized in a variety of ways with the objective of gaining information on heterogeneity in cell lines. Within a few weeks of cloning, distinct cellular morphologies and growth patterns became apparent in the four sublines. Growth rate measurements made over 3 months did not show any significant differences between the sublines. Surface protein profiles obtained by radioimmunoprecipitation using antisera in conjunction with extracts from (/sup 35/S)Met and /sup 125/I-labeled cells revealed differences between the sublines. Analysis of total cell DNA showed that one of the sublines possessed only half the chromosome complement of the other sublines and the parental line. Karyotyping confirmed this result and, in addition, demonstrated that chromosome numbers fluctuated around a mean value for each subline. Karyotypic variability became apparent within 2 months of cloning and tended to increase with time in culture. G-banding analysis showed that the analyzed cell populations contained distinctive cytogenetic aberrations. Properties of the cloned sublines were monitored over a 9-month period. One of the sublines that had shown heterogeneous morphology even after 6 weeks maintained the heterogeneity throughout this time. Another subline underwent a marked change in morphology (round to irregular) and growth habit (single cells to large clumps) with increasing time in culture. Interestingly, several alterations to surface proteins accompanied these growth changes. A third subline had relatively stable morphology and chromosome number throughout the 9-month period. The modal chromosome number was hypotetraploid for three sublines and the parent line, but was diploid for another subline.

  5. Comparative Proteomic Profiling of Pancreatic Ductal Adenocarcinoma Cell Lines

    PubMed Central

    Kim, Yikwon; Han, Dohyun; Min, Hophil; Jin, Jonghwa; Yi, Eugene C.; Kim, Youngsoo

    2014-01-01

    Pancreatic cancer is one of the most fatal cancers and is associated with limited diagnostic and therapeutic modalities. Currently, gemcitabine is the only effective drug and represents the preferred first-line treatment for chemotherapy. However, a high level of intrinsic or acquired resistance of pancreatic cancer to gemcitabine can contribute to the failure of gemcitabine treatment. To investigate the underlying molecular mechanisms for gemcitabine resistance in pancreatic cancer, we performed label-free quantification of protein expression in intrinsic gemcitabine-resistant and - sensitive human pancreatic adenocarcinoma cell lines using our improved proteomic strategy, combined with filter-aided sample preparation, single-shot liquid chromatography-mass spectrometry, enhanced spectral counting, and a statistical method based on a power law global error model. We identified 1931 proteins and quantified 787 differentially expressed proteins in the BxPC3, PANC-1, and HPDE cell lines. Bioinformatics analysis identified 15 epithelial to mesenchymal transition (EMT) markers and 13 EMT-related proteins that were closely associated with drug resistance were differentially expressed. Interestingly, 8 of these proteins were involved in glutathione and cysteine/methionine metabolism. These results suggest that proteins related to the EMT and glutathione metabolism play important roles in the development of intrinsic gemcitabine resistance by pancreatic cancer cell lines. PMID:25518923

  6. Connexin 43 enhances paclitaxel cytotoxicity in colorectal cancer cell lines

    PubMed Central

    Wang, Siqi; Zhang, Shiwu; Zhao, Zhenying; Zhang, Chunze; Yang, Xiaoyun; Wang, Yijia

    2017-01-01

    Colorectal cancer has a relatively low sensitivity to paclitaxel. The purpose of this study was to investigate the role of connexin 43 (Cx43), which is a structural component of gap junctional communication (GJC), in paclitaxel cytotoxicity in colorectal cancer cells. Three colorectal cancer cell lines (HCT106, HCT116 and LoVo) were transfected with Cx43 and used to examine paclitaxel cytotoxicity. A western blot assay was used to confirm Cx43 expression in transfected cell lines as well as the expression of several proteins that are associated with paclitaxel cytotoxicity. A parachute dye-coupling assay was used to measure GJC function. An MTT assay was used to analyze the viability of paclitaxel-treated cells. Cx43 expression level and GJC function were significantly upregulated by the transfection (P<0.05). The viability of transfected cells was significantly inhibited compared with that of untransfected cells when treated with paclitaxel (20 or 80 nM) at high culture density but not at low culture density (P<0.05). Cx43 transfection significantly increased the mitotic arrest, tubulin polymerization and apoptosis effects of paclitaxel (P<0.05). It was also found that paclitaxel had an inhibitory effect on GJC function after 12 h of treatment in LoVo cells (P<0.05). These results indicate that Cx43 may serve as a target of paclitaxel chemotherapy for colorectal cancer. PMID:28810580

  7. Spontaneous malignant transformation in two epithelial cell lines of rat liver cells.

    PubMed

    Masuji, H; Sato, J

    1976-10-01

    The cellular morphology, chromosomal structure, and tumorigenicity of two lines (B and J-13) of rat epithelial cells were examined serially during in vitro cultivation. The cells for such cultures were derived from the hepatic tissues of two 7-day-old male rats of the Donryu strain. The cultured cells were first inoculated into newborn syngenetic rats on the 641st day in vitro (80th subcultures) for line B, and on the 446th day (58 subcultures) for line J-13. The inoculated cells produced tumors with hemorrhagic ascites in rats after long latent periods, viz, 215-599 days in line B and 170-369 days in line J-13. All the tumors were undifferentiated hepatocarcinomas. The pleomorphism in shape and size of the cultured cells gradually became obvious with time of cultivation and was more pronounced in recultured tumor cells. Chromosomes of the culured cells were a normal diploid pattern until about the 200th day in vitro, but thereafter the modal chromsome number shifted to hypodiploid or hypotriploid via hypodiploid stages. The chromosome constitution of recultured tumor cells resembled that of inoculated cells in number distribution, but had changed to a more complicated karyotype. In experiments with line B, the same marker chromosome was detected in all tumor cells analyzed as had been present in inoculated cells.

  8. Bryostatin analogue-induced apoptosis in mantle cell lymphoma cell lines.

    PubMed

    Lopez-Campistrous, Ana; Song, Xiaohua; Schrier, Adam J; Wender, Paul A; Dower, Nancy A; Stone, James C

    2012-08-01

    The anti-cancer effects of bryostatin-1, a potent diacylglycerol analogue, have traditionally been attributed to its action on protein kinase C. However, we previously documented apoptosis in a B non-Hodgkin lymphoma cell line involving diacylglycerol analogue stimulation of Ras guanyl-releasing protein, a Ras activator, and Bim, a proapoptotic Bcl-2 family protein. To further explore the role of Bim, we examined several Bim-deficient B non-Hodgkin lymphoma cells for their responses to pico, a synthetic bryostatin-1-like compound. The Bim(-) mantle cell lymphoma cell lines Jeko-1, Mino, Sp53, UPN1, and Z138 and the Bim(+) cell line Rec-1, as well as the Burkitt lymphoma cells lines BL2 (Bim(-)) and Daudi (Bim(+)), were examined for their response to pico using assays for proliferation and apoptosis as well as biochemical methods for Ras guanyl-releasing proteins and Bcl-2 family members. With the exception of UPN1, mantle cell lymphoma cell lines underwent pico-induced apoptosis, as did BL2. In some cases, hallmarks of apoptosis were substantially diminished in the presence of mitogen-activated protein kinase kinase inhibitors. Pico treatment generally led to increased expression of proapoptotic Bik, although the absolute levels of Bik varied considerably between cell lines. A pico-resistant variant of Z138 exhibited decreased Bik induction compared to parental Z138 cells. Pico also generally decreased expression of anti-apoptotic Bcl-XL and Mcl1. Although, these changes in Bcl-2 family members seem unlikely to fully account for the differential behavior of the cell lines, our demonstration of a potent apoptotic process in most cell lines derived from mantle cell lymphoma encourages a re-examination of diacylglycerol analogues in the treatment of this subset of B non-Hodgkin lymphoma cases. Copyright © 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  9. Cytotoxicity evaluation of silica nanoparticles using fish cell lines.

    PubMed

    Vo, Nguyen T K; Bufalino, Mary R; Hartlen, Kurtis D; Kitaev, Vladimir; Lee, Lucy E J

    2014-01-01

    Nanoparticles (NPs) have extensive industrial, biotechnological, and biomedical/pharmaceutical applications, leading to concerns over health risks to humans and biota. Among various types of nanoparticles, silica nanoparticles (SiO2 NPs) have become popular as nanostructuring, drug delivery, and optical imaging agents. SiO2 NPs are highly stable and could bioaccumulate in the environment. Although toxicity studies of SiO2 NPs to human and mammalian cells have been reported, their effects on aquatic biota, especially fish, have not been significantly studied. Twelve adherent fish cell lines derived from six species (rainbow trout, fathead minnow, zebrafish, goldfish, haddock, and American eel) were used to comparatively evaluate viability of cells by measuring metabolic impairment using Alamar Blue. Toxicity of SiO2 NPs appeared to be size-, time-, temperature-, and dose-dependent as well as tissue-specific. However, dosages greater than 100 μg/mL were needed to achieve 24 h EC50 values (effective concentrations needed to reduce cell viability by 50%). Smaller SiO2 NPs (16 nm) were relatively more toxic than larger sized ones (24 and 44 nm) and external lining epithelial tissue (skin, gills)-derived cells were more sensitive than cells derived from internal tissues (liver, brain, intestine, gonads) or embryos. Higher EC50 values were achieved when toxicity assessment was performed at higher incubation temperatures. These findings are in overall agreement with similar human and mouse cell studies reported to date. Thus, fish cell lines could be valuable for screening emerging contaminants in aquatic environments including NPs through rapid high-throughput cytotoxicity bioassays.

  10. Isolation, Characterization, and Establishment of Spontaneously Immortalized Cell Line HRPE-2S With Stem Cell Properties.

    PubMed

    Shams Najafabadi, Hoda; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Ranaei Pirmardan, Ehsan; Masoumi, Maryam

    2016-12-12

    The retinal pigment epithelium is a monolayer of highly specialized pigmented cells located between the neural retina and the Bruch's membrane of the choroid. RPE cells play a crucial role in the maintenance and function of the underlying photoreceptors. This study introduces a spontaneously arising human retinal pigment epithelial cell line, HRPE-2S, which was isolated from primary RPE cell culture of 2 days old male donor. We characterized morphology and functional properties of the new cell line. The immortalized cell line was maintained in culture for more than 70 passages and 240 divisions. The average doubling time of the cells was approximately 22 h and got freezed at 26th passage. The cell line expressed RPE-specific markers RPE65 and cell junction protein ZO1 as an epithelial cell marker. It also expressed CHX10, PAX6, Nestin, SOX2 as stem and retinal progenitor cell markers. Ki67 as a marker of cell proliferation was expressed in all HRPE-2S cells. It represented typical epithelial cobblestone morphology and did not phenotypically change through several passages. Stem cell-like aggregations (neurospheres) were observed in SEM microscopy. The cells represented high mitotic index. They could be viable under hypoxic conditions and serum deprivation. According to functional studies, the cell line exhibited stem cell-like behaviors with particular emphasis on its self-renewal capacity. LDH isoenzymes expression pattern confirmed the same cellular source for both of the HRPE-2S cells and primary RPE cells. Characteristics of HRPE-2S cells promise it as an in vitro model for RPE stem cell-based researches. J. Cell. Physiol. 9999: 1-15, 2016. © 2016 Wiley Periodicals, Inc.

  11. Radiation sensitivities of 31 human oesophageal squamous cell carcinoma cell lines

    PubMed Central

    Ban, Sadayuki; Michikawa, Yuichi; Ishikawa, Ken-ichi; Sagara, Masashi; Watanabe, Koji; Shimada, Yutaka; Inazawa, Johji; Imai, Takashi

    2005-01-01

    The purpose of this study was to determine the radiosensitivities of 31 human oesophageal squamous cell carcinoma cell lines with a colony-formation assay. A large variation in radiosensitivity existed among 31 cell lines. Such a large variation may partly explain the poor result of radiotherapy for this cancer. One cell line (KYSE190) demonstrated an unusual radiosensitivity. Ataxia-telangiectasia-mutated (ATM) gene in these cells had five missense mutations, and ATM protein was truncated or degraded. Inability to phosphorylate Chk2 in the irradiated KYSE190 cells suggests that the ATM protein in these cells had lost its function. The dysfunctional ATM protein may be a main cause of unusual radiosensitivity of KYSE190 cells. Because the donor of these cells was not diagnosed with ataxia telangiectasia, mutations in ATM gene might have occurred during the initiation and progression of cancer. Radiosensitive cancer developed in non-hereditary diseased patients must be a good target for radiotherapy. PMID:16045545

  12. Bioenergetic Analysis of Ovarian Cancer Cell Lines: Profiling of Histological Subtypes and Identification of a Mitochondria-Defective Cell Line

    PubMed Central

    Dier, Usawadee; Shin, Dong-Hui; Hemachandra, L. P. Madhubhani P.; Uusitalo, Larissa M.; Hempel, Nadine

    2014-01-01

    Epithelial ovarian cancer (EOC) is the most lethal of all gynecological cancers, and encompasses distinct histological subtypes that have specific genetic and tissues-of-origin differences. Ovarian clear cell carcinoma (OCCC) represents approximately 10% of cases and has been termed a stress responsive cancer. OCCC is characterized by increased expression of oxidative stress and glycolysis-related genes. In the present study, we hypothesized that bioenergetic profiling might uniquely distinguish OCCC from other EOC histological subtypes. Using an extracellular flux analyzer, OCCC lines (ES-2, TOV-21-G) were shown to be highly metabolically active, with high oxygen consumption rate (OCR) and high extracellular acidification rate (ECAR), indicative of enhanced mitochondrial oxidative phosphorylation and glycolytic rate, respectively. A high bioenergetics profile was associated with the cell lines' ability to form anchorage independent spheroids. Given their high glycolytic and mitochondrial activity, OCCC cells displayed strong sensitivity to 2-deoxy-D-glucose and Rotenone growth inhibition, although this chemosensitivity profile was not specific to only OCCC cells. Bioenergetic profiling also identified a non-OCCC cell line, OVCA420, to have severely compromised mitochondrial function, based on low OCR and a lack of stimulation of maximal respiration following application of the uncoupler FCCP. This was accompanied by mitochondrial morphology changes indicative of enhanced fission, increased expression of the mitochondrial fission protein Drp1, a loss of mitochondrial membrane potential and dependence on glycolysis. Importantly, this loss of mitochondrial function was accompanied by the inability of OVCA420 cells to cope with hypoxic stress, and a compromised ability to stabilize HIF-1α in response to 1% O2 hypoxia. This knowledge may be imperative for researchers planning to utilize this cell line for further studies of metabolism and hypoxia, and suggests that

  13. Bioenergetic analysis of ovarian cancer cell lines: profiling of histological subtypes and identification of a mitochondria-defective cell line.

    PubMed

    Dier, Usawadee; Shin, Dong-Hui; Hemachandra, L P Madhubhani P; Uusitalo, Larissa M; Hempel, Nadine

    2014-01-01

    Epithelial ovarian cancer (EOC) is the most lethal of all gynecological cancers, and encompasses distinct histological subtypes that have specific genetic and tissues-of-origin differences. Ovarian clear cell carcinoma (OCCC) represents approximately 10% of cases and has been termed a stress responsive cancer. OCCC is characterized by increased expression of oxidative stress and glycolysis-related genes. In the present study, we hypothesized that bioenergetic profiling might uniquely distinguish OCCC from other EOC histological subtypes. Using an extracellular flux analyzer, OCCC lines (ES-2, TOV-21-G) were shown to be highly metabolically active, with high oxygen consumption rate (OCR) and high extracellular acidification rate (ECAR), indicative of enhanced mitochondrial oxidative phosphorylation and glycolytic rate, respectively. A high bioenergetics profile was associated with the cell lines' ability to form anchorage independent spheroids. Given their high glycolytic and mitochondrial activity, OCCC cells displayed strong sensitivity to 2-deoxy-D-glucose and Rotenone growth inhibition, although this chemosensitivity profile was not specific to only OCCC cells. Bioenergetic profiling also identified a non-OCCC cell line, OVCA420, to have severely compromised mitochondrial function, based on low OCR and a lack of stimulation of maximal respiration following application of the uncoupler FCCP. This was accompanied by mitochondrial morphology changes indicative of enhanced fission, increased expression of the mitochondrial fission protein Drp1, a loss of mitochondrial membrane potential and dependence on glycolysis. Importantly, this loss of mitochondrial function was accompanied by the inability of OVCA420 cells to cope with hypoxic stress, and a compromised ability to stabilize HIF-1α in response to 1% O2 hypoxia. This knowledge may be imperative for researchers planning to utilize this cell line for further studies of metabolism and hypoxia, and suggests that

  14. Epstein–Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines

    PubMed Central

    Zhao, Bo; Mar, Jessica C.; Maruo, Seiji; Lee, Sungwook; Gewurz, Benjamin E.; Johannsen, Eric; Holton, Kristina; Rubio, Renee; Takada, Kenzo; Quackenbush, John; Kieff, Elliott

    2011-01-01

    EBV nuclear antigen 3C (EBNA3C) is an essential transcription factor for EBV transformed lymphoblast cell line (LCL) growth. To identify EBNA3C-regulated genes in LCLs, microarrays were used to measure RNA abundances in each of three different LCLs that conditionally express EBNA3C fused to a 4-OH-Tamoxifen–dependent estrogen receptor hormone binding domain (EBNA3CHT). At least three RNAs were assayed for each EBNA3CHT LCL under nonpermissive conditions, permissive conditions, and nonpermissive conditions with wild-type EBNA3C transcomplementation. Using a two-way ANOVA model of EBNA3C levels, we identified 550 regulated genes that were at least 1.5-fold up- or down-regulated with false discovery rates < 0.01. EBNA3C-regulated genes overlapped significantly with genes regulated by EBNA2 and EBNA3A consistent with coordinated effects on cell gene transcription. Of the 550 EBNA3C-regulated genes, 106 could be placed in protein networks. A seeded Bayesian network analysis of the 80 most significant EBNA3C-regulated genes suggests that RAC1, LYN, and TNF are upstream of other EBNA3C-regulated genes. Gene set enrichment analysis found enrichment for MAP kinase signaling, cytokine–cytokine receptor interactions, JAK-STAT signaling, and cell adhesion molecules, implicating these pathways in EBNA3C effects on LCL growth or survival. EBNA3C significantly up-regulated the CXCL12 ligand and its CXCR4 receptor and increased LCL migration. CXCL12 up-regulation depended on EBNA3C's interaction with the cell transcription factor, RBPJ, which is essential for LCL growth. EBNA3C also up-regulated MYC 1.3-fold and down-regulated CDKN2A exons 2 and 3, shared by p16 and p14, 1.4-fold, with false discovery rates < 5 × 10−4. PMID:21173222

  15. Neurofilament protein aggregation in a cell line model system.

    PubMed

    Hull, Elizabeth; Spoja, Christoffer; Cordova, Matt; Cohlberg, Jeffrey A

    2008-02-01

    Protein aggregates are associated with many diseases and even aggregates of proteins that have no role in disease are inherently toxic to both neuronal and non-neuronal cells. We have developed a model system to explore the mechanism of protein aggregation using a mouse muscle cell line expressing chimeric neurofilament (NF) proteins, a constituent of the protein aggregates in ALS, Lewy body dementia, and Charcot-Marie-Tooth disease. Formation of protein aggregates in these cells leads to reduced cell viability and activated caspases. Aggregates contained both chimeric NF proteins and ubiquitin by immunolocalization and were predominately cytosolic when proteins were expressed at low levels or for shorter periods of time but were present in the nucleus when expression levels increased. This system represents a flexible, new tool to decipher the molecular mechanism of protein aggregation and the contributions of aggregation to cell toxicity.

  16. Whole-genome sequencing of nine esophageal adenocarcinoma cell lines

    PubMed Central

    Contino, Gianmarco; Eldridge, Matthew D.; Secrier, Maria; Bower, Lawrence; Fels Elliott, Rachael; Weaver, Jamie; Lynch, Andy G.; Edwards, Paul A.W.; Fitzgerald, Rebecca C.

    2016-01-01

    Esophageal adenocarcinoma (EAC) is highly mutated and molecularly heterogeneous. The number of cell lines available for study is limited and their genome has been only partially characterized. The availability of an accurate annotation of their mutational landscape is crucial for accurate experimental design and correct interpretation of genotype-phenotype findings. We performed high coverage, paired end whole genome sequencing on eight EAC cell lines—ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4—all verified against original patient material, and one esophageal high grade dysplasia cell line, CP-D. We have made available the aligned sequence data and report single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations, identified by comparison with the human reference genome and known single nucleotide polymorphisms (SNPs). We compare these putative mutations to mutations found in primary tissue EAC samples, to inform the use of these cell lines as a model of EAC. PMID:27594985

  17. Viral and host cellular transcription in Autographa californica nuclear polyhedrosis virus-infected gypsy moth cell lines.

    PubMed Central

    Guzo, D; Rathburn, H; Guthrie, K; Dougherty, E

    1992-01-01

    Infection of two gypsy moth cell lines (IPLB-Ld652Y and IPLB-LdFB) by the Autographa californica multiple-enveloped nuclear polyhedrosis virus (AcMNPV) is characterized by extremely attenuated viral protein synthesis followed by a total arrest of all viral and cellular protein production. In this study, AcMNPV- and host cell-specific transcription were examined. Overall levels of viral RNAs in infected gypsy moth cells were, at most measured times, comparable to RNA levels from an infected cell line (TN-368) permissive for AcMNPV replication. Northern blot (RNA) analyses using viral and host gene-specific probes revealed predominantly normal-length virus- and cell-specific transcripts postinfection. Transport of viral RNAs from the nucleus to the cytoplasm and transcript stability in infected gypsy moth cells also appeared normal compared with similar parameters for AcMNPV-infected TN-368 cells. Host cellular and viral mRNAs extracted from gypsy moth and TN-368 cells at various times postinfection and translated in vitro yielded similar spectra of host and viral proteins. Treatment of infected gypsy moth cells with the DNA synthesis inhibitor aphidicolin eliminated the total protein synthesis shutoff in infected IPLB-LdFB cells but had no effect on protein synthesis inhibition in infected IPLB-Ld652Y cells. The apparent selective block in the translation of viral transcripts early in infection and the absence of normal translation or transcription of host cellular genes at later times is discussed. Images PMID:1560533

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

    PubMed

    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

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

    USDA-ARS?s Scientific Manuscript database

    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,...

  20. A murine stromal cell line promotes the proliferation of the human factor-dependent leukemic cell line UT-7.

    PubMed

    Auffray, I; Dubart, A; Izac, B; Vainchenker, W; Coulombel, L

    1994-05-01

    In long-term human bone marrow cultures, stromal cells of human origin are usually used on the assumption that human primitive progenitor cells do not respond to cytokines produced by stromal cells from other species. There is accumulating evidence, however, that murine stromal cells also promote maintenance and differentiation of very primitive human stem cells, which suggests the existence of novel stromal activities that cross species barriers. In this study, we show that a murine bone marrow-derived stromal cell line, MS-5, allows the proliferation of the human leukemic cell line UT-7. The long-term growth of UT-7 is usually supported only by human interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo). None of these three cytokines was involved in the observed effect, since murine GM-CSF and IL-3 do not act on human cells and MS-5 cells do not produce Epo. Soluble stem cell factor (SCF) induced UT-7 cell proliferation. However, S1/S1 mutant fibroblasts also supported UT-7 cell growth and anti-c-kit antibodies only partially abolished UT-7 cell proliferative response to MS-5 cells. These observations excluded a major role of SCF in this system. MS-5-derived growth-promoting activity was diffusible, but attempts to grow UT-7 cells in high levels of known soluble murine stromal-derived cytokines active on human cells showed no or minimal response, suggesting that MS-5's proliferative effect was not mediated by known cytokines. Finally, involvement of an autocrine loop of activation induced by MS-5 was excluded: RT-PCR analysis did not detect increased transcripts for GM-CSF, IL-3, IL-6, SCF, or Epo in UT-7 cells cocultured for 2 to 6 days with MS-5. In addition, UT-7 cell proliferation on MS-5 was not inhibited by neutralizing antibodies against the human GM-CSF receptor or the human IL-6 receptor alpha chain. Whether UT-7 cell proliferation triggered by MS-5 reflects the existence of novel stromal cytokines or

  1. Rosiglitazone inhibits cell proliferation by inducing G1 cell cycle arrest and apoptosis in ADPKD cyst-lining epithelia cells.

    PubMed

    Liu, Yawei; Dai, Bing; Fu, Lili; Jia, Jieshuang; Mei, Changlin

    2010-06-01

    Abnormal proliferation is an important pathological feature of autosomal dominant polycystic kidney disease (ADPKD). Many drugs inhibiting cell proliferation have been proved to be effective in slowing the disease progression in ADPKD. Recent evidence has suggested that peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have anti-neoplasm effects through inhibiting cell growth and inducing cell apoptosis in various cancer cells. In the present study, we examined the expression of PPARgamma in human ADPKD kidney tissues and cyst-lining epithelial cell line, and found that the expression of PPARgamma was greater in ADPKD kidney tissues and cyst-lining epithelial cell line than in normal kidney tissues and human kidney cortex (HKC) cell line. Rosiglitazone inhibited significantly proliferation of cyst-lining epithelial cells in a concentration- and time-dependent manner. These effects were diminished by GW9662, a specific PPARgamma antagonist. Cell cycle analysis showed a G0/G1 arrest in human ADPKD cyst-lining epithelial cells with rosiglitazone treatment. Analysis of cell cycle regulatory proteins revealed that rosiglitazone decreased the protein levels of proliferating cell nuclear antigen, pRb, cyclin D1, cyclin D2 and Cdk4 but increased the levels of p21 and p27 in a dose-dependent manner. Rosiglitazone also induced apoptosis in cyst-lining epithelial cells, which was correlated with increased bax expression and decreased bcl-2 expression. These results suggest PPARgamma agonist might serve as a promising drug for the treatment of ADPKD.

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

    PubMed

    Yslas, Edith I; Cavallo, Pablo; Acevedo, Diego F; Barbero, César A; Rivarola, Viviana A

    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.

  3. Fibronectin synthesized by a human hepatoma cell line

    SciTech Connect

    Glasgow, J.E.; Colman, R.W.

    1984-07-01

    Fibronectin is a family of immunologically similar glycoproteins which mediate a variety of cell-cell and cell-substratum interactions. It is a constituent of the extracellular matrix of connective tissue and circulates in plasma. When suspension and adherent cultures of a human hepatoma cell line (SK-HEP-1) were incubated in serum-free medium, the resulting conditioned medium contained material which was specifically immunoprecipitated by antisera to human plasma fibronectin. By double immunodiffusion, a component in the conditioned culture medium was shown to form a line of identity with fibronectin in human plasma and to migrate as an alpha 2- to beta-globulin during immunoelectrophoresis. Human fibronectin was quantified in conditioned medium by electroimmunodiffusion, and was found to increase for at least three days at about 0.1 micrograms/10(6) cells/day. Adherent cultures of SK-HEP-1 cells were incubated with L-(/sup 35/S)methionine to label newly synthesized proteins. Labeled fibronectin in conditioned medium or in cell extracts comigrated with fibronectin in human plasma as shown by autoradiography following crossed-immunoelectrophoresis. Fibronectin was demonstrated in the extra-cellular matrix of adherent SK-HEP-1 cultures by immunofluorescence. It was shown previously that SK-HEP-1 cells synthesize alpha 1-protease inhibitor, one of the products of normal hepatocytes. The finding that these hepatoma cells also synthesize fibronectin supports the concept that the hepatocyte may be one source of circulating fibronectin, a possibility consistent with the established role of this cell type in blood plasma protein synthesis.

  4. A preliminary study of side population cells in human gastric cancer cell line HGC-27.

    PubMed

    Gao, Ganglong; Sun, Zhenliang; Wenyong, Liu; Dongxia, Ye; Zhao, Runjia; Zhang, Xueli

    2015-03-16

    Cancer stem cell-like side population (SP) cells, which may be responsible for recurrence, tumor metastasis, and resistance to cancer therapy, have been identified and characterized in several types of cell lines from gastric cancer. However, there is no report on isolation of SP cells from human gastric cancer cell line HGC-27. This study aims to analyze the proportion of SP cells in HGC-27 cell line, differentiate SP from non-side population (NSP) cells, and determine whether the SP cells have certain biological properties of stem cells. (1) HGC-27 suspension was prepared and stained with Hoechst33342 and PI for flow cytometric isolation of SP (2). Differences in proliferation and stemness-related gene expression profiles (CD133, CD44, OCT-4, MDR1, EpCAM, and ABCG2) between SP and NSP cells were detected by gastric formation assay and quantitative real-time PCR (3). Oncogenicity of SP and NSP cells was determined in nude mice in vivo. (1) SP cells accounted for 0.1-1.0% of HGC-27 cells, and decreased to 0% after verapamil inhibition. Using flow cytometry, we sorted 7.5×10⁵ SP cells and most HGC-27 cells were NSP cells (2). Gastric formation assay and MTT demonstrated that there was a significant difference in proliferation between SP and NSP cells. Gene expression analysis showed that the expression of genes was significantly higher in SP cells (3). The oncogenicity experiment in nude mice revealed that 105 SP cells were able to form tumors, which demonstrated higher tumorigenicity than non-SP cells. These results collectively suggested that SP cells from HGC-27 cell line have some cancer stem cell properties and could be used for studying the pathogenesis of gastric cancer, which may contribute to discovery of novel therapeutic targets.

  5. The dual role of TLR3 in metastatic cell line.

    PubMed

    Matijevic, Tanja; Pavelic, Jasminka

    2011-10-01

    Toll-like receptors (TLRs) are members of transmembrane proteins that recognize conserved molecular motifs of viral and bacterial origin and initiate innate immune response. As the role of TLRs in tumors cells is still not clear, our aim was to investigate the role of TLR3 in primary tumor and metastatic cells (SW480, SW620, FaDu and Detroit 562). We have reported here on the dual role of TLR3 in pharynx metastatic cell line (Detroit 562); on one hand TLR3 activation drove cells to apoptosis while on the other its stimulation contributed to tumor progression by altering the expression of tumor promoting genes (PLAUR, RORB) and enhancing the cell migration potential. In addition, we have shown TLR3 signaling pathway is functional in another metastatic cancer cell line (SW620) suggesting TLR3 might be important in the process of tumor metastasis. Since TLR3 agonists have been used in tumor therapy with the aim to activate immune system, scientific contribution of this work is drawing attention to the importance of further work on this topic, especially pro-tumor effect of TLR3, in order to avoid possible side-effects.

  6. Toxicity of Calcium Hydroxide Nanoparticles on Murine Fibroblast Cell Line

    PubMed Central

    Dianat, Omid; Azadnia, Sina; Mozayeni, Mohammad Ali

    2015-01-01

    Introduction: One of the major contributing factors, which may cause failure of endodontic treatment, is the presence of residual microorganisms in the root canal system. For years, most dentists have been using calcium hydroxide (CH) as the intracanal medicament between treatment sessions to eliminate remnant microorganisms. Reducing the size of CH particles into nanoparticles enhances the penetration of this medicament into dentinal tubules and increases their antimicrobial efficacy. This in vitro study aimed to compare the cytotoxicity of CH nanoparticles and conventional CH on fibroblast cell line using the Mosmann’s Tetrazolium Toxicity (MTT) assay. Methods and Materials: This study was conducted on L929 murine fibroblast cell line by cell culture and evaluation of the direct effect of materials on the cultured cells. Materials were evaluated in two groups of 10 samples each at 24, 48 and 72 h. At each time point, 10 samples along with 5 positive and 5 negative controls were evaluated. The samples were transferred into tubes and exposed to fibroblast cells. The viability of cells was then evaluated. The Two-way ANOVA was used for statistical analysis and the level of significance was set at 0.05. Results: Cytotoxicity of both materials decreased over time and for conventional CH was lower than that of nanoparticles. However, this difference was not statistically significant (P>0.05). Conclusion: The cytotoxicity of CH nanoparticles was similar to that of conventional CH. PMID:25598810

  7. Role of glutathione in cisplatin resistance in osteosarcoma cell lines.

    PubMed

    Komiya, S; Gebhardt, M C; Mangham, D C; Inoue, A

    1998-01-01

    This study was designed to examine whether and how glutathione and catalase increase the resistance of osteosarcoma cells to the toxicity of cisplatin. Eight osteosarcoma cell lines were exposed to varying concentrations of cisplatin, and a [3H]thymidine incorporation study then estimated their drug sensitivity. Cells were pretreated with aminotriazole and buthionine sulfoximine to depress catalase and glutathione activities and then entered into the same protocol to assess their sensitivity to cisplatin. Intracytoplasmic levels of catalase and glutathione were measured before and after the treatments. Cisplatin-glutathione conjugates were created to examine how glutathione might depress the toxicity of cisplatin. Although the cell lines differed in the magnitude of their response to cisplatin, there was a statistical correlation between intrinsic glutathione content and cisplatin resistance. Pretreatment with aminotriazole reduced catalase activity by 84% but did not change the sensitivity to cisplatin. Depletion of glutathione activity by 70% increased the sensitivity of the cells to the cytotoxicity of cisplatin. In addition, cisplatin was detoxified following conjugation with glutathione. The increased sensitization to cisplatin toxicity caused by the depletion of glutathione and cisplatin detoxification after the in vitro reaction of glutathione to cisplatin indicated that the formation of the glutathione-cisplatin conjugate was an important mechanism in the cellular resistance to cisplatin. These data also demonstrated that catalase activity did not contribute to resistance to cisplatin and suggested that H2O2-induced oxidative stress did not significantly contribute to the cytotoxicity of cisplatin in osteosarcoma cells.

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

    PubMed Central

    Biskup, Edyta; Manfé, Valentina; Kamstrup, Maria R.; Gniadecki, Robert

    2010-01-01

    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 (doubling 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. PMID:25386244

  9. Bovine viral diarrhea virus (BVDV) in cell lines used for somatic cell cloning.

    PubMed

    Stringfellow, David A; Riddell, Kay P; Givens, M Daniel; Galik, Patricia K; Sullivan, Eddie; Dykstra, Christine C; Robl, James; Kasinathan, Poothapillai

    2005-03-01

    Culture of cell lines from fetuses or postnatal animals is an essential part of somatic cell cloning. Fetal bovine serum (FBS) is commonly used in media for propagation of these cells. Unfortunately, bovine fetuses and postnatal animals as well as FBS are all possible sources of non-cytopathic bovine viral diarrhea virus (BVDV) which is widely distributed among cattle. This study was prompted when screening of samples sent to veterinary diagnostic labs revealed that 15 of 39 fetal fibroblast cell lines used in cloning research were positive for BVDV as determined by various assays including reverse transcription-polymerase chain reaction (RT-PCR). Goals of the research were to use both virus isolation and reverse transcription-nested polymerase chain reaction (RT-nPCR) to confirm which of the cell lines were actually infected with BVDV and to assay samples of media, FBS and the earliest available passages of each cell line in an attempt to determine the source of the viral infections. Sequence analysis of amplified cDNA from all isolates was performed to provide a definitive link between possible sources of virus and infected cell lines. Only 5 of the 39 cell lines were actually infected with BVDV. Three of these five lines were not infected at the earliest cryopreserved passage, leading to the conclusion that they likely became infected after culture in media containing contaminated FBS. In fact, sequence comparison of the amplified cDNA from one lot of FBS confirmed that it was the source of infection for one of these cell lines. Since BVDV was isolated from the remaining two cell lines at the earliest available passage, the fetuses from which they were established could not be ruled out as the source of the virus.

  10. Genetic instability in lymphoblastoid cell lines expressing biallelic and monoallelic variants in the human MUTYH gene.

    PubMed

    Grasso, Francesca; Giacomini, Elisa; Sanchez, Massimo; Degan, Paolo; Gismondi, Viviana; Mazzei, Filomena; Varesco, Liliana; Viel, Alessandra; Bignami, Margherita

    2014-07-15

    The MUTYH DNA glycosylase counteracts mutagenesis by removing adenine misincorporated opposite DNA 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Biallelic germline mutations in MUTYH cause the autosomal recessive MUTYH-associated polyposis (MAP). The impact on genetic instability of the p.Tyr179Cys and p.Arg245His MUTYH variants was evaluated in lymphoblastoid cell lines (LCLs) derived from MAP patients and their relatives in comparison to wild-type LCLs. No difference in MUTYH expression was identified between wild type and LCLs with the p.Tyr179Cys, while the p.Arg245His mutation was associated with an unstable MUTYH protein. LCLs homozygous for the p.Tyr179Cys or the p.Arg245His variant contained increased DNA 8-oxodG levels and exhibited a mutator phenotype at the PIG-A gene. The extent of the increased spontaneous mutation frequency was 3-fold (range 1.6- to 4.6-fold) in four independent LCLs carrying the p.Tyr179Cys variant, while a larger increase (6-fold) was observed in two p.Arg245His LCLs. A similar hypermutability and S-phase delay following treatment with KBrO3 was observed in LCLs homozygous for either variant. When genetic instability was investigated in monoallelic p.Arg245His carriers, mutant frequencies showed an increase which is intermediate between wild-type and homozygous cells, whereas the mutator effect in heterozygous p.Tyr179Cys LCLs was similar to that in homozygotes. These findings indicate that the type of MUTYH mutation can affect the extent of genome instability associated with MUTYH inactivation. In addition, the mild spontaneous mutator phenotype observed in monoallelic carriers highlights the biological importance of this gene in the protection of the genome against endogenous DNA damage. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. Designing of promiscuous inhibitors against pancreatic cancer cell lines

    NASA Astrophysics Data System (ADS)

    Kumar, Rahul; Chaudhary, Kumardeep; Singla, Deepak; Gautam, Ankur; Raghava, Gajendra P. S.

    2014-04-01

    Pancreatic cancer remains the most devastating disease with worst prognosis. There is a pressing need to accelerate the drug discovery process to identify new effective drug candidates against pancreatic cancer. We have developed QSAR models for predicting promiscuous inhibitors using the pharmacological data. Our models achieved maximum Pearson correlation coefficient of 0.86, when evaluated on 10-fold cross-validation. Our models have also successfully validated the drug-to-oncogene relationship and further we used these models to screen FDA approved drugs and tested them in vitro. We have integrated these models in a webserver named as DiPCell, which will be useful for screening and designing novel promiscuous drug molecules. We have also identified the most and least effective drugs for pancreatic cancer cell lines. On the other side, we have identified resistant pancreatic cancer cell lines, which need investigative scanner on them to put light on resistant mechanism in pancreatic cancer.

  12. Regulation of alkaline phosphatase expression in human choriocarcinoma cell lines.

    PubMed Central

    Hamilton, T A; Tin, A W; Sussman, H H

    1979-01-01

    The coincident expression of two structurally distinct isoenzymes of human alkaline phosphatase was demonstrated in two independently derived gestational choriocarcinoma cell lines. These proteins were shown to have enzymatic, antigenic, and physical-chemical properties resembling those of isoenzymes from term placenta and adult liver. The regulation of these isoenzymes has been studied during the exposure of both cell lines to 5-bromodeoxyuridine and dibutyryl cyclic AMP. The responses of the alkaline phosphatase isoenzymes to these agents have also been compared with the response of another protein phenotypic to placenta, the alpha subunit of chorionic gonadotropin. The results show that (i) the separate structural genes coding for placental and liver alkaline phosphatases are regulated in a noncoordinate fashion; (ii) both alkaline phosphatase genes respond independently of the alpha subunit; and (iii) the induction of the placental type isoenzyme occurs via at least two independent pathways. Images PMID:218197

  13. Bisphosphonates induce apoptosis in human breast cancer cell lines

    PubMed Central

    Senaratne, S G; Pirianov, G; Mansi, J L; Arnett, T R; Colston, K W

    2000-01-01

    Breast cancer has a prodigious capacity to metastasize to bone. In women with advanced breast cancer and bone metastases, bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that bisphosphonates may have a direct effect on breast cancer cells. We have investigated the in vitro effects of bisphosphonates zoledronate, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines (MDA-MB-231, Hs 578T and MCF-7). Cell growth was monitored by crystal violet dye assay, and cell viability was quantitated by MTS dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax proteins and identification of the proteolytic cleavage of Poly (ADP)-ribose polymerase (PARP). All four bisphosphonates significantly reduced cell viability in all three cell lines. Zoledronate was the most potent bisphosphonate with IC50values of 15, 20 and 3 μM respectively in MDA-MB-231, MCF-7 and Hs 578T cells. Corresponding values for pamidronate were 40, 35 and 25 μM, whereas clodronate and EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all indicate that bisphosphonates have direct anti-tumour effects on human breast cancer cells. © 2000 Cancer Research Campaign PMID:10780527

  14. Efficient Genetic Method for Establishing Drosophila Cell Lines Unlocks the Potential to Create Lines of Specific Genotypes

    PubMed Central

    Truesdell, Sharon; Paul, Litty; Chen, Ting; Butchar, Jonathan P.; Justiniano, Steven

    2008-01-01

    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 RasV12 (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 RasV12 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. PMID:18670627

  15. Effect of a tsA mutation of simian virus 40 late gene expression: variations between host cell lines.

    PubMed Central

    Alwine, J C; Khoury, G

    1980-01-01

    Infection of AGMK or CV-1 cells by the early simian virus 40 mutant tsA58 at the permissive temperature (32 degrees C) followed by a shift to the nonpermissive temperature (41 degrees C) caused a substantial decrease in the levels of late viral RNA in the cytoplasm of AGMK cells but not CV-1 cells. At the translational level, this depression of late viral RNA levels was reflected by a decrease in late viral protein synthesis. Thus, in AGMK cells, an early region gene product (presumably large T-antigen) appeared to be continuously required for efficient expression of the late viral genes. In contrast, late simian virus 40 gene expression, once it is initiated in CV-1 cells, continued efficiently regardless of the tsA mutation. The difference in expression of the late simian virus 40 genes in these tsA mutant-infected monkey kidney cell lines may reflect a difference in host cell proteins which regulate viral gene expression in conjunction with early viral proteins. Images PMID:6251258

  16. 21 CFR 610.17 - Permissible combinations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Permissible combinations. 610.17 Section 610.17 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS General Provisions § 610.17 Permissible combinations...

  17. 21 CFR 610.17 - Permissible combinations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Permissible combinations. 610.17 Section 610.17 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS General Provisions § 610.17 Permissible combinations...

  18. 47 CFR 101.703 - Permissible communications.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 5 2010-10-01 2010-10-01 false Permissible communications. 101.703 Section 101.703 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES FIXED MICROWAVE SERVICES Common Carrier Fixed Point-to-Point Microwave Service § 101.703 Permissible...

  19. 47 CFR 101.703 - Permissible communications.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 5 2011-10-01 2011-10-01 false Permissible communications. 101.703 Section 101.703 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES FIXED MICROWAVE SERVICES Common Carrier Fixed Point-to-Point Microwave Service § 101.703 Permissible...

  20. 47 CFR 27.802 - Permissible communications.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 2 2014-10-01 2014-10-01 false Permissible communications. 27.802 Section 27.802 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES 1.4 GHz Band § 27.802 Permissible communications. Licensees in...

  1. 47 CFR 27.802 - Permissible communications.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 2 2011-10-01 2011-10-01 false Permissible communications. 27.802 Section 27.802 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES 1.4 GHz Band § 27.802 Permissible communications. Licensees in...

  2. 47 CFR 27.802 - Permissible communications.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 2 2010-10-01 2010-10-01 false Permissible communications. 27.802 Section 27.802 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES 1.4 GHz Band § 27.802 Permissible communications. Licensees in...

  3. 47 CFR 27.802 - Permissible communications.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 2 2012-10-01 2012-10-01 false Permissible communications. 27.802 Section 27.802 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES 1.4 GHz Band § 27.802 Permissible communications. Licensees in...

  4. 47 CFR 27.802 - Permissible communications.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 2 2013-10-01 2013-10-01 false Permissible communications. 27.802 Section 27.802 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES 1.4 GHz Band § 27.802 Permissible communications. Licensees in...

  5. 47 CFR 27.2 - Permissible communications.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 2 2010-10-01 2010-10-01 false Permissible communications. 27.2 Section 27.2 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES General Information § 27.2 Permissible communications. (a) Miscellaneous...

  6. 46 CFR 565.8 - Special permission.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 9 2010-10-01 2010-10-01 false Special permission. 565.8 Section 565.8 Shipping FEDERAL MARITIME COMMISSION REGULATIONS AND ACTIONS TO ADDRESS RESTRICTIVE FOREIGN MARITIME PRACTICES CONTROLLED CARRIERS § 565.8 Special permission. Section 8(d) of the Shipping Act of 1984 (46 U.S.C. 40501(e...

  7. 47 CFR 90.1205 - Permissible operations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 5 2010-10-01 2010-10-01 false Permissible operations. 90.1205 Section 90.1205... § 90.1205 Permissible operations. (a) Unattended and continuous operation is permitted. (b) Voice, data and video operations are permitted. (c) Aeronautical mobile operations are prohibited....

  8. 47 CFR 27.902 - Permissible communications.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 2 2010-10-01 2010-10-01 false Permissible communications. 27.902 Section 27.902 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES 1670-1675 MHz Band § 27.902 Permissible communications....

  9. 47 CFR 27.902 - Permissible communications.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 2 2013-10-01 2013-10-01 false Permissible communications. 27.902 Section 27.902 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES 1670-1675 MHz Band § 27.902 Permissible communications....

  10. 47 CFR 27.902 - Permissible communications.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 2 2012-10-01 2012-10-01 false Permissible communications. 27.902 Section 27.902 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES 1670-1675 MHz Band § 27.902 Permissible communications....

  11. 47 CFR 27.902 - Permissible communications.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 2 2011-10-01 2011-10-01 false Permissible communications. 27.902 Section 27.902 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES 1670-1675 MHz Band § 27.902 Permissible communications....

  12. 47 CFR 27.902 - Permissible communications.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 2 2014-10-01 2014-10-01 false Permissible communications. 27.902 Section 27.902 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES MISCELLANEOUS WIRELESS COMMUNICATIONS SERVICES 1670-1675 MHz Band § 27.902 Permissible communications....

  13. 12 CFR 28.4 - Permissible activities.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... the FRB's Regulation K, 12 CFR part 211. (c) Foreign operations guarantees. A national bank may... 12 Banks and Banking 1 2014-01-01 2014-01-01 false Permissible activities. 28.4 Section 28.4 Banks... Foreign Operations of National Banks § 28.4 Permissible activities. (a) General. Subject to the applicable...

  14. 12 CFR 28.4 - Permissible activities.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... the FRB's Regulation K, 12 CFR part 211. (c) Foreign operations guarantees. A national bank may... 12 Banks and Banking 1 2010-01-01 2010-01-01 false Permissible activities. 28.4 Section 28.4 Banks... Foreign Operations of National Banks § 28.4 Permissible activities. (a) General. Subject to the applicable...

  15. 12 CFR 28.4 - Permissible activities.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... the FRB's Regulation K, 12 CFR part 211. (c) Foreign operations guarantees. A national bank may... 12 Banks and Banking 1 2012-01-01 2012-01-01 false Permissible activities. 28.4 Section 28.4 Banks... Foreign Operations of National Banks § 28.4 Permissible activities. (a) General. Subject to the applicable...

  16. 21 CFR 610.17 - Permissible combinations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Permissible combinations. 610.17 Section 610.17 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS General Provisions § 610.17 Permissible combinations...

  17. 21 CFR 610.17 - Permissible combinations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Permissible combinations. 610.17 Section 610.17 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS General Provisions § 610.17 Permissible combinations...

  18. Antitumoral effect of vanadium compounds in malignant melanoma cell lines.

    PubMed

    Rozzo, Carla; Sanna, Daniele; Garribba, Eugenio; Serra, Maria; Cantara, Alessio; Palmieri, Giuseppe; Pisano, Marina

    2017-09-01

    In this study we evaluated the anticancer activity against malignant melanoma (MM) of four different vanadium species: the inorganic anion vanadate(V) (indicated with VN), and three oxidovanadium(IV) complexes, [V(IV)O(dhp)2] where dhp(-) is the anion 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS2), [V(IV)O(mpp)2] where mpp(-) is 1-methyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS3), and [V(IV)O(ppp)2] where ppp(-) is 1-phenyl-2-methyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS4). The antitumor effects of these compounds were studied against two different MM cell lines (A375 and CN-mel) and a fibroblast cell line (BJ) as normal control. All tested V compounds exert antiproliferative activity on MM cells in a dose dependent manner (IC50 ranges from 2.4μM up to 14μM) being A375 the most sensitive cell line. VN and VS2 were the two most active compounds against A375 (IC50 of 4.7 and 2.6μM, respectively), causing apoptosis and cell cycle block. The experimental data indicate that the cell cycle arrest occurs at different phases for the two V species analyzed (G2 checkpoint for VN and G0/G1 for VS2), showing the importance of the chemical form in determining their mechanism of action. These results add more insights into the landscape of vanadium versatility in biological systems and into its role as a potential cancer therapeutic agent. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    PubMed

    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.

  20. Characterization of stem-like cells in a new astroblastoma cell line.

    PubMed

    Coban, Esra Aydemir; Kasikci, Ezgi; Karatas, Omer Faruk; Suakar, Oznur; Kuskucu, Aysegul; Altunbek, Mine; Türe, Uğur; Sahin, Fikrettin; Bayrak, Omer Faruk

    2017-03-15

    Cell lines established from tumors are the most commonly used models in cancer research, and their use in recent years has enabled a greater understanding of the biology of cancer and the means to develop effective treatment strategies. Astroblastomas are uncommon neuroepithelial tumors of glial origin, predominantly affecting young people, mainly teenagers and children, predominantly females. To date, only a single study has reported that astroblastomas contain a large number of neural stem-like cells, which had only a partial proliferation capacity and differentiation. Our objective was to establish an astroblastoma cell line to investigate the presence of astroblastic cells and cancer stem-like cells. The migratory and invasion abilities of the cells were quantified with invasion and migration assays and compared to a glioblastoma cell line. The presence of stem cells was detected with surface-marker analysis by using flow cytometry, and measuring the differentiation ability with a differentiation assay and the self-renewal capacity with a sphere-forming assay. These characteristics may determine whether this novel cell line is a model for astroblastomas that may have stem-cell characteristics. With this novel cell line, scientists can investigate the molecular pathways underlying astroblastomas and develop new therapeutic strategies for patients with these tumors. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Cholangiocarcinoma cell line TK may be useful for the pharmacokinetic study of the chemotherapeutic agent gemcitabine.

    PubMed

    Kamada, Minori; Akiyoshi, Kohei; Akiyama, Nobutake; Funamizu, Naotake; Watanabe, Michiko; Fujioka, Kouki; Ikeda, Kei-Ichi; Manome, Yoshinobu

    2014-08-01

    Cholangiocarcinoma is a disease with a poor prognosis. A human cholangiocarcinoma cell line, TK, was previously established to enable further understanding of the disease. We conducted this investigation to determine whether or not the TK line is useful for pharmacokinetic study of the chemotherapeutic agent gemcitabine (GEM). Along with the BXPC3 human pancreatic adenocarcinoma cell line, the sensitivity to and effects on the TK cell line of GEM were compared. The influence of deoxycytidine kinase (dCK) transduction was also comparatively investigated. The effects of GEM in terms of drug sensitivity of the TK cell line, cell cycle and levels of transcripts of key enzymes were comparable to the BXPC3 cell line. Responses to the drug were similar in both cell lines. In contrast to pancreatic carcinoma, cell lines for research on cholangiocarcinoma have been limited. This study suggests the application of the TK cell line to the pharmacokinetic study of the chemosensitization of therapeutic drugs, such as GEM.

  2. Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines.

    PubMed

    Tomiya, Noboru; Narang, Someet; Lee, Yuan C; Betenbaugh, Michael J

    2004-01-01

    In the past decades, a large number of studies in mammalian cells have revealed that processing of glycoproteins is compartmentalized into several subcellular organelles that process N-glycans to generate complex-type oligosaccharides with terminal N -acetlyneuraminic acid. Recent studies also suggested that processing of N-glycans in insect cells appear to follow a similar initial pathway but diverge at subsequent processing steps. N-glycans from insect cell lines are not usually processed to terminally sialylated complex-type structures but are instead modified to paucimannosidic or oligomannose structures. These differences in processing between insect cells and mammalian cells are due to insufficient expression of multiple processing enzymes including glycosyltransferases responsible for generating complex-type structures and metabolic enzymes involved in generating appropriate sugar nucleotides. Recent genomics studies suggest that insects themselves may include many of these complex transferases and metabolic enzymes at certain developmental stages but expression is lost or limited in most lines derived for cell culture. In addition, insect cells include an N -acetylglucosaminidase that removes a terminal N -acetylglucosamine from the N-glycan. The innermost N -acetylglucosamine residue attached to asparagine residue is also modified with alpha(1,3)-linked fucose, a potential allergenic epitope, in some insect cells. In spite of these limitations in N-glycosylation, insect cells have been widely used to express various recombinant proteins with the baculovirus expression vector system, taking advantage of their safety, ease of use, and high productivity. Recently, genetic engineering techniques have been applied successfully to insect cells in order to enable them to produce glycoproteins which include complex-type N-glycans. Modifications to insect N-glycan processing include the expression of missing glycosyltransferases and inclusion of the metabolic

  3. Formation of coding joints in V(D)J recombination-inducible severe combined immune deficient pre-B cell lines

    PubMed Central

    Chang, Yung; Brown, Matthew L.

    1999-01-01

    Characterization of the severe combined immune deficient (scid) defect in the recombination process has provided many insights into the underlying mechanisms of variable (diversity) joining recombination. By using recombination-inducible scid pre-B cell lines transformed with the temperature-sensitive Abelson-murine leukemia virus, we show that large quantities of recombination intermediates can be generated, and their resolution can be followed during further cell culture. In this study, we demonstrate that the ability of these scid pre-B cell lines to resolve coding ends depends on the cell culture temperature. At the nonpermissive temperature of 39°C, scid pre-B cell lines fail to form coding joints and contain mostly unresolved hairpin-coding ends. Once the cell culture is returned to the permissive temperature of 33°C, these same cells make a significant amount of coding joints concomitant with the disappearance of hairpin-coding ends. Thus, the scid cells are capable of resolving coding ends under certain culture conditions. However, the majority of the recovered coding joints contains extensive deletions, indicating that the temperature-dependent resolution of coding ends is still scid-like. Our results suggest that the inability of scid cells to promptly nick hairpin-coding ends may lead to aberrant joining in these cells. PMID:9874794

  4. Hepatitis C virus infection of cholangiocarcinoma cell lines.

    PubMed

    Fletcher, Nicola F; Humphreys, Elizabeth; Jennings, Elliott; Osburn, William; Lissauer, Samantha; Wilson, Garrick K; van IJzendoorn, Sven C D; Baumert, Thomas F; Balfe, Peter; Afford, Simon; McKeating, Jane A

    2015-06-01

    Hepatitis C virus (HCV) infects the liver and hepatocytes are the major cell type supporting viral replication. Hepatocytes and cholangiocytes derive from a common hepatic progenitor cell that proliferates during inflammatory conditions, raising the possibility that cholangiocytes may support HCV replication and contribute to the hepatic reservoir. We screened cholangiocytes along with a panel of cholangiocarcinoma-derived cell lines for their ability to support HCV entry and replication. While primary cholangiocytes were refractory to infection and lacked expression of several entry factors, two cholangiocarcinoma lines, CC-LP-1 and Sk-ChA-1, supported efficient HCV entry; furthermore, Sk-ChA-1 cells supported full virus replication. In vivo cholangiocarcinomas expressed all of the essential HCV entry factors; however, cholangiocytes adjacent to the tumour and in normal tissue showed a similar pattern of receptor expression to ex vivo isolated cholangiocytes, lacking SR-BI expression, explaining their inability to support infection. This study provides the first report that HCV can infect cholangiocarcinoma cells and suggests that these heterogeneous tumours may provide a reservoir for HCV replication in vivo.

  5. Biotechnology and the chicken B cell line DT40.

    PubMed

    Bachl, J; Caldwell, R B; Buerstedde, J-M

    2007-01-01

    Protein optimization is a major focus of the biotech and pharmaceutical industry. Various in vitro technologies have been developed to accelerate protein evolution and to achieve protein optimization of functional characteristics such as substrate specificity, enzymatic activity and thermostability. The chicken B cell line DT40 diversifies its immunoglobulin (Ig) gene by gene conversion and somatic hypermutation. This machinery can be directed to almost any gene inserted into the Ig locus. Enormously diverse protein libraries of any gene of interest can be quickly generated in DT40 by utilizing random shuffling of complex genetic domains (gene conversion) and by the introduction of novel non-templated genetic information (random mutagenesis). The unique characteristics of the chicken cell line DT40 make it a powerful in-cell diversification system to improve proteins of interest within living cells. One essential advantage of the DT40 protein optimization approach is the fact that variants are generated within an in-cell system thus allowing the direct screening for desired features in the context of intracellular networks. Utilizing specially designed selection strategies, such as the powerful fluorescent protein technology, enables the reliable identification of protein variants exhibiting the most desirable traits. Thus, DT40 is well positioned as a biotechnological tool to generate optimized proteins by applying a powerful combination of gene specific hypermutation, gene conversion and mutant selection. Copyright 2007 S. Karger AG, Basel.

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

    PubMed Central

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

    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 vivo 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-l cell line) and cholangiocarcinoma cell lines (QBC939, KMBC and OZ cell lines) treated with various doses of TSA were detected by methylthiazol tetrazolium (MTT) assay. A nude mouse model of transplanted gallbladder carcinoma (Mz-ChA-l cell line) was successfully established, and changes in the growth of transplanted tumor after treated with TSA were measured. RESULTS: TSA could inhibit the proliferation of gallbladder carcinoma cell line (Mz-ChA-l 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-l 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. PMID:18442209

  7. Identification and Characterization of CD133(pos) Subpopulation Cells From a Human Laryngeal Cancer Cell Line.

    PubMed

    Qiu, Hai-ou; Wang, Huifang; Che, Na; Li, Dong; Mao, Yong; Zeng, Qiao; Ge, Rongming

    2016-04-06

    Recent research indicates that CD133 are expressed in several kinds of stem cells, among which, its high expression in laryngeal carcinoma has caused wide concern. To further explore efficaciously targeting drugs to laryngeal carcinoma stem cells (CSCs), we transplanted a solid tumor from CSCs into abdominal subcutaneous tissue of nude mice, and then compared the biological characteristics of laryngeal solid tumors with or without cisplatin intervention. In this study, the expression of CD133 was detected in the Hep-2 cell line by flow cytometry. By applying magnetic cell sorting (MACS) technology, we reported the results of purifying CD133-positive cells from a Hep-2 cell line. Cell proliferation, colony formation, and tumor-forming ability were examined in vitro and in vivo to identify the marker of CSCs in Hep-2 cell line. Upon flow cytometry analysis, CD133 was expressed constantly on 40.12±1.32% in Hep-2 cell line. Cell proliferation and colony formation ability were higher in CD133-positive cells compared to CD133-negative cells, and the in vivo tumorigenesis experiment showed the same results as in vitro assay. The 2 subpopulations cells were both sensitive to DDP, among which, the effect of DPP on proliferation ability and tumor-forming ability of CD133-positive cells was obviously greater than that of CD133-negative cells. Above all, our study revealed that CD133-positive cells have properties of higher proliferation, colony formation, and tumorigenesis in Hep-2 cell line, indicating that CD133 could be a marker to characterize laryngeal cancer stem cells.

  8. dsRNA formed as an intermediate during Coxsackievirus infection does not induce NO production in a beta-cell line with or without addition of IFN-gamma.

    PubMed

    Berg, Anna-Karin; Elshebani, Asma; Andersson, Arne; Frisk, Gun

    2005-02-18

    Virus infection is one environmental factor that has been implicated as a precipitating event initiating beta-cell damage during the development of type 1 diabetes. One aim of this study was to investigate how permissive an insulin-producing beta-cell line, RINm5F, is to enterovirus (EV) infections. A second aim was to study if the viral replicative intermediate, double-stranded RNA (dsRNA), together with IFN-gamma results in nitric oxide (NO) production. Monolayer cultures of RINm5F cells were not permissive to infection with seven different strains of EV. However, when the growth pattern of the beta-cell line changed and the cells started to grow as free-floating RIN cell clusters (RCC), all EV strains replicated. Immunostaining for the Coxsackie-adenovirus-receptor (CAR) detected the protein on the free-floating RIN cell clusters, but not on the RINm5F cells cultured as a monolayer of beta-cells. This shows that the CAR expression can change and/or the CAR protein can be redistributed on the cell surface as a consequence of altered growth pattern thus allowing viral replication in a previously non-permissive beta-cell line. As expected, NO production was significantly increased (p<0.05) by addition of synthetic dsRNA and IFN-gamma to the RCC. In contrast, the dsRNA formed during virus infection with a Coxsackievirus B4 strain (E2) with or without addition of IFN-gamma did not induce NO production in these cells. This indicates that synthetic dsRNA does not mimic a real viral infection in that respect, and suggests an NO-independent mechanism for virus-induced beta-cell damage.

  9. Reversal of diabetes following transplantation of an insulin-secreting human liver cell line: Melligen cells

    PubMed Central

    Lawandi, Janet; Tao, Chang; Ren, Binhai; Williams, Paul; Ling, Dora; Swan, M Anne; Nassif, Najah T; Torpy, Fraser R; O’Brien, Bronwyn A; Simpson, Ann M

    2015-01-01

    As an alternative to the transplantation of islets, a human liver cell line has been genetically engineered to reverse type 1 diabetes (TID). The initial liver cell line (Huh7ins) commenced secretion of insulin in response to a glucose concentration of 2.5 mmol/l. After transfection of the Huh7ins cells with human islet glucokinase, the resultant Melligen cells secreted insulin in response to glucose within the physiological range; commencing at 4.25 mmol/l. Melligen cells exhibited increased glucokinase enzymatic activity in response to physiological glucose concentrations, as compared with Huh7ins cells. When transplanted into diabetic immunoincompetent mice, Melligen cells restored normoglycemia. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that both cell lines expressed a range of β-cell transcription factors and pancreatic hormones. Exposure of Melligen and Huh7ins cells to proinflammatory cytokines (TNF-α, IL-1β, and IFN-γ) affected neither their viability nor their ability to secrete insulin to glucose. Gene expression (microarray and qRT-PCR) analyses indicated the survival of Melligen cells in the presence of known β-cell cytotoxins was associated with the expression of NF-κB and antiapoptotic genes (such as BIRC3). This study describes the successful generation of an artificial β-cell line, which, if encapsulated to avoid allograft rejection, may offer a clinically applicable cure for T1D. PMID:26029722

  10. HIV-1 latency in actively dividing human T cell lines

    PubMed Central

    Jeeninga, Rienk E; Westerhout, Ellen M; van Gerven, Marja L; Berkhout, Ben

    2008-01-01

    Background Eradication of HIV-1 from an infected individual cannot be achieved by current drug regimens. Viral reservoirs established early during the infection remain unaffected by anti-retroviral therapy and are able to replenish systemic infection upon interruption of the treatment. Therapeutic targeting of viral latency will require a better understanding of the basic mechanisms underlying the establishment and long-term maintenance of HIV-1 in resting memory CD4 T cells, the most prominent reservoir of transcriptional silent provirus. However, the molecular mechanisms that permit long-term transcriptional control of proviral gene expression in these cells are still not well understood. Exploring the molecular details of viral latency will provide new insights for eventual future therapeutics that aim at viral eradication. Results We set out to develop a new in vitro HIV-1 latency model system using the doxycycline (dox)-inducible HIV-rtTA variant. Stable cell clones were generated with a silent HIV-1 provirus, which can subsequently be activated by dox-addition. Surprisingly, only a minority of the cells was able to induce viral gene expression and a spreading infection, eventhough these experiments were performed with the actively dividing SupT1 T cell line. These latent proviruses are responsive to TNFα treatment and alteration of the DNA methylation status with 5-Azacytidine or genistein, but not responsive to the regular T cell activators PMA and IL2. Follow-up experiments in several T cell lines and with wild-type HIV-1 support these findings. Conclusion We describe the development of a new in vitro model for HIV-1 latency and discuss the advantages of this system. The data suggest that HIV-1 proviral latency is not restricted to resting T cells, but rather an intrinsic property of the virus. PMID:18439275

  11. Biomarkers in Tumorigenesis Using Cancer Cell Lines: A Systematic Review

    PubMed

    Raju K, Lizbeth; Augustine, Dominic; Rao, Roopa S; S V, Sowmya; Haragannavar, Vanishri C; Nambiar, Shwetha; Prasad, Kavitha; Awan, Kamran Habib; Patil, Shankargouda

    2017-09-27

    Cancer is a leading cause of death worldwide. Despite many research advancements in the field, the genetic changes regulating the transformation of normal oral cells into malignant cells have not been fully elucidated. Several studies have evaluated carcinogenesis at the molecular level. Cancer cell lines are commonly used in biomedical research because they provide an unlimited source of cells and represent various stages of initiation and progression of carcinogenesis in vitro. Aims: The objective of the study was to review original research articles using cancer cell lines as a tool to understand carcinogenesis and to identify the genes involved in tumor development. Additionally, we also examined the application of the genes as predictive biomarkers. Methods and Materials: Several databases, including PubMed, Google Scholar, Ebsco, and Science Direct, were searched from 1985 to December 2016 using various combinations of the following key words: “mouth neoplasm”, “cell lines”, and “tumorigenesis”. Original experimental studies published in English were included. We excluded letters to the editor, historic reviews, and unpublished data from the analysis. Results: There were 17 studies (in vitro) included in the analysis. There were 14 genes and 4 miRNAs involved in malignant transformation of oral keratinocytes into cancer cells. The most commonly studied genes were p53, cyclin D1, and hTERT. Conclusion: Additional reviews and studies are needed to identify a panel of genes specific to various potentially malignant disorders and to aid in the early detection of oral squamous cell carcinoma (OSCC) because tumorigenesis involves the mutation of multiple genes. Furthermore, improving advanced cost-effective diagnostic methods may benefit the public health sector. Creative Commons Attribution License

  12. Bombesin stimulates insulin secretion by a pancreatic islet cell line.

    PubMed Central

    Swope, S L; Schonbrunn, A

    1984-01-01

    The amphibian tetradecapeptide, bombesin (BBS) has been shown to stimulate insulin secretion both in vivo and by pancreatic islet cells in vitro. To determine whether BBS can act directly on pancreatic beta cells, we examined its effects on insulin secretion by HIT-T15 cells (HIT cells), a clonal islet cell line. Addition of 100 nM BBS to HIT cells stimulated insulin release 25-fold within 30 sec. The rapid stimulatory effect of BBS on insulin release was short-lived: the secretory rate returned to basal levels after 90 min of BBS treatment. The decrease in the rate of insulin release in the continued presence of BBS was due not to depletion of intracellular insulin stores but to specific desensitization to this peptide. Stimulation of insulin secretion by BBS was dose dependent with an ED50 value (0.51 +/- 0.15 nM) similar to the concentration of BBS-like immunoreactive material in rat plasma. Five BBS analogs, including porcine gastrin-releasing peptide, were as powerful as BBS in stimulating insulin release. The relative potencies of the analogs tested indicated that the COOH-terminal octapeptide sequence in BBS was sufficient for stimulation of release. In contrast, 14 peptides structurally unrelated to BBS did not alter insulin secretion. BBS action was synergistic with that of glucagon; insulin secretion in the presence of maximal concentrations of both peptides was greater than the additive effects of the two peptides added individually. Somatostatin inhibited BBS-stimulated release by 69 +/- 1% with an ID50 value of 3.2 +/- 0.3 nM. These results show that BBS stimulation of insulin secretion by a clonal pancreatic cell line closely parallels its effects in vivo and support the hypothesis that BBS stimulates insulin secretion by a direct effect on the pancreatic beta cell. The clonal HIT cell line provides a homogeneous cell preparation amenable for studies on the biochemical mechanisms of BBS action in the endocrine pancreas. PMID:6143320

  13. Cell culture methods for the establishment of the NCI series of lung cancer cell lines.

    PubMed

    Oie, H K; Russell, E K; Carney, D N; Gazdar, A F

    1996-01-01

    More than 200 human small cell lung cancer and non-small cell lung cancer cell lines were established over 15 years mainly by utilizing the serum-free, hormone and growth factor supplemented, defined media HITES and ACL4. Use of modified, established cell culture techniques such as the mechanical spillout method for the releasing of cell aggregates from tumor tissue, ficoll gradient centrifugation for the separation of tumor cells from erythrocytes and tissue debris, and an apparatue consisting of a platinum tubing attached to a suction flask for removal of spent medium have greatly contributed to the success in culturing tumor cells. Characterization of these lung cancer cell lines have extended our knowledge of lung cell biology. Studies elucidating the nutritional requirements of lung cancer cell growth may be helpful for the manipulation of these tumors in patients.

  14. Restoration of WNT4 inhibits cell growth in leukemia-derived cell lines

    PubMed Central

    2013-01-01

    Background WNT signaling pathways are significantly altered during cancer development. Vertebrates possess two classes of WNT signaling pathways: the “canonical” WNT/β-catenin signaling pathway, and the “non-canonical” pathways including WNT/Ca2+ and WNT/Planar cell polarity [PCP] signaling. WNT4 influences hematopoietic progenitor cell expansion and survival; however, WNT4 function in cancer development and the resulting implications for oncogenesis are poorly understood. The aim of this study was twofold: first, to determine the expression of WNT4 in mature peripheral blood cells and diverse leukemia-derived cells including cell lines from hematopoietic neoplasms and cells from patients with leukemia; second, to identify the effect of this ligand on the proliferation and apoptosis of the blast-derived cell lines BJAB, Jurkat, CEM, K562, and HL60. Methods We determined WNT4 expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in peripheral blood mononuclear cells (PBMCs) and T- and B-lymphocytes from healthy individuals, as well as from five leukemia-derived cell lines and blasts derived from patients with leukemia. To analyze the effect of WNT4 on cell proliferation, PBMCs and cell lines were exposed to a commercially available WNT4 recombinant human protein. Furthermore, WNT4 expression was restored in BJAB cells using an inducible lentiviral expression system. Cell viability and proliferation were measured by the addition of WST-1 to cell cultures and counting cells; in addition, the progression of the cell cycle and the amount of apoptosis were analyzed in the absence or presence of WNT4. Finally, the expression of WNT-pathway target genes was measured by qRT-PCR. Results WNT4 expression was severely reduced in leukemia-derived cell lines and blasts derived from patients with leukemia. The exposure of cell lines to WNT4 recombinant protein significantly inhibited cell proliferation; inducing WNT4 expression in BJAB

  15. Innervation regulates synaptic ribbons in lateral line mechanosensory hair cells.

    PubMed

    Suli, Arminda; Pujol, Remy; Cunningham, Dale E; Hailey, Dale W; Prendergast, Andrew; Rubel, Edwin W; Raible, David W

    2016-06-01

    Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse. © 2016. Published by The Company of Biologists Ltd.

  16. Innervation regulates synaptic ribbons in lateral line mechanosensory hair cells

    PubMed Central

    Pujol, Remy; Cunningham, Dale E.; Hailey, Dale W.; Prendergast, Andrew; Rubel, Edwin W.; Raible, David W.

    2016-01-01

    ABSTRACT Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse. PMID:27103160

  17. Cytogenetic instability of dental pulp stem cell lines.

    PubMed

    Duailibi, Monica Talarico; Kulikowski, Leslie Domenici; Duailibi, Silvio Eduardo; Lipay, Monica Vannucci Nunes; Melaragno, Maria Isabel; Ferreira, Lydia Masako; Vacanti, Joseph Phillip; Yelick, Pamela Crotty

    2012-02-01

    Human adult stem cells (hASCs) offer a potentially renewable source of cell types that are easily isolated and rapidly expanded for use in regenerative medicine and cell therapies without the complicating ethical problems that are associated with embryonic stem cells. However, the eventual therapeutic use of hASCs requires that these cells and their derivatives maintain their genomic stability. There is currently a lack of systematic studies that are aimed at characterising aberrant chromosomal changes in cultured ASCs over time. However, the presence of mosaicism and accumulation of karyotypic abnormalities within cultured cell subpopulations have been reported. To investigate cytogenetic integrity of cultured human dental stem cell (hDSC) lines, we analysed four expanded hDSC cultures using classical G banding and fluorescent in situ hybridisation (FISH) with X chromosome specific probe. Our preliminary results revealed that about 70% of the cells exhibited karyotypic abnormalities including polyploidy, aneuploidy and ring chromosomes. The heterogeneous spectrum of abnormalities indicates a high frequency of chromosomal mutations that continuously arise upon extended culture. These findings emphasise the need for the careful analysis of the cytogenetic stability of cultured hDSCs before they can be used in clinical therapies.

  18. [Mechanisms of gamma-inducible death of Jurkat cells line].

    PubMed

    Gamkrelidze, M M; Bezhitashvili, N D; Pavliashvili, A T; Mchedlishvili, T V; Sanikidze, T V

    2008-06-01

    Mechanisms of radio-inducible death of Jurkat cells were investigated. Human lymphoblastoid T-cell line Jurkat is widely established model for studying apoptosis mechanisms. The cell was radiated by "Teragam" (Czech Republic) by dose 2 g during 1 minute. After radiation cells were incubated at standard conditions during 24 hours. After gamma radiation in cell population amount of cells in gaplois (apoptotic G 0) stage was increased 8,2 folds, in diplois (G 0/G1) stage - by 17%, in synthetic (S) stage decreased by 35% and tetraploid (G2/M) stage by 73% in comparison to control group. It was revealed intensive production of free radicals of oxygen and nitric oxide and decreasing activity of antioxidant enzymes (superoxidismutasa, catalasa and glutathione peroxidase). Revealed dependence between intensification of apoptosis and radiation-induced arrest of cell cycle G2/M phase may be determined by excess amount of free oxygen and nitrogen radicals generated in Jurkat cells as a result of nondirect effects of low doses of gamma radiation.

  19. Pleomorphism and drug resistant cancer stem cells are characteristic of aggressive primary meningioma cell lines.

    PubMed

    Khan, Ishaq; Baeesa, Saleh; Bangash, Mohammed; Schulten, Hans-Juergen; Alghamdi, Fahad; Qashqari, Hanadi; Madkhali, Nawal; Carracedo, Angel; Saka, Mohamad; Jamal, Awatif; Al-Maghrabi, Jaudah; AlQahtani, Mohammed; Al-Karim, Saleh; Damanhouri, Ghazi; Saini, Kulvinder; Chaudhary, Adeel; Abuzenadah, Adel; Hussein, Deema

    2017-01-01

    Meningioma tumors arise in arachnoid membranes, and are the most reported central nervous system (CNS) tumors worldwide. Up to 20% of grade I meningioma tumors reoccur and currently predictive cancer stem cells (CSCs) markers for aggressive and drug resistant meningiomas are scarce. Meningioma tissues and primary cell lines were investigated using whole transcriptome microarray analysis, immunofluorescence staining of CSCs markers (including CD133, Sox2, Nestin, and Frizzled 9), and drug treatment with cisplatin or etoposide. Unsupervised hierarchical clustering of six meningioma samples separated tissues into two groups. Analysis identified stem cells related pathways to be differential between the two groups and indicated the de-regulation of the stem cell associated genes Reelin (RELN), Calbindin 1 (CALB1) and Anterior Gradient 2 Homolog (AGR2). Immunofluorescence staining for four tissues confirmed stemness variation in situ. Biological characterization of fifteen meningioma primary cell lines concordantly separated cells into two functionally distinct sub-groups. Pleomorphic cell lines (NG type) grew significantly faster than monomorphic cell lines (G type), had a higher number of cells that express Ki67, and were able to migrate aggressively in vitro. In addition, NG type cell lines had a lower expression of nuclear Caspase-3, and had a significantly higher number of CSCs co-positive for CD133+ Sox2+ or AGR2+ BMI1+. Importantly, these cells were more tolerant to cisplatin and etoposide treatment, showed a lower level of nuclear Caspase-3 in treated cells and harbored drug resistant CSCs. Collectively, analyses of tissues and primary cell lines revealed stem cell associated genes as potential targets for aggressive and drug resistant meningiomas.

  20. Establishment of lal-/- myeloid lineage cell line that resembles myeloid-derived suppressive cells.

    PubMed

    Ding, Xinchun; Wu, Lingyan; Yan, Cong; Du, Hong

    2015-01-01

    Myeloid-derived suppressor cells (MDSCs) in mouse are inflammatory cells that play critical roles in promoting cancer growth and metastasis by directly stimulating cancer cell proliferation and suppressing immune surveillance. In order to facilitate characterization of biochemical and cellular mechanisms of MDSCs, it is urgent to establish an "MDSC-like" cell line. By cross breeding of immortomouse (simian virus 40 large T antigen transgenic mice) with wild type and lysosomal acid lipase (LAL) knock-out (lal-/-) mice, we have established a wild type (HD1A) and a lal-/- (HD1B) myeloid cell lines. Compared with HD1A cells, HD1B cells demonstrated many characteristics similar to lal-/- MDSCs. HD1B cells exhibited increased lysosomes around perinuclear areas, dysfunction of mitochondria skewing toward fission structure, damaged membrane potential, and increased ROS production. HD1B cells showed increased glycolytic metabolism during blockage of fatty acid metabolism to fuel the energy need. Similar to lal-/- MDSCs, the mTOR signal pathway in HD1B cells is overly activated. Rapamycin treatment of HD1B cells reduced ROS production and restored the mitochondrial membrane potential. HD1B cells showed much stronger immunosuppression on CD4+ T cell proliferation and function in vitro, and enhanced cancer cells proliferation. Knockdown of mTOR with siRNA reduced the HD1B cell ability to immunosuppress T cells and stimulate cancer cell proliferation. Therefore, the HD1B myeloid cell line is an "MDSC-like" cell line that can be used as an alternative in vitro system to study how LAL controls various myeloid cell functions.

  1. Rapid micropatterning of cell lines and human pluripotent stem cells on elastomeric membranes.

    PubMed

    Paik, Isha; Scurr, David J; Morris, Bryan; Hall, Graham; Denning, Chris; Alexander, Morgan R; Shakesheff, Kevin M; Dixon, James E

    2012-10-01

    Tissue function during development and in regenerative medicine completely relies on correct cell organization and patterning at micro and macro scales. We describe a rapid method for patterning mammalian cells including human embryonic stem cells (HESCs) and induced pluripotent stem cells (iPSCs) on elastomeric membranes such that micron-scale control of cell position can be achieved over centimeter-length scales. Our method employs surface engineering of hydrophobic polydimethylsiloxane (PDMS) membranes by plasma polymerization of allylamine. Deposition of plasma polymerized allylamine (ppAAm) using our methods may be spatially restricted using a micro-stencil leaving faithful hydrophilic ppAAm patterns. We employed airbrushing to create aerosols which deposit extracellular matrix (ECM) proteins (such as fibronectin and Matrigel™) onto the same patterned ppAAm rich regions. Cell patterns were created with a variety of well characterized cell lines (e.g., NIH-3T3, C2C12, HL1, BJ6, HESC line HUES7, and HiPSC line IPS2). Individual and multiple cell line patterning were also achieved. Patterning remains faithful for several days and cells are viable and proliferate. To demonstrate the utility of our technique we have patterned cells in a variety of configurations. The ability to rapidly pattern cells at high resolution over macro scales should aid future tissue engineering efforts for regenerative medicine applications and in creating in vitro stem cell niches.

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    PubMed

    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. © The Author(s) 2014.

  4. Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line

    PubMed Central

    2012-01-01

    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. PMID:22273551

  5. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    SciTech Connect

    Itoigawa, Yoshiaki; Kishimoto, Koshi N.; Okuno, Hiroshi; Sano, Hirotaka; Kaneko, Kazuo; Itoi, Eiji

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  6. Construction and characterization of deltaretrovirus indicator cell lines.

    PubMed

    Jewell, Nancy A; Mansky, Louis M

    2005-01-01

    The deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2), replicate poorly in culture and the molecular details of their life cycles are limited. To facilitate the analysis of virus replication, mammalian cell lines were created with the long terminal repeats (LTRs) of each virus driving expression of the enhanced green fluorescent protein gene (egfp). The BLGFP, H1GFP and H2GFP cell lines detect virus infection by the expression of GFP via the transactivation of the LTR via the Tax protein of BLV, HTLV-1 or HTLV-2, respectively. GFP expression was measured by flow cytometry, yielding sensitive and rapid detection of virus infectivity. Interestingly, we observed that the Tax proteins of HTLV-1 and HTLV-2 could transactivate the BLV LTR at levels that were comparable to that of BLV Tax. In contrast, the BLV Tax showed low levels of transactivation in H1GFP and H2GFP cells. HTLV-1 and HTLV-2 Tax proteins efficiently transactivated both the HTLV-1 and HTLV-2 LTRs. Finally, spinoculation of BLV resulted in only a two-fold increase in viral titer.

  7. The comparison of glycosphingolipids isolated from an epithelial ovarian cancer cell line and a nontumorigenic epithelial ovarian cell line using MALDI-MS and MALDI-MS/MS.

    PubMed

    Rajanayake, Krishani K; Taylor, William R; Isailovic, Dragan

    2016-08-05

    Glycosphingolipids (GSLs) are important biomolecules, which are linked to many diseases such as GSL storage disorders and cancer. Consequently, the expression of GSLs may be altered in ovarian cancer cell lines in comparison to apparently healthy cell lines. Here, differential expressions of GSLs in an epithelial ovarian cancer cell line SKOV3 and a nontumorigenic epithelial ovarian cell line T29 were studied using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and MALDI-MS/MS. The isolation of GSLs from SKOV3 and T29 cell lines was carried out using Folch partition. GSLs were successfully detected by MALDI-MS, and structurally assigned by a comparison of their MALDI-MS/MS fragmentation patterns with MS/MS data found in SimLipid database. Additionally, LIPID MAPS was used to assign GSL ion masses in MALDI-MS spectra. Seventeen neutral GSLs were identified in Folch partition lower (chloroform/methanol) phases originating from both cell lines, while five globo series neutral GSLs were identified only in the Folch partition lower phase of SKOV3 cell line. Several different sialylated GSLs were detected in Folch partition upper (water/methanol) phases of SKOV3 and T29 cell lines. Overall, this study demonstrates the alteration and increased glycosylation of GSLs in an epithelial ovarian cancer cell line in comparison to a nontumorigenic epithelial ovarian cell line.

  8. Single-walled carbon nanohorn (SWNH) aggregates inhibited proliferation of human liver cell lines and promoted apoptosis, especially for hepatoma cell lines

    PubMed Central

    Zhang, Jinqian; Sun, Qiang; Bo, Jian; Huang, Rui; Zhang, Mengran; Xia, Zhenglin; Ju, Lili; Xiang, Guoan

    2014-01-01

    Single-walled carbon nanohorns (SWNHs) may be useful as carriers for anticancer drugs due to their particular structure. However, the interactions between the material itself and cancerous or normal cells have seldom been studied. To address this problem, the effects of raw SWNH material on the biological functions of human liver cell lines were studied. Our results showed that unmodified SWNHs inhibited mitotic entry, growth, and proliferation of human liver cell lines and promoted their apoptosis, especially in hepatoma cell lines. Individual spherical SWNH particles were found inside the nuclei of human hepatoma HepG2 cells and the lysosomes of normal human liver L02 cells, implying that SWNH particles could penetrate into human liver cells_and the different interacted mechanisms on human normal cell lines compared to hepatoma cell lines. Further research on the mechanisms and application in treatment of hepatocellular carcinoma with SWNHs is needed. PMID:24523586

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

    PubMed

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

    2016-01-07

    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.

  10. Establishment of a novel human medulloblastoma cell line characterized by highly aggressive stem-like cells.

    PubMed

    Silva, Patrícia Benites Gonçalves da; Rodini, Carolina Oliveira; Kaid, Carolini; Nakahata, Adriana Miti; Pereira, Márcia Cristina Leite; Matushita, Hamilton; Costa, Silvia Souza da; Okamoto, Oswaldo Keith

    2016-08-01

    Medulloblastoma is a highly aggressive brain tumor and one of the leading causes of morbidity and mortality related to childhood cancer. These tumors display differential ability to metastasize and respond to treatment, which reflects their high degree of heterogeneity at the genetic and molecular levels. Such heterogeneity of medulloblastoma brings an additional challenge to the understanding of its physiopathology and impacts the development of new therapeutic strategies. This translational effort has been the focus of most pre-clinical studies which invariably employ experimental models using human tumor cell lines. Nonetheless, compared to other cancers, relatively few cell lines of human medulloblastoma are available in central repositories, partly due to the rarity of these tumors and to the intrinsic difficulties in establishing continuous cell lines from pediatric brain tumors. Here, we report the establishment of a new human medulloblastoma cell line which, in comparison with the commonly used and well-established cell line Daoy, is characterized by enhanced proliferation and invasion capabilities, stem cell properties, increased chemoresistance, tumorigenicity in an orthotopic metastatic model, replication of original medulloblastoma behavior in vivo, strong chromosome structural instability and deregulation of genes involved in neural development. These features are advantageous for designing biologically relevant experimental models in clinically oriented studies, making this novel cell line, named USP-13-Med, instrumental for the study of medulloblastoma biology and treatment.

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

    SciTech Connect

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    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.

  12. Cross-contamination of cell lines as revealed by DNA fingerprinting in the IFO animal cell bank.

    PubMed

    Satoh, M; Takeuchi, M

    1993-01-01

    For quality control of cell lines, the Institute for Fermentation, Osaka (IFO) animal cell bank recently introduced DNA fingerprinting analysis, which enables verification of cell lines at the individual level, to detect cross-culture contamination. By using this analysis, we found two cases of cross-contamination of cell lines.

  13. Antioxidant enzymes in malignant prostate cell lines and in primary cultured prostatic cells.

    PubMed

    Jung, K; Seidel, B; Rudolph, B; Lein, M; Cronauer, M V; Henke, W; Hampel, G; Schnorr, D; Loening, S A

    1997-01-01

    The antioxidant enzymes catalase, glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were determined in the androgen-response LNCaP and androgen-nonresponsive PC-3 and DU 145 cells as well as in prostatic epithelial cell cultures of benign and malignant human prostatic tissue. There were no differences between the enzyme activities of the human primary cell cultures from cancerous tissue and their normal counterparts. The enzyme activities of the three permanent cell lines were either higher (SOD, catalase, GR) or lower (GST, GPx) than in the primary cell cultures. In LNCaP cells catalase and GR were significantly higher, GST, in contrast, was significantly lower than in PC-3 and DU 145 cells. GST in PC-3 and DU 145 cells, and SOD in all the three cell lines showed no significant differences. Catalase, GPx and GR values were significantly different in the three permanent cell lines. The different enzymatic equipment of the prostate cancer cell lines provides the basis for experimental testing of new concepts of cancer treatment with the help of systematic modulations of the antioxidant defence systems in prostate cancer.

  14. Astaxanthin Inhibits Proliferation of Human Gastric Cancer Cell Lines by Interrupting Cell Cycle Progression.

    PubMed

    Kim, Jung Ha; Park, Jong-Jae; Lee, Beom Jae; Joo, Moon Kyung; Chun, Hoon Jai; Lee, Sang Woo; Bak, Young-Tae

    2016-05-23

    Astaxanthin is a carotenoid pigment that has antioxidant, antitumoral, and anti-inflammatory properties. In this in vitro study, we investigated the mechanism of anticancer effects of astaxanthin in gastric carcinoma cell lines. The human gastric adenocarcinoma cell lines AGS, KATO-III, MKN-45, and SNU-1 were treated with various concentrations of astaxanthin. A cell viability test, cell cycle analysis, and immunoblotting were performed. The viability of each cancer cell line was suppressed by astaxanthin in a dose-dependent manner with significantly decreased proliferation in KATO-III and SNU-1 cells. Astaxanthin increased the number of cells in the G0/G1 phase but reduced the proportion of S phase KATO-III and SNU-1 cells. Phosphorylated extracellular signal-regulated kinase (ERK) was decreased in an inverse dose-dependent correlation with astaxanthin concentration, and the expression of p27(kip-1) increased the KATO-III and SNU-1 cell lines in an astaxanthin dose-dependent manner. Astaxanthin inhibits proliferation by interrupting cell cycle progression in KATO-III and SNU-1 gastric cancer cells. This may be caused by the inhibition of the phosphorylation of ERK and the enhanced expression of p27(kip-1).

  15. Susceptibility of a minipig kidney cell line (MPK) to hog cholera virus.

    PubMed

    Buonavoglia, C; Falcone, E; Pestalozza, S; Iovane, G; Rivero, V B

    1988-07-01

    A comparitive study on the different susceptibility of MPK cells (Minipig Kidney cell line) and PK15 cells (Pig Kidney cell line) to the Hog Cholera Virus (HCV) was conducted. Higher HCV titres (3 log10) were reached on MPK cells compared with PK15 cells.

  16. Perfluorooctane sulfonate induces apoptosis in N9 microglial cell line.

    PubMed

    Zhang, Ling; Li, Yuan-yuan; Zeng, Huai-cai; Li, Miao; Wan, Yan-Jian; Schluesener, Hermann J; Zhang, Zhi-yuan; Xu, Shun-qing

    2011-03-01

    Perfluorooctane sulfonate (PFOS) is an environmental persistent acid found at low levels in human, wildlife, and environmental media samples. To study the apoptosis effects of PFOS on microglia, murine N9 cell line was used as a model in current research. The results showed that PFOS could reduce the cell viability significantly, and the cellular apoptosis induced by PFOS was closely accompanied with dissipation of mitochondria membrane potential, upregulation messenger RNAs (mRNAs) of p53, Bax, caspase 9, and caspase 3, and decreased expression of Bcl-2 mRNA. These results suggested that PFOS could disturb homeostasis of N9 cells, impact mitochondria, and affect gene expression of apoptotic regulators, all of which resulted in a start-up of apoptosis.

  17. In vitro comparative cytotoxic effect of Nimbolide: A limonoid from Azadirachta indica (Neem tree) on cancer cell lines and normal cell lines through MTT assay.

    PubMed

    Kashif, Muhammad; Hwang, Yawon; Hong, Gyeongmi; Kim, Gonhyung

    2017-05-01

    The present study was conducted to find the cytotoxicity in vitro of nimbolide, limonoids derivative of flowers and leaves from Azadirachta indica (neem tree) on the selected cell lines of cancer (Du-145, PC-3, A-549) and normal fibroblast cell lines (NIH3T3, CCD-18Co) using MTT assay. The cells were seeded in 96 multi-well tissue plate using different concentrations of nimbolide for 24hrs and 48hrs. The percentage of viability of cell lines was calculated by optical density obtained by micro plate reader and cytotoxic effect in term of IC50 value was determined by using linear regression analysis. The percentages of viability of cells treated with different concentrations of nimbolide were significantly lower (P<0.05) than the untreated cancer cell lines while in normal cell lines no significant difference (P>0.05) between treated and the non-treated cells was observed. Nimbolide exerted time and dose dependent cytotoxic effect on the cancer lines and mild effect on the normal cell lines. It was further confirmed through PKH 26. Results of the present study suggested nimbolide as a potent chemotherapeutic and chemopreventive agent as it exerted a more cytotoxic effect on cancer cell lines as compared with the normal cell lines. Nimbolide may be a new hope as an anticancer drug in future.

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

    SciTech Connect

    Lee, Jae-Eun; Hong, Eun-Jung; Nam, Hye-Young; Hwang, Meeyul; Kim, Ji-Hyun; Han, Bok-Ghee; Jeon, Jae-Pil

    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.

  19. Genetically-defined novel oral squamous cell carcinoma cell lines for the development of molecular therapies

    PubMed Central

    Fadlullah, Muhammad Zaki Hidayatullah; Chiang, Ivy Kim-Ni; Dionne, Kalen R.; Yee, Pei San; Gan, Chai Phei; Sam, Kin Kit; Tiong, Kai Hung; Ng, Adrian Kwok Wen; Martin, Daniel; Lim, Kue Peng; Kallarakkal, Thomas George; Mustafa, Wan Mahadzir Wan; Lau, Shin Hin; Abraham, Mannil Thomas; Zain, Rosnah Binti; Rahman, Zainal Ariff Abdul; Molinolo, Alfredo; Patel, Vyomesh; Gutkind, J. Silvio; Tan, Aik Choon; Cheong, Sok Ching

    2016-01-01

    Emerging biological and translational insights from large sequencing efforts underscore the need for genetically-relevant cell lines to study the relationships between genomic alterations of tumors, and therapeutic dependencies. Here, we report a detailed characterization of a novel panel of clinically annotated oral squamous cell carcinoma (OSCC) cell lines, derived from patients with diverse ethnicity and risk habits. Molecular analysis by RNAseq and copy number alterations (CNA) identified that the cell lines harbour CNA that have been previously reported in OSCC, for example focal amplications in 3q, 7p, 8q, 11q, 20q and deletions in 3p, 5q, 8p, 18q. Similarly, our analysis identified the same cohort of frequently mutated genes previously reported in OSCC including TP53, CDKN2A, EPHA2, FAT1, NOTCH1, CASP8 and PIK3CA. Notably, we identified mutations (MLL4, USP9X, ARID2) in cell lines derived from betel quid users that may be associated with this specific risk factor. Gene expression profiles of the ORL lines also aligned with those reported for OSCC. By focusing on those gene expression signatures that are predictive of chemotherapeutic response, we observed that the ORL lines broadly clustered into three groups (cell cycle, xenobiotic metabolism, others). The ORL lines noted to be enriched in cell cycle genes responded preferentially to the CDK1 inhibitor RO3306, by MTT cell viability assay. Overall, our in-depth characterization of clinically annotated ORL lines provides new insight into the molecular alterations synonymous with OSCC, which can facilitate in the identification of biomarkers that can be used to guide diagnosis, prognosis, and treatment of OSCC. PMID:27050151

  20. Integrative proteomic profiling of ovarian cancer cell lines reveals precursor cell associated proteins and functional status

    PubMed Central

    Coscia, F.; Watters, K. M.; Curtis, M.; Eckert, M. A.; Chiang, C. Y.; Tyanova, S.; Montag, A.; Lastra, R. R.; Lengyel, E.; Mann, M.

    2016-01-01

    A cell line representative of human high-grade serous ovarian cancer (HGSOC) should not only resemble its tumour of origin at the molecular level, but also demonstrate functional utility in pre-clinical investigations. Here, we report the integrated proteomic analysis of 26 ovarian cancer cell lines, HGSOC tumours, immortalized ovarian surface epithelial cells and fallopian tube epithelial cells via a single-run mass spectrometric workflow. The in-depth quantification of >10,000 proteins results in three distinct cell line categories: epithelial (group I), clear cell (group II) and mesenchymal (group III). We identify a 67-protein cell line signature, which separates our entire proteomic data set, as well as a confirmatory publicly available CPTAC/TCGA tumour proteome data set, into a predominantly epithelial and mesenchymal HGSOC tumour cluster. This proteomics-based epithelial/mesenchymal stratification of cell lines and human tumours indicates a possible origin of HGSOC either from the fallopian tube or from the ovarian surface epithelium. PMID:27561551

  1. Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

    PubMed

    Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

    2012-04-01

    Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was

  2. Red Cell Apheresis with Automated In-Line Filtration

    PubMed Central

    Matthes, Gert; Ingilizov, Marin; Dobao, Maria Luz; Marques, Susana; Callaert, Martine

    2014-01-01

    Summary Background The aim of this study was to provide data on concurrent red blood cell (RBC) and platelet (PLT) apheresis with RBC in-line leukoreduction and automated addition of saline-adenine-glucose-mannitol (SAGM) using the new version (V6.0) of Trima Accel®. Methods In this two-center paired study, each subject completed a test and a control procedure with an interval of 9 weeks between procedures. In the test arm, single RBC and PLT units were collected on the Trima Accel V6.0 (in-line leukofiltration and automated addition of SAGM). In the control arm, they were collected on Trima Accel V5.1/V5.2 (post-collection leukoreduction, manual SAGM addition). RBC percent hemolysis, potassium concentration and adenosine triphosphate over storage, hemoglobin (Hb) yield, and residual white blood cells (WBC) were determined. Results 34 subjects successfully completed both test and control procedures. Post-storage hemolysis was similar in both groups, and all values were less than 0.8% for both arms. Residual WBC counts in all RBC units were less than 1 × 106/unit. In-line processed RBC units (V6.0) have a significantly higher volume and more Hb/unit due to filtration recovery improvements. All procedures were well tolerated by the subjects. Conclusion In-line filtration and automated addition of storage solution on Trima Accel V6.0 allows collection of ready-to-use RBC units that meet EU requirements. PMID:24847185

  3. Functional somatostatin receptors on a rat pancreatic acinar cell line

    SciTech Connect

    Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.; Clerc, P.; Logsdon, C.; Svoboda, M.; Susini, C.; Vaysse, N.; Ribet, A. Mount Zion Hospital and Medical Center, San Francisco, CA Universite Libre de Bruxelles, Brussels )

    1988-07-01

    Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of {sup 125}I-(Tyr{sup 11})Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 {plus minus} 20 fmol/10{sup 6} cells. Somatostatin receptor structure was analyzed by covalently cross-linking {sup 125}I-(Tyr{sup 11})somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibition of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N{sub i} to inhibit adenylate cyclase.

  4. Phototoxic effect of fluoroquinolones on two human cell lines.

    PubMed

    Trisciuoglio, D; Krasnowska, E; Maggi, A; Pozzi, R; Parasassi, T; Sapora, O

    2002-08-01

    Photosensitization induced by the fluoroquinolone ofloxacin (OFLX) has been studied using two human cell lines, HL60 and K562, two UV wavelengths, 290 and 330 nm, and two different exposure protocols, acute and protracted. The examined endpoints are the cellular lethality and recovery and the membrane changes produced by the oxidative damage, studied using cloning and counting techniques and the measurement of the generalized polarization (GP) of the fluorescent membrane probe 2-dimethylamino-6-lauroyl-naphthalene (Laurdan). The results show that: (i) the photosensitizing effect is detectable at concentrations similar to those found in patients treated with OFLX only when the cells are irradiated with 330 nm; (ii) the amount of photodamage is a function of the drug concentration and of UV dose and persists also after the removal of the drug; (iii) during the first 24 h after OFLX treatment, a large decrease of the cell number can be observed due to cell lysis; (iv) the OFLX is inserted in the cell membranes at concentrations directly related with the drug concentration and incubation time; (v) the OFLX produces an increase in the GP values similar to that produced by membrane lipid oxidation which persists for hours after the removal of the drug. The overall results suggest the cell membrane as the main target of the OFLX adverse action, with a possible mechanism involving the formation of reactive oxygen species (ROS), which triggers, in turn, the lipid peroxidation chain reaction.

  5. Adhesion of Actinobacillus actinomycetemcomitans to a human oral cell line.

    PubMed Central

    Mintz, K P; Fives-Taylor, P M

    1994-01-01

    Two quantitative, rapid assays were developed to study the adhesion of Actinobacillus actinomycetemcomitans, an oral bacterium associated with periodontal disease, to human epithelial cells. The human oral carcinoma cell line KB was grown in microtiter plates, and adherent bacteria were detected by an enzyme-linked immunosorbent assay with purified anti-A. actinomycetemcomitans serum and horseradish peroxidase-conjugated secondary antibody or [3H]thymidine-labeled bacteria. Adhesion was found to be time dependent and increased linearly with increasing numbers of bacteria added. Variation in the level of adhesion was noted among strains of A. actinomycetemcomitans. Adhesion was not significantly altered by changes in pH (from pH 5 to 9) but was sensitive to sodium chloride concentrations greater than 0.15 M. Pooled human saliva was inhibitory for adhesion when bacteria were pretreated with saliva before being added to the cells. Pretreatment of the KB cells with saliva did not inhibit adhesion. Protease treatment of A. actinomycetemcomitans reduced adhesion of the bacteria to KB cells. The data are consistent with the hypothesis that a protein(s) is required for bacterial adhesion and that host components may play a role in modulating adhesion to epithelial cells. Images PMID:8063383

  6. DNA repair in human promyelocytic cell line, HL-60.

    PubMed Central

    Farzaneh, F; Feon, S; Lebby, R A; Brill, D; David, J C; Shall, S

    1987-01-01

    The human promyelocytic cell line, HL-60, shows large changes in endogenous poly(ADP-ribose) and in nuclear ADP-ribosyl transferase activity (ADPRT) during its induced myelocytic differentiation. DNA strand-breaks are an essential activator for this enzyme; and transient DNA strand breaks occur during the myelocytic differentiation of HL-60 cells. We have tested the hypothesis that these post-mitotic, terminally differentiating cells are less efficient in DNA repair, and specifically in DNA strand rejoining, than their proliferating precursor cells. We have found that this hypothesis is not tenable. We observe that there is no detectable reduction in the efficiency of DNA excision repair after exposure to either dimethyl sulphate or gamma-irradiation in HL-60 cells induced to differentiate by dimethyl sulphoxide. Moreover, the efficient excision repair of either dimethyl sulphate or gamma-irradiation induced lesions, both in the differentiated and undifferentiated HL-60 cells, is blocked by the inhibition of ADPRT activity. Images PMID:3106934

  7. Production of skeletal muscle elements by cell lines derived from neoplastic rat mammary epithelial stem cells.

    PubMed

    Rudland, P S; Dunnington, D J; Gusterson, B; Monaghan, P; Hughes, C M

    1984-05-01

    Single-cell-cloned cell lines intermediate in morphology between the cuboidal epithelial and fully elongated myoepithelial-like cells have been isolated from the single-cell-cloned epithelial stem cell lines Rama 25 and Rama 37 originally obtained from dimethylbenz(a)anthracene-induced mammary tumors from Sprague-Dawley and Wistar-Furth rats, respectively. These are designated Rama 25-l1, Rama 25-l2, Rama 25-l4 (Sprague-Dawley) and Rama 50-55, Rama 59, and Rama 60 (Wistar-Furth), respectively. When growing as tumors in nude mice or syngeneic Wistar-Furth rats, respectively, many of the newly cloned cell lines give rise to spindle and giant, multinucleated cells which stain immunocytochemically with antisera to myoglobin and myosin and contain longitudinal fibrils, some of which contain phosphotungstic acid-hematoxylin-staining cross-striations. Ultrastructural analysis demonstrates the presence of A-, l-, and H-bands and Z-discs and the hexagonal arrangement of thick and thin filaments characteristic of skeletal muscle. Similar results are obtained with selected cloned cell lines growing on floating collagen gels in vitro. Thus, a developmentally committed mammary epithelial cell can give rise, under suitable conditions, to a well-differentiated mesenchymal lineage, that of skeletal muscle. It is suggested that such cells may be responsible for the generation of the well-differentiated mesenchymal elements seen in the mixed (epithelial and myoepithelial) tumors of glandular origin.

  8. Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation

    PubMed Central

    Taylor, AW; Dixit, S; Yu, J

    2015-01-01

    The eye is an immune privileged tissue with multiple mechanisms of immunosuppression to protect the light gathering tissues from the damage of inflammation. One of theses mechanisms involves retinal pigment epithelial cell suppression of phagosome activation in macrophages. The objective of this work is to determine if the human RPE cell line ARPE-19 is capable of suppressing the activation of the phagolysosome in macrophages in a manner similar to primary RPE. The conditioned media of RPE eyecups, sub-confluent, just confluent cultures, or established confluent cultures of human ARPE-19 cells were generated. These condition media were used to treat macrophages phagocytizing pHrodo bioparticles. After 24 hours incubation the macrophages were imaged by fluorescent microscopy, and fluorescence was measured. The fluorescent intensity is proportional to the amount of bioparticles phagocytized and are in an activated phagolysosome. The conditioned media of in situ mouse RPE eyecups significantly suppressed the activation of phagolysosome. The conditioned media from cultures of human ARPE-19 cells, grown to sub-confluence (50%) or grown to confluence had no effect on phagolysosome activation. In contrast, the conditioned media from established confluent cultures significantly suppressed phagolysosome activation. The neuropeptides alpha-MSH and NPY were depleted from the conditioned media of established confluent ARPE-19 cell cultures. This depleted conditioned media had diminished suppression of phagolysosome activation while promoting macrophage cell death. In addition, the condition media from cultures of ARPE-19 monolayers wounded with a bisecting scrape was diminished in suppressing phagolysosome activation. This technical report suggests that like primary RPE monolayers, established confluent cultures of ARPE-19 cells produce soluble factors that suppress the activation of macrophages, and can be used to study the molecular mechanisms of retinal immunobiology. In

  9. Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation.

    PubMed

    Taylor, A W; Dixit, S; Yu, J

    2015-01-29

    The eye is an immune privileged tissue with multiple mechanisms of immunosuppression to protect the light gathering tissues from the damage of inflammation. One of theses mechanisms involves retinal pigment epithelial cell suppression of phagosome activation in macrophages. The objective of this work is to determine if the human RPE cell line ARPE-19 is capable of suppressing the activation of the phagolysosome in macrophages in a manner similar to primary RPE. The conditioned media of RPE eyecups, sub-confluent, just confluent cultures, or established confluent cultures of human ARPE-19 cells were generated. These condition media were used to treat macrophages phagocytizing pHrodo bioparticles. After 24 hours incubation the macrophages were imaged by fluorescent microscopy, and fluorescence was measured. The fluorescent intensity is proportional to the amount of bioparticles phagocytized and are in an activated phagolysosome. The conditioned media of in situ mouse RPE eyecups significantly suppressed the activation of phagolysosome. The conditioned media from cultures of human ARPE-19 cells, grown to sub-confluence (50%) or grown to confluence had no effect on phagolysosome activation. In contrast, the conditioned media from established confluent cultures significantly suppressed phagolysosome activation. The neuropeptides alpha-MSH and NPY were depleted from the conditioned media of established confluent ARPE-19 cell cultures. This depleted conditioned media had diminished suppression of phagolysosome activation while promoting macrophage cell death. In addition, the condition media from cultures of ARPE-19 monolayers wounded with a bisecting scrape was diminished in suppressing phagolysosome activation. This technical report suggests that like primary RPE monolayers, established confluent cultures of ARPE-19 cells produce soluble factors that suppress the activation of macrophages, and can be used to study the molecular mechanisms of retinal immunobiology. In

  10. Ferulic acid promoting apoptosis in human osteosarcoma cell lines

    PubMed Central

    Zhang, Xu-dong; Wu, Qiang; Yang, Shu-hua

    2017-01-01

    Objective: To explore the promoting apoptosis and antitumor activities of ferulic acid (FA) in human osteosarcoma and its potential mechanism. Methods: The SaOS-2 and MG63 osteosarcoma cell lines were opted to experiment and these cells were, respectively, cultured with various concentrations of FA (0 μM, 10 μM, 20 μM, 40 μM) for 72 hours at 37°C. The viabilities of the FA treated cells were monitored by MTT. Apoptosis cells were evaluated using annexin V/PI by flow cytometry. Apoptosis proteins caspase-3, procaspase-3, Bcl-2 and Bax were detected by western blot. Expressions of apoptotic genes Bcl-2 and Bax were quantified by qPCR. Results: The cell viabilities were critically declined in the concentration-dependent manner in FA groups (P < 0.01). The apoptosis cells were increased proportionately with the concentration of FA (P < 0.05). The procaspase-3 protein contents, and Bcl-2 mRNA and protein contents were significantly decreased while caspase-3 protein contents, and Bax mRNA and protein contents were concomitantly increased in the concentration-dependent manner in FA groups (P < 0.05). The response to FA by the SaOS-2 osteosarcoma cell was similar with the MG63 osteosarcoma cell (P > 0.05). Conclusion: Ferulic acid could significantly descend osteosarcoma cell viability through the promoting apoptosis pathway in which FA activates both caspase-3 and Bax and inactivates Bcl-2. PMID:28367185

  11. Metabolism of inorganic arsenic in intestinal epithelial cell lines.

    PubMed

    Calatayud, M; Vélez, D; Devesa, V

    2012-11-19

    This study evaluates the metabolism of inorganic arsenic (iAs) [As(III) and As(V)] in human intestinal cells as a function of cell type, differentiation stage, type of support used for cell growth, and exposure time. Additionally, mRNA expression of arsenic (+3 oxidation state) methyltransferase (AS3MT) was evaluated. For this purpose, Caco-2 (absorptive type) and HT29-MTX (goblet type) cells were exposed at various stages of differentiation (5, 15, and 21 days post-seeding) with different concentrations of As(III) and As(V) (1 and 10 μM) and exposure times (24, 48, and 72 h), using multiwell plates or Transwells. The results show that both cell lines express AS3MT at all stages of differentiation and in all culture conditions. Caco-2 cells are capable of metabolizing iAs, As(III) metabolism being greater than that observed for As(V). Metabolism depends on the stage of differentiation, reaching 36% after 48 h of exposure of differentiated cells (15 days post-seeding), with the monomethylated species as the major metabolite. Analysis of the cell interior shows that the metabolites are present predominantly in trivalent form. The type of support is also an important factor, metabolism being greater in multiwell plates than in Transwells (36 ± 6% vs 11 ± 3%). Neither monomethylated arsenic species (MMA) nor dimethylated arsenic species (DMA) are detected in HT29-MTX cells after exposure to iAs, possibly because most of the iAs is retained in the mucus layer and does not internalize. These results show that the intestine is an organ that may take part in presystemic metabolism of iAs. Moreover, the transformation of iAs into more toxic species indicates the need to study the effects of this species on the intestinal epithelium.

  12. Generation of a conditionally immortalized myeloid progenitor cell line requiring the presence of both interleukin-3 and stem cell factor to survive and proliferate.

    PubMed

    Lee, Candy; Evans, Caroline A; Spooncer, Elaine; Pierce, Andrew; Mottram, Rachel; Whetton, Anthony D

    2003-09-01

    The H-2Kappab temperature-sensitive (ts) A58 transgenic (Immorto) mouse has been used previously to generate conditionally immortalized cells from a number of tissues. The present study aimed to investigate characteristics of primitive myeloid precursor cells derived from H-2Kappab-tsA58 bone marrow. Cell populations were enriched for granulocyte/macrophage progenitors by centrifugal elutriation, and were cultured in the presence and absence of cytokines at the permissive and restrictive temperatures for the A58 oncogene. Cells derived from H-2Kappab-tsA58 mice required both A58 activation and the growth factors, stem cell factor (SCF) and interleukin-3 (IL-3), for long-term cell survival and growth; cells were maintained for > 300 d in culture under these conditions. IL-3- and SCF-dependent clonal cell lines were derived with a phenotype (lin-, Sca-1+, CD34+, ER-MP 58+, ER-MP 12+, ER-MP 20-) characteristic of primitive myeloid progenitors. These cells differentiated on addition of granulocyte/macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) and acquired mature cell morphology with some upregulation of differentiation markers. In conclusion, the A58 oncogene can immortalize haemopoietic progenitor cells. These cells require two cytokines for growth, IL-3 and SCF; as such, they constitute a useful resource for the study of synergistic interactions between growth factors. The ability to develop monocytic cell characteristics also permits the investigation of cytokine-mediated early haemopoietic progenitor cell development.

  13. Current status of publicly available sarcoma cell lines for use in proteomic studies.

    PubMed

    Pan, Xiaoqing; Yoshida, Akihiko; Kawai, Akira; Kondo, Tadashi

    2016-01-01

    Cell lines are valuable resources for proteomic studies and can be used as tools to verify the significance of proteomic findings. Here, the authors overview the current status of the publicly available sarcoma cell lines. The authors surveyed seven major cell banks and found that the diversity observed in the sarcoma cell banks was largely insufficient; sarcoma cell lines are available for only a limited histological subtype. They also observed a number of issues with the pathological diagnosis of the cell lines, limitations in their behavioral diversity, and various unmet needs. Well characterized cell lines with accurate diagnosis based on modern diagnosis criteria should be available from public cell banks. The authors conclude that additional cell lines, along with detailed genetic and pathological analyses, should be prepared and deposited in order to promote sarcoma-specific proteomic research. The authors focused on sarcoma cell lines, but their discussion can be applied to the other cancers.

  14. Glycoprotein VI oligomerization in cell lines and platelets.

    PubMed

    Berlanga, Oscar; Bori-Sanz, Teresa; James, John R; Frampton, Jon; Davis, Simon J; Tomlinson, Michael G; Watson, Steve P

    2007-05-01

    Glycoprotein VI (GPVI) is a physiologic receptor for collagen expressed at the surface of platelets and megakaryocytes. Constitutive dimerization of GPVI has been proposed as being necessary for the interaction with collagen, although direct evidence of dimerization has not been reported in cell lines or platelets. To investigate oligomerization of GPVI in transfected cell lines and in platelets under non-stimulated conditions. By using a combination of molecular and biochemical techniques, we demonstrate that GPVI association occurs at the surface of transfected 293T cells under basal conditions, through an interaction at the extracellular domain of the receptor. Bioluminescence resonance energy transfer was used to confirm oligomerization of GPVI under these conditions. A chemical crosslinker was used to detect constitutive oligomeric forms of GPVI at the surface of platelets, which contain the Fc receptor (FcR) gamma-chain. The present results directly demonstrate GPVI-FcR gamma-chain oligomerization at the surface of the platelet, and thereby add to the growing evidence that oligomerization of GPVI may be a prerequisite for binding of the receptor to collagen, and therefore for proper functioning of platelets upon vascular damage.

  15. APOPTOSIS INDUCTION OF EPIFRIEDELINOL ON HUMAN CERVICAL CANCER CELL LINE.

    PubMed

    Yang, Jie; Fa, Jing; Li, Bingxing

    2017-01-01

    Present investigation evaluates the antitumor activity of epifriedelinol for the management of cervical cancer by inducing process of apoptosis. Human Cervical Cancer Cell Line, C33A and HeLa were selected for study and treated with epifriedelinol at a concentration of (50-1000 µg/ml). Cytotoxicity of epifriedelinol was estimated by MTT assay and induction of apoptosis was assessed by estimating the activity of caspase 3, 8 and 9 enzyme, apoptosis assay and translocation of cytochrome c. Moreover an expression of several proteins that plays role in the apoptosis process was estimated by western blot method. Result of the study suggested that treatment with epifriedelinol significantly decrease the viability count of cancerous cell in a dose perndent manner and also enhances the formation of oligonucleosome in both the cell lines. However activity of caspase enzymes and translocation of cytochrome c were enhanced after treatment with epifriedelinol. It was also observed that epifriedelinol treatment alters the ratio of pro-apoptotic to anti-apoptotic proteins and enhances the expressions of inhibitor of apoptosis proteins (IAP). Result of our study proves the anticancer activity of epifriedelinol in cervical cancer by inducing apoptosis as treatment with it enhances the production of oligonucleosomes, translocation of cytochrome c and activity caspase enzymes.

  16. New Model for Gastroenteropancreatic Large-Cell Neuroendocrine Carcinoma: Establishment of Two Clinically Relevant Cell Lines

    PubMed Central

    Krieg, Andreas; Mersch, Sabrina; Boeck, Inga; Dizdar, Levent; Weihe, Eberhard; Hilal, Zena; Krausch, Markus; Möhlendick, Birte; Topp, Stefan A.; Piekorz, Roland P.; Huckenbeck, Wolfgang; Stoecklein, Nikolas H.; Anlauf, Martin; Knoefel, Wolfram T.

    2014-01-01

    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. PMID:24551139

  17. Transformation of Drosophila cell lines: an alternative approach to exogenous protein expression.

    PubMed

    Cherbas, Lucy; Cherbas, Peter

    2007-01-01

    Techniques and experimental applications are described for exogenous protein expression in Drosophila cell lines. Ways in which the Drosophila cell lines and the baculovirus expression vector system differ in their applications are emphasized.

  18. Is parainfluenza virus a threatening virus for human cancer cell lines?

    PubMed

    Danjoh, Inaho; Sone, Hiyori; Noda, Nahomi; Iimura, Emi; Nagayoshi, Mariko; Saijo, Kaoru; Hiroyama, Takashi; Nakamura, Yukio

    2009-08-01

    Immortalized cell lines, such as human cancer cell lines, are an indispensable experimental resource for many types of biological and medical research. However, unless the cell line has been authenticated prior to use, interpretation of experimental results may be problematic. The potential problems this may cause are illustrated by studies in which authentication of cell lines has not been carried out. For example, immortalized cell lines may unknowingly be infected with viruses that alter their characteristics. In fact, parainfluenza virus type 5 (PIV5) poses a threat to the use of immortalized cell lines in biological and medical research; PIV5 infection significantly alters cellular physiology associated with the response to interferon. If PIV5 infection is widespread in immortalized cell lines, then a very large number of published studies might have to be re-evaluated. Fortunately, analyses of a large number of immortalized cell lines indicate that PIV5 infection is not widespread.

  19. Hodgkin's disease cell lines: a model for interleukin-1-independent accessory cell function.

    PubMed

    McKenzie, J L; Egner, W; Calder, V L; Hart, D N

    1992-11-01

    The haemopoietic origins of the Hodgkin's disease (HD)-derived cell lines L428, KM-H2 and HDLM-2 remain controversial. Analysis of T-cell receptor (TcR) and Ig rearrangements cannot resolve this, and lineage promiscuity limits the interpretation of isolated surface antigen expression. Nonetheless the cell marker profile of L428 has similarities with human dendritic cells (DC), and L428 strongly stimulates in the mixed leucocyte reaction (MLR). We therefore undertook an extended immunophenotypic comparison of the HD lines with that recently defined for DC, prior to examining their ability to stimulate allogenic T lymphocytes, and comparing the molecular interactions involved with those of primary MLR stimulatory cells. The immunophenotype of the HD lines failed to establish either a lymphoid or monocytoid derivation. The profile of L428 appeared similar to the human DC. All three lines were potent stimulators in the primary MLR, and each expressed relevant adhesion and signal-transducing molecules important for co-stimulati