WorldWideScience

Sample records for cell line derived

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

    Science.gov (United States)

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

    2007-12-21

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

  2. Derivation of the human embryonic stem cell line RCM1

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

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

  3. Nestin expression in the cell lines derived from glioblastoma multiforme

    International Nuclear Information System (INIS)

    Veselska, Renata; Kuglik, Petr; Cejpek, Pavel; Svachova, Hana; Neradil, Jakub; Loja, Tomas; Relichova, Jirina

    2006-01-01

    Nestin is a protein belonging to class VI of intermediate filaments that is produced in stem/progenitor cells in the mammalian CNS during development and is consecutively replaced by other intermediate filament proteins (neurofilaments, GFAP). Down-regulated nestin may be re-expressed in the adult organism under certain pathological conditions (brain injury, ischemia, inflammation, neoplastic transformation). Our work focused on a detailed study of the nestin cytoskeleton in cell lines derived from glioblastoma multiforme, because re-expression of nestin together with down-regulation of GFAP has been previously reported in this type of brain tumor. Two cell lines were derived from the tumor tissue of patients treated for glioblastoma multiforme. Nestin and other cytoskeletal proteins were visualized using imunocytochemical methods: indirect immunofluorescence and immunogold-labelling. Using epifluorescence and confocal microscopy, we described the morphology of nestin-positive intermediate filaments in glioblastoma cells of both primary cultures and the derived cell lines, as well as the reorganization of nestin during mitosis. Our most important result came through transmission electron microscopy and provided clear evidence that nestin is present in the cell nucleus. Detailed information concerning the pattern of the nestin cytoskeleton in glioblastoma cell lines and especially the demonstration of nestin in the nucleus represent an important background for further studies of nestin re-expression in relationship to tumor malignancy and invasive potential

  4. Establishment of mesenchymal cell line derived from human developing odontoma.

    Science.gov (United States)

    Hatano, H; Kudo, Y; Ogawa, I; Shimasue, H; Shigeishi, H; Ohta, K; Higashikawa, K; Takechi, M; Takata, T; Kamata, N

    2012-11-01

    An odontoma, which shows proliferating odontogenic epithelium and mesenchymal tissue, is one of the most common odontogenic tumors encountered. These are commonly found in tooth-bearing regions, although the etiology remains unknown. There are no previous reports of an established line of immortalized human odontoma cells. Using odontoma fragments obtained from a girl treated at our department, we established an immortalized human odontoma cell line and investigated cell morphology, dynamic proliferation, the presence of contamination, and karyotype. Moreover, cell characterization was examined using osteogenic and odontogenic markers. We successfully established a mesenchymal odontoma cell (mOd cells). The cells were found to be fibroblastic and had a high level of telomerase activity. Cell growth was confirmed after more than 200 population doublings without significant growth retardation. mOd cells expressed mRNA for differentiation markers, including collagen type I (COLI), alkaline phosphatase, bone sialoprotein, osteopontin, osteocalcin, cementum-derived protein (CP-23), dentin sialophosphoprotein (DSPP), and distal-less homeobox 3 (DLX3), as well as bone morphogenetic proteins (BMPs). In addition, they showed a high level of calcified nodule formation activity in vitro. We successfully established a cell line that may be useful for investigating the mechanisms of normal odontogenesis as well as characteristics of odontoma tumors. © 2012 John Wiley & Sons A/S.

  5. Derivation of Ethnically Diverse Human Induced Pluripotent Stem Cell Lines.

    Science.gov (United States)

    Chang, Eun Ah; Tomov, Martin L; Suhr, Steven T; Luo, Jiesi; Olmsted, Zachary T; Paluh, Janet L; Cibelli, Jose

    2015-10-20

    The human genome with all its ethnic variations contributes to differences in human development, aging, disease, repair, and response to medical treatments and is an exciting area of research and clinical study. The availability of well-characterized ethnically diverse stem cell lines is limited and has not kept pace with other advances in stem cell research. Here we derived xenofree ethnically diverse-human induced pluripotent stem cell (ED-iPSC) lines from fibroblasts obtained from individuals of African American, Hispanic-Latino, Asian, and Caucasian ethnic origin and have characterized the lines under a uniform platform for comparative analysis. Derived ED-iPSC lines are low passage number and evaluated in vivo by teratoma formation and in vitro by high throughput microarray analysis of EB formation and early differentiation for tri-lineage commitment to endoderm, ectoderm and mesoderm. These new xenofree ED-iPSC lines represent a well-characterized valuable resource with potential for use in future research in drug discovery or clinical investigations.

  6. Myelinating cocultures of rodent stem cell line-derived neurons and immortalized Schwann cells.

    Science.gov (United States)

    Ishii, Tomohiro; Kawakami, Emiko; Endo, Kentaro; Misawa, Hidemi; Watabe, Kazuhiko

    2017-10-01

    Myelination is one of the most remarkable biological events in the neuron-glia interactions for the development of the mammalian nervous system. To elucidate molecular mechanisms of cell-to-cell interactions in myelin synthesis in vitro, establishment of the myelinating system in cocultures of continuous neuronal and glial cell lines are desirable. In the present study, we performed co-culture experiments using rat neural stem cell-derived neurons or mouse embryonic stem (ES) cell-derived motoneurons with immortalized rat IFRS1 Schwann cells to establish myelinating cultures between these cell lines. Differentiated neurons derived from an adult rat neural stem cell line 1464R or motoneurons derived from a mouse ES cell line NCH4.3, were mixed with IFRS1 Schwann cells, plated, and maintained in serum-free F12 medium with B27 supplement, ascorbic acid, and glial cell line-derived neurotrophic factor. Myelin formation was demonstrated by electron microscopy at 4 weeks in cocultures of 1464R-derived neurons or NCH4.3-derived motoneurons with IFRS1 Schwann cells. These in vitro coculture systems utilizing the rodent stable stem and Schwann cell lines can be useful in studies of peripheral nerve development and regeneration. © 2017 Japanese Society of Neuropathology.

  7. Dysfunctional p53 deletion mutants in cell lines derived from Hodgkin's lymphoma

    DEFF Research Database (Denmark)

    Feuerborn, Alexander; Moritz, Constanze; von Bonin, Frederike

    2006-01-01

    derived from cHL are rare and therefore not notably involved in the pathogenesis of the malignant H&RS cells. Re-evaluating the expression in cHL-derived cell lines, we found that in 3/6 of these cell lines, TP53 transcripts are characterized by deletions within exon 4 (L428 cells) and nearly a complete...

  8. Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene

    DEFF Research Database (Denmark)

    Efrat, S; Linde, S; Kofod, Hans

    1988-01-01

    Three pancreatic beta-cell lines have been established from insulinomas derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene. The beta tumor cell (beta TC) lines maintain the features of differentiated beta cells for about 50 passages in culture...... by glucose, although with a lower threshold for maximal stimulation than that for normal beta cells. beta TC lines can be repeatedly derived from primary beta-cell tumors that heritably arise in the transgenic mice. Thus, targeted expression of an oncogene with a cell-specific regulatory element can be used...

  9. Electrophysiological Characteristics of Embryonic Stem Cell-Derived Cardiomyocytes are Cell Line-Dependent

    Directory of Open Access Journals (Sweden)

    Tobias Hannes

    2015-01-01

    Full Text Available Background: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs of different murine ESC lines. Methods: Two wild-type (D3 and R1 and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7 were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC promoter. Action potentials (APs were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Results: Spontaneous AP frequency and AP duration (APD as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Conclusion: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.

  10. Macrophage cell lines derived from major histocompatibility complex II-negative mice

    Science.gov (United States)

    Beharka, A. A.; Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

  11. Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

    Science.gov (United States)

    Gardell, Alison M.; Qin, Qin; Rice, Robert H.; Li, Johnathan; Kültz, Dietmar

    2014-01-01

    Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish. PMID:24797371

  12. Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus cell lines.

    Directory of Open Access Journals (Sweden)

    Alison M Gardell

    Full Text Available Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus cell lines from brain (OmB and lip epithelium (OmL, and compared them to a previously immortalized bulbus arteriosus (TmB cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5, we characterized the proteomes of the newly derived cell lines (OmB and OmL using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.

  13. Establishment and characterization of 7 novel hepatocellular carcinoma cell lines from patient-derived tumor xenografts.

    Directory of Open Access Journals (Sweden)

    Hong Xin

    Full Text Available Hepatocellular carcinoma (HCC is a common cancer with poor prognosis worldwide and the molecular mechanism is not well understood. This study aimed to establish a collection of human HCC cell lines from patient-derived xenograft (PDX models. From the 20 surgical HCC sample collections, 7 tumors were successfully developed in immunodeficient mice and further established 7 novel HCC cell lines (LIXC002, LIXC003, LIXC004, LIXC006, LIXC011, LIXC012 and CPL0903 by primary culture. The characterization of cell lines was defined by morphology, growth kinetics, cell cycle, chromosome analysis, short tandem repeat (STR analysis, molecular profile, and tumorigenicity. Additionally, response to clinical chemotherapeutics was validated both in vitro and in vivo. STR analysis indicated that all cell lines were unique cells different from known cell lines and free of contamination by bacteria or mycoplasma. The other findings were quite heterogeneous between individual lines. Chromosome aberration could be found in all cell lines. Alpha-fetoprotein was overexpressed only in 3 out of 7 cell lines. 4 cell lines expressed high level of vimentin. Ki67 was strongly stained in all cell lines. mRNA level of retinoic acid induced protein 3 (RAI3 was decreased in all cell lines. The 7 novel cell lines showed variable sensitivity to 8 tested compounds. LIXC011 and CPL0903 possessed multiple drug resistance property. Sorafenib inhibited xenograft tumor growth of LIXC006, but not of LIXC012. Our results indicated that the 7 novel cell lines with low passage maintaining their clinical and pathological characters could be good tools for further exploring the molecular mechanism of HCC and anti-cancer drug screening.

  14. Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos

    International Nuclear Information System (INIS)

    Fang, Zhen F.; Gai, Hui; Huang, You Z.; Li, Shan G.; Chen, Xue J.; Shi, Jian J.; Wu, Li; Liu, Ailian; Xu, Ping; Sheng, Hui Z.

    2006-01-01

    Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ES cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines

  15. Derivation of Huntington Disease affected Genea046 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea046 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying HTT gene CAG expansion of 45 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 85% of cells expressed Nanog, 92% Oct4, 75% Tra1–60 and 99% SSEA4 and demonstrated Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and visible contamination.

  16. Benzodiazepinone derivatives protect against endoplasmic reticulum stress-mediated cell death in human neuronal cell lines.

    Science.gov (United States)

    Zou, Haixia; Limpert, Allison S; Zou, Jiwen; Dembo, Anna; Lee, Pooi-San; Grant, Daniel; Ardecky, Robert; Pinkerton, Anthony B; Magnuson, Gavin K; Goldman, Mark E; Rong, Juan; Teriete, Peter; Sheffler, Douglas J; Reed, John C; Cosford, Nicholas D P

    2015-03-18

    Endoplasmic reticulum (ER) stress causes neuronal dysfunction followed by cell death and is recognized as a feature of many neurodegenerative diseases. Using a phenotypic screen, we recently identified benzodiazepinone derivatives that reduce ER stress-mediated apoptosis in a rat neuronal progenitor cell line (CSM14.1). Herein we describe how structure-activity relationship (SAR) studies around these screening hits led to compounds that display robust cytoprotective activity against thapsigargin-induced ER stress in SH-SY5Y and H4 human neuronal cell lines. We demonstrate that the most potent of these derivatives, compound 4hh, inhibits the activation of p38 MAP kinase (p38) and c-Jun N-terminal kinase (JNK), protein kinases that are downstream signal effectors of the unfolded protein response (UPR). Compound 4hh specifically protects against thapsigargin-induced cell death and displays no protection against other insults known to induce cellular stress or activate p38. However, compound 4hh provides moderate inhibition of p38 activity stimulated by compounds that disrupt calcium homeostasis. Our data indicate that probe compound 4hh is a valuable small molecule tool that can be used to investigate the effects of ER stress on human neurons. This approach may provide the basis for the future development of therapeutics for the treatment of neurodegenerative diseases.

  17. Derivation of the human embryonic stem cell line RCe007-A (RC-3

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe007-A (RC-3 was derived from a blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and HLA and blood group typing data is available.

  18. Derivation of the human embryonic stem cell line RCe008-A (RC-4

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe008-A (RC-4 was derived from a blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to ectoderm and mesoderm in vitro. It has a mixed 46XX/45X female karyotype and microsatellite PCR identity and blood group typing data is available.

  19. Establishment and Characterization of 7 Novel Hepatocellular Carcinoma Cell Lines from Patient-Derived Tumor Xenografts

    OpenAIRE

    Xin, Hong; Wang, Ke; Hu, Gang; Xie, Fubo; Ouyang, Kedong; Tang, Xuzhen; Wang, Minjun; Wen, Danyi; Zhu, Yizhun; Qin, Xiaoran

    2014-01-01

    Hepatocellular carcinoma (HCC) is a common cancer with poor prognosis worldwide and the molecular mechanism is not well understood. This study aimed to establish a collection of human HCC cell lines from patient-derived xenograft (PDX) models. From the 20 surgical HCC sample collections, 7 tumors were successfully developed in immunodeficient mice and further established 7 novel HCC cell lines (LIXC002, LIXC003, LIXC004, LIXC006, LIXC011, LIXC012 and CPL0903) by primary culture. The character...

  20. In vivo assay for the developmental competence of embryo-derived zebrafish cell lines

    NARCIS (Netherlands)

    Speksnijder, JE; Hage, WJ; Lanser, PH; Collodi, P; Zivkovic, D

    We have produced chimeric zebrafish embryos by transplanting permanent embryo-derived cell lines into blastula-stage embryos. Furthermore, we have established a fluorescent in vivo assay to monitor the developmental effects and fate of such transplanted cells using confocal laser scanning

  1. Tumor-Derived Cell Lines as Molecular Models of Cancer Pharmacogenomics.

    Science.gov (United States)

    Goodspeed, Andrew; Heiser, Laura M; Gray, Joe W; Costello, James C

    2016-01-01

    Compared with normal cells, tumor cells have undergone an array of genetic and epigenetic alterations. Often, these changes underlie cancer development, progression, and drug resistance, so the utility of model systems rests on their ability to recapitulate the genomic aberrations observed in primary tumors. Tumor-derived cell lines have long been used to study the underlying biologic processes in cancer, as well as screening platforms for discovering and evaluating the efficacy of anticancer therapeutics. Multiple -omic measurements across more than a thousand cancer cell lines have been produced following advances in high-throughput technologies and multigroup collaborative projects. These data complement the large, international cancer genomic sequencing efforts to characterize patient tumors, such as The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC). Given the scope and scale of data that have been generated, researchers are now in a position to evaluate the similarities and differences that exist in genomic features between cell lines and patient samples. As pharmacogenomics models, cell lines offer the advantages of being easily grown, relatively inexpensive, and amenable to high-throughput testing of therapeutic agents. Data generated from cell lines can then be used to link cellular drug response to genomic features, where the ultimate goal is to build predictive signatures of patient outcome. This review highlights the recent work that has compared -omic profiles of cell lines with primary tumors, and discusses the advantages and disadvantages of cancer cell lines as pharmacogenomic models of anticancer therapies. ©2015 American Association for Cancer Research.

  2. Derivation of the clinical grade human embryonic stem cell line RCe015-A (RC-11

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-07-01

    Full Text Available The human embryonic stem cell line RCe015-A (RC-11 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a fragmented cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe015-A (RC-11 shows normal pluripotency marker expression and differentiation to the three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  3. Comparative study of therapeutic antibody candidates derived from mini-pool and clonal cell lines.

    Science.gov (United States)

    Fan, Lianchun; Rizzi, Giovanni; Bierilo, Kathleen; Tian, Jun; Yee, Joon Chong; Russell, Reb; Das, Tapan K

    2017-11-01

    The long journey of developing a drug from initial discovery target identification to regulatory approval often leaves many patients with missed window of opportunities. Both regulatory agencies and biopharmaceutical industry continue to develop creative approaches to shorten the time of new drug development in order to deliver life-saving medicine to patients. Generally, drug substance materials to support the toxicology and early phase clinical study can only be manufactured after creating the final Master Cell Bank (MCB) of the clonally derived cell line, which normally takes 1-2 years. With recent advances in cell line development, cell culture process and analytical technologies, generating more homogeneous bulk/mini-pool population with higher productivity and acceptable quality attributes has become a norm, thereby making it possible to shorten the timeline to initiate First in Human (FIH) trial by using bulk/mini-pool generated materials to support toxicology and FIH studies. In this study, two monoclonal antibodies of different subclasses (IgG1 and IgG4) were expressed from the mini-pool cells as well as clonally derived cell lines generated from the same mini-pool. Cell growth, productivity, and product quality were compared between the materials generated from the mini-pool and clonally derived cell line. The results demonstrate the similarity of the antibody products generated from mini-pool cells and clonally derived cell lines from the same mini-pool, and strongly support the concept and feasibility of using antibody materials produced from mini-pool cultures for toxicology and FIH studies. The strategy to potentially shorten the FIH timeline is discussed. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1456-1462, 2017. © 2017 American Institute of Chemical Engineers.

  4. Heterogeneity of cell lines derived after transformation of early passage rodent cells by the Ha-ras1 human oncogene.

    Science.gov (United States)

    Spandidos, D A; Freshney, M; Wilkie, N M

    1985-01-01

    The chromosome patterns of Chinese hamster cell lines derived after immortalization or tumorigenic conversion of early passage cells with recombinants carrying the mutated T24 or the normal human Ha-ras1 gene have been characterized by trypsin-Giemsa banding. Whereas immortalized Chinese hamster cell lines exhibited a near normal karyotype, tumorigenic cell lines were found to have abnormal karyotypes carrying marker chromosomes. Moreover, chromosomal patterns correlated with growth in semisolid media and tumourigenicity in nude mice. Similarly, malignant conversion of early passage Syrian hamster cells, with a recombinant carrying the mutated T24 human Ha-ras1 gene, resulted in cells with a near diploid karyotype. On the other hand, tumorigenic conversion of early passage Wistar rat cells with the same oncogene produced cell lines with heteroploid karyotypes. More chromosomal alterations have been observed during further growth of these cells. It is suggested that the transformed phenotype in these cells may be dependent on the chromosomal instability.

  5. Derivation of the human embryonic stem cell line RCe006-A (RC-2

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe006-A (RC-2 was derived from a frozen and thawed blastocyst voluntarily donated as surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line exhibits expression of expected pluripotency markers and in vitro differentiation potential to three germinal lineage representative cell populations. It has a male trisomy 12 karyotype (47XY, +12. Microsatellite DNA marker identity and HLA and blood group typing data are available.

  6. Establishment and characterization of a new cell line derived from human colorectal laterally spreading tumor

    Science.gov (United States)

    Wang, Xin-Ying; Lai, Zhou-Sheng; Yeung, Chung-Man; Wang, Ji-De; Deng, Wen; Li, Hoi Yee; Han, Yu-Jing; Kung, Hsiang-Fu; Jiang, Bo; Lin, Marie Chia-mi

    2008-01-01

    AIM: To study the molecular mechanism of laterally spreading tumor (LST), a cell line [Laterally Spreading Tumor-Rectum 1 (LST-R1)] was derived and the characteristics of this cell line were investigated. METHODS: A new cell line (LST-R1) originated from laterally spreading tumor was established. Properties of the cell line were characterized using scanning and transmission electron microscopy, immunohistochemistry method, cytogenetic analysis and nude mice xenograft experiments. In vitro invasion assay, cDNA microarray and Western blotting were used to compare the difference between the LST-R1 and other colorectal cancer cell lines derived from prudent colon cancer. RESULTS: Our study demonstrated that both epithelial special antigen (ESA) and cytokeratin-20 (CK20) were expressed in LST-R1. The cells presented microvilli and tight junction with large nuclei. The karyotypic analysis showed hyperdiploid features with structural chromosome aberrations. The in vivo tumorigenicity was also demonstrated in nude mice xenograft experiments. The invasion assay suggested this cell line has a higher invasive ability. cDNA microarray and Western blotting show the loss of the expression of E-cadherin in LST-R1 cells. CONCLUSION: We established and characterized a colorectal cancer cell line, LST-R1 and LST-R1 has an obvious malignant tendency, which maybe partially attributed to the changes of the expression of some adhesion molecules, such as E-cadherin. It is also a versatile tool for exploring the original and progressive mechanisms of laterally spreading tumor and the early colon cancer genesis. PMID:18300345

  7. Karyotype characterization of in vivo- and in vitro-derived porcine parthenogenetic cell lines.

    Science.gov (United States)

    Liu, Qiang; Zhang, Manling; Hou, Dongxia; Han, Xuejie; Jin, Yong; Zhao, Lihua; Nie, Xiaowei; Zhou, Xin; Yun, Ting; Zhao, Yuhang; Huang, Xianghua; Hou, Daorong; Yang, Ning; Wu, Zhaoqiang; Li, Xueling; Li, Rongfeng

    2014-01-01

    Mammalian haploid cell lines provide useful tools for both genetic studies and transgenic animal production. To derive porcine haploid cells, three sets of experiments were conducted. First, genomes of blastomeres from 8-cell to 16-cell porcine parthenogenetically activated (PA) embryos were examined by chromosome spread analysis. An intact haploid genome was maintained by 48.15% of blastomeres. Based on this result, two major approaches for amplifying the haploid cell population were tested. First, embryonic stem-like (ES-like) cells were cultured from PA blastocyst stage embryos, and second, fetal fibroblasts from implanted day 30 PA fetuses were cultured. A total of six ES-like cell lines were derived from PA blastocysts. No chromosome spread with exactly 19 chromosomes (the normal haploid complement) was found. Four cell lines showed a tendency to develop to polyploidy (more than 38 chromosomes). The karyotypes of the fetal fibroblasts showed different abnormalities. Cells with 19-38 chromosomes were the predominant karyotype (59.48-60.91%). The diploid cells were the second most observed karyotype (16.17%-22.73%). Although a low percentage (3.45-8.33%) of cells with 19 chromosomes were detected in 18.52% of the fetus-derived cell lines, these cells were not authentic haploid cells since they exhibited random losses or gains of some chromosomes. The haploid fibroblasts were not efficiently enriched via flow cytometry sorting. On the contrary, the diploid cells were efficiently enriched. The enriched parthenogenetic diploid cells showed normal karyotypes and expressed paternally imprinted genes at extremely low levels. We concluded that only a limited number of authentic haploid cells could be obtained from porcine cleavage-stage parthenogenetic embryos. Unlike mouse, the karyotype of porcine PA embryo-derived haploid cells is not stable, long-term culture of parthenogenetic embryos, either in vivo or in vitro, resulted in abnormal karyotypes. The porcine PA

  8. Axon Guidance of Sympathetic Neurons to Cardiomyocytes by Glial Cell Line-Derived Neurotrophic Factor (GDNF)

    NARCIS (Netherlands)

    Miwa, Keiko; Lee, Jong-Kook; Takagishi, Yoshiko; Opthof, Tobias; Fu, Xianming; Hirabayashi, Masumi; Watabe, Kazuhiko; Jimbo, Yasuhiko; Kodama, Itsuo; Komuro, Issei

    2013-01-01

    Molecular signaling of cardiac autonomic innervation is an unresolved issue. Here, we show that glial cell line-derived neurotrophic factor (GDNF) promotes cardiac sympathetic innervation in vitro and in vivo. In vitro, ventricular myocytes (VMs) and sympathetic neurons (SNs) isolated from neonatal

  9. Identification of two distinct chromosome 12-derived amplification units in neuroblastoma cell line NGP

    NARCIS (Netherlands)

    van Roy, N.; Forus, A.; Myklebost, O.; Cheng, N. C.; Versteeg, R.; Speleman, F.

    1995-01-01

    The neuroblastoma cell line NGP contains two homogeneously staining regions (hsr). One of these hsrs contains MYCN sequences. Reverse painting experiments demonstrated that the second HSR consisted of two chromosome 12-derived amplification units, located at 12q14-15 and 12q24. Southern blot and

  10. Derivation of NEM2 affected human embryonic stem cell line Genea079

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea079 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying compound heterozygous mutations in the NEB gene, exon 55 deletion & c.15110dupA, indicative of Nemaline Myopathy Type 2 (NEM2. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male Allele pattern. The hESC line had pluripotent cell morphology, 86% of cells expressed Nanog, 95% Oct4, 54% Tra1-60 and 98% SSEA4 and gave a PluriTest Pluripotency score of 30.25, Novelty of 1.21. The cell line was negative for Mycoplasma and visible contamination.

  11. Establishment and cryopreservation of a giant panda skeletal muscle-derived cell line.

    Science.gov (United States)

    Yu, Fang-Jian; Zeng, Chang-Jun; Zhang, Yan; Wang, Cheng-Dong; Xiong, Tie-Yi; Fang, Sheng-Guo; Zhang, He-Min

    2015-06-01

    The giant panda Ailuropoda melanoleuca is an endangered species and is a symbol for wildlife conservation. Although efforts have been made to protect this rare and endangered species through breeding and conservative biology, the long-term preservation of giant panda genome resources (gametes, tissues, organs, genomic libraries, etc.) is still a practical option. In this study, the giant panda skeletal muscle-derived cell line was successfully established via primary explants culture and cryopreservation techniques. The population doubling time of giant panda skeletal cells was approximately 33.8 h, and this population maintained a high cell viability before and after cryopreservation (95.6% and 90.7%, respectively). The two skeletal muscle-specific genes SMYD1 and MYF6 were expressed and detected by RT-PCR in the giant panda skeletal muscle-derived cell line. Karyotyping analysis revealed that the frequencies of giant panda skeletal muscle cells showing a chromosome number of 2n=42 ranged from 90.6∼94.2%. Thus, the giant panda skeletal muscle-derived cell line provides a vital resource and material platform for further studies and is likely to be useful for the protection of this rare and endangered species.

  12. Derivation of the human embryonic stem cell line RCe010-A (RC-6

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe010-A (RC-6 was derived from a frozen and thawed blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    a retinal ganglion cell line (RGC5) bioassay. Indirect ophthalmoscopy, intraocular pressure assessment, and fundus photography were performed before enucleation. There was initial variability in the cGDNF, but after 24h GDNF was cleared in a monoexponential fashion with a half-life of 37 h (CL 33-43 h......The purpose of this study was to establish the intravitreal (ITV) pharmacokinetics of glial cell line-derived neurotrophic factor (GDNF) and observe possible complications after ITV injection. Twenty Danish landrace pigs and 34 eyes were included in the study; 30 were injected with 100 ng of GDNF...

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    The purpose of this study was to establish the intravitreal (ITV) pharmacokinetics of glial cell line-derived neurotrophic factor (GDNF) and observe possible complications after ITV injection. Twenty Danish landrace pigs and 34 eyes were included in the study; 30 were injected with 100 ng of GDNF...... a retinal ganglion cell line (RGC5) bioassay. Indirect ophthalmoscopy, intraocular pressure assessment, and fundus photography were performed before enucleation. There was initial variability in the cGDNF, but after 24h GDNF was cleared in a monoexponential fashion with a half-life of 37 h (CL 33-43 h...

  15. Derivation of the human embryonic stem cell line RCe011-A (RC-7

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe011-A (RC-7 was derived from a failed to fertilise oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  16. Derivation of the human embryonic stem cell line RCe014-A (RC-10

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe014-A (RC-10 was derived from a fresh oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XY and 47XY +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  17. Derivation of the human embryonic stem cell line RCe012-A (RC-8

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe012-A (RC-8 was derived from a frozen and thawed day 5 embryo cultivated to the blastocyst stage. The embryo was voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  18. Ultra-fast vitrification of patient-derived circulating tumor cell lines.

    Science.gov (United States)

    Sandlin, Rebecca D; Wong, Keith H K; Tessier, Shannon N; Swei, Anisa; Bookstaver, Lauren D; Ahearn, Bennett E; Maheswaran, Shyamala; Haber, Daniel A; Stott, Shannon L; Toner, Mehmet

    2018-01-01

    Emerging technologies have enabled the isolation and characterization of rare circulating tumor cells (CTCs) from the blood of metastatic cancer patients. CTCs represent a non-invasive opportunity to gain information regarding the primary tumor and recent reports suggest CTCs have value as an indicator of disease status. CTCs are fragile and difficult to expand in vitro, so typically molecular characterization must be performed immediately following isolation. To ease experimental timelines and enable biobanking, cryopreservation methods are needed. However, extensive cellular heterogeneity and the rarity of CTCs complicates the optimization of cryopreservation methods based upon cell type, necessitating a standardized protocol. Here, we optimized a previously reported vitrification protocol to preserve patient-derived CTC cell lines using highly conductive silica microcapillaries to achieve ultra-fast cooling rates with low cryoprotectant concentrations. Using this vitrification protocol, five CTC cell lines were cooled to cryogenic temperatures. Thawed CTCs exhibited high cell viability and expanded under in vitro cell culture conditions. EpCAM biomarker expression was maintained for each CTC cell line. One CTC cell line was selected for molecular characterization, revealing that RNA integrity was maintained after storage. A qPCR panel showed no significant difference in thawed CTCs compared to fresh controls. The data presented here suggests vitrification may enable the standardization of cryopreservation methods for CTCs.

  19. Genetic and Genomic Characterization of 462 Melanoma Patient-Derived Xenografts, Tumor Biopsies, and Cell Lines

    Directory of Open Access Journals (Sweden)

    Bradley Garman

    2017-11-01

    Full Text Available Summary: Tumor-sequencing studies have revealed the widespread genetic diversity of melanoma. Sequencing of 108 genes previously implicated in melanomagenesis was performed on 462 patient-derived xenografts (PDXs, cell lines, and tumors to identify mutational and copy number aberrations. Samples came from 371 unique individuals: 263 were naive to treatment, and 108 were previously treated with targeted therapy (34, immunotherapy (54, or both (20. Models of all previously reported major melanoma subtypes (BRAF, NRAS, NF1, KIT, and WT/WT/WT were identified. Multiple minor melanoma subtypes were also recapitulated, including melanomas with multiple activating mutations in the MAPK-signaling pathway and chromatin-remodeling gene mutations. These well-characterized melanoma PDXs and cell lines can be used not only as reagents for a large array of biological studies but also as pre-clinical models to facilitate drug development. : Garman et al. have characterized melanoma PDXs and cell lines described in Krepler et al. (see the related paper in this issue of Cell Reports, identifying major and minor subtypes, some of which were previously not well defined, targeted and immunotherapy resistance, and tumor heterogeneity, creating a set of reagents for future drug discovery and biological studies. Keywords: melanoma, patient-derived xenografts, massively parallel sequencing, cell lines

  20. Chromosomal imbalances in four new uterine cervix carcinoma derived cell lines

    Science.gov (United States)

    Hidalgo, Alfredo; Monroy, Alberto; Arana, Rosa Ma; Taja, Lucía; Vázquez, Guelaguetza; Salcedo, Mauricio

    2003-01-01

    Background Uterine cervix carcinoma is the second most common female malignancy worldwide and a major health problem in Mexico, representing the primary cause of death among the Mexican female population. High risk human papillomavirus (HPV) infection is considered to be the most important risk factor for the development of this tumor and cervical carcinoma derived cell lines are very useful models for the study of viral carcinogenesis. Comparative Genomic Hybridization (CGH) experiments have detected a specific pattern of chromosomal imbalances during cervical cancer progression, indicating chromosomal regions that might contain genes that are important for cervical transformation. Methods We performed HPV detection and CGH analysis in order to initiate the genomic characterization of four recently established cervical carcinoma derived cell lines from Mexican patients. Results All the cell lines were HPV18 positive. The most prevalent imbalances in the cell lines were gains in chromosomes 1q23-q32, 3q11.2-q13.1, 3q22-q26.1, 5p15.1-p11.2, this alteration present as a high copy number amplification in three of the cell lines, 7p15-p13, 7q21, 7q31, 11q21, and 12q12, and losses in 2q35-qter, 4p16, 6q26-qter, 9q34 and 19q13.2-qter. Conclusions Analysis of our present findings and previously reported data suggest that gains at 1q31-q32 and 7p13-p14, as well as losses at 6q26-q27 are alterations that might be unique for HPV18 positive cases. These chromosomal regions, as well as regions with high copy number amplifications, coincide with known fragile sites and known HPV integration sites. The general pattern of chromosomal imbalances detected in the cells resembled that found in invasive cervical tumors, suggesting that the cells represent good models for the study of cervical carcinoma. PMID:12659655

  1. Chromosomal imbalances in four new uterine cervix carcinoma derived cell lines

    Directory of Open Access Journals (Sweden)

    Vázquez Guelaguetza

    2003-03-01

    Full Text Available Abstract Background Uterine cervix carcinoma is the second most common female malignancy worldwide and a major health problem in Mexico, representing the primary cause of death among the Mexican female population. High risk human papillomavirus (HPV infection is considered to be the most important risk factor for the development of this tumor and cervical carcinoma derived cell lines are very useful models for the study of viral carcinogenesis. Comparative Genomic Hybridization (CGH experiments have detected a specific pattern of chromosomal imbalances during cervical cancer progression, indicating chromosomal regions that might contain genes that are important for cervical transformation. Methods We performed HPV detection and CGH analysis in order to initiate the genomic characterization of four recently established cervical carcinoma derived cell lines from Mexican patients. Results All the cell lines were HPV18 positive. The most prevalent imbalances in the cell lines were gains in chromosomes 1q23-q32, 3q11.2-q13.1, 3q22-q26.1, 5p15.1-p11.2, this alteration present as a high copy number amplification in three of the cell lines, 7p15-p13, 7q21, 7q31, 11q21, and 12q12, and losses in 2q35-qter, 4p16, 6q26-qter, 9q34 and 19q13.2-qter. Conclusions Analysis of our present findings and previously reported data suggest that gains at 1q31-q32 and 7p13-p14, as well as losses at 6q26-q27 are alterations that might be unique for HPV18 positive cases. These chromosomal regions, as well as regions with high copy number amplifications, coincide with known fragile sites and known HPV integration sites. The general pattern of chromosomal imbalances detected in the cells resembled that found in invasive cervical tumors, suggesting that the cells represent good models for the study of cervical carcinoma.

  2. Derivation of the clinical grade human embryonic stem cell line RCe015-A (RC-11).

    Science.gov (United States)

    De Sousa, P A; Tye, B J; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; McDonald, K; Bradburn, H; Laurie, A; Canham, M; Kunath, T; Downie, J M; Bateman, M; Courtney, A

    2016-05-07

    The human embryonic stem cell line RCe015-A (RC-11) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a fragmented cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe015-A (RC-11) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Chromosomal imbalances in four new uterine cervix carcinoma derived cell lines

    OpenAIRE

    Hidalgo, Alfredo; Monroy, Alberto; Arana, Rosa Ma; Taja, Lucía; Vázquez, Guelaguetza; Salcedo, Mauricio

    2003-01-01

    Abstract Background Uterine cervix carcinoma is the second most common female malignancy worldwide and a major health problem in Mexico, representing the primary cause of death among the Mexican female population. High risk human papillomavirus (HPV) infection is considered to be the most important risk factor for the development of this tumor and cervical carcinoma derived cell lines are very useful models for the study of viral carcinogenesis. Comparative Genomic Hybridization (CGH) experim...

  4. Identification and characterisation in vitro of cells with a non-SCLC cell-like phenotype derived from a continuous SCLC cell line.

    Science.gov (United States)

    Khan, M Z; Freshney, R I; Murray, A M; Merry, S; Plumb, J A; McNicol, A M

    1991-01-01

    Two adherent sublines, H69V and H69VZ, have been isolated from the classic SCLC cell line NCI-H69. Significant morphological differences were observed between the parental and the derivative cell lines. While NCI-H69 grew as densely packed free floating cellular aggregates the derivative lines grew as a monolayer of epithelioid cells. The growth rates of both the derivative lines were faster than the parental line with doubling times closer to non-SCLC cell lines in the derivative lines. Both H69V and H69VZ either express very low levels or do not express neuroendocrine cell markers including L-dopa-decarboxylase (DDC), creatine kinase-BB isoenzyme (CK-BB), bombesin-like immunoreactivity (BLI), neuron specific enolase (NSE), and neurosecretory type dense core granules (DGCs), compared to the parental cell line. All the lines stained positive for epithelial markers such as CAM5.2. LDH isoenzyme and chromosome analyses confirmed the human origin of all the cell lines. Therefore, it appears that cell line NCI-H69 contains stem cell subpopulation capable of generating cells of both small and non-small cell like phenotypes.

  5. Derivation of Human Induced Pluripotent Stem Cell (iPSC) Lines and Mechanism of Pluripotency: Historical Perspective and Recent Advances.

    Science.gov (United States)

    Chhabra, Arvind

    2017-12-01

    Derivation of human embryonic stem cell (hES) lines in 1998 was not only a major technological breakthrough in the field of regenerative medicine; it also triggered a passionate debate about the ethical issues associated with the utilization of human embryos for derivation of hESC lines. Successful derivation of induced pluripotent stem cell (iPS) lines from human somatic cells with defined reprogramming factors by Shinya Yamanaka`s group in 2007 was another breakthrough that generated enormous excitement and hope for the development of donor-specific personalized cell replacement therapies (CRT) without the ethical dilemma associated with it. As we approach twentieth anniversary of derivation of hESC lines and the tenth anniversary of isolation of donor-specific iPSC lines, this manuscript summarizes the key advances in pluripotent stem cell (PSC) research field that led to derivation of human iPSC lines, different methodologies for derivation iPSC lines and characterization of the mechanism of reprogramming. We will also review progress towards generating donor-specific somatic cell lineages from iPSC lines, especially the functional immune cell lineages, and progress towards advancing these findings to the clinic. Finally, we will discuss the challenges, such as genome instability and inherent immunogenicity of hPSC lines that need to be addressed to develop safe and effective iPSC-based CRT.

  6. The procurement of cells for the derivation of human embryonic stem cell lines for therapeutic use: recommendations for good practice.

    Science.gov (United States)

    Murdoch, Alison; Braude, Peter; Courtney, Aidan; Brison, Daniel; Hunt, Charles; Lawford-Davies, James; Moore, Harry; Stacey, Glyn; Sethe, Sebastian

    2012-03-01

    The donation of human embryos for the derivation of embryonic stem cell lines that may be used in the development of therapeutic products raises more complex ethical, practical and regulatory problems than the donation of embryos for non-clinical research. This review considers these issues and offers recommendations for good practice.

  7. Glial cell line-derived neurotrophic factor induced the differentiation of amniotic fluid-derived stem cells into vascular endothelial-like cells in vitro.

    Science.gov (United States)

    Zhang, Ruyu; Lu, Ying; Li, Ju; Wang, Jia; Liu, Caixia; Gao, Fang; Sun, Dong

    2016-02-01

    Amniotic fluid-derived stem cells (AFSCs) are a novel source of stem cells that are isolated and cultured from second trimester amniocentesis. Glial cell line-derived neurotrophic factor (GDNF) acts as a tissue morphogen and regulates stem cell proliferation and differentiation. This study investigated the effect of an adenovirus-mediated GDNF gene, which was engineered into AFSCs, on the cells' biological properties and whether GDNF in combination with AFSCs can be directionally differentiated into vascular endothelial-like cells in vitro. AFSCs were isolated and cultured using the plastic adherence method in vitro and identified by the transcription factor Oct-4, which is the primary marker of pluripotent stem cells. AFSCs were efficiently transfected by a GFP-labeled plasmid system of an adenovirus vector carrying the GDNF gene (Ad-GDNF-GFP). Transfected AFSCs stably expressed GDNF. Transfected AFSCs were cultured in endothelial growth medium-2 containing vascular endothelial growth factor. After 1 week, AFSCs were positive for von Willebrand factor (vWF) and CD31, which are markers of endothelial cells, and the recombinant GDNF group was significantly higher than undifferentiated controls and the GFP only group. These results demonstrated that AFSCs differentiated into vascular endothelial-like cells in vitro, and recombinant GDNF promoted differentiation. The differentiation-induced AFSCs may be used as seed cells to provide a new manner of cell and gene therapies for transplantation into the vascular injury site to promote angiogenesis.

  8. Chemo-radioresistance of small cell lung cancer cell lines derived from untreated primary tumors obtained by diagnostic bronchofiberscopy

    International Nuclear Information System (INIS)

    Tanio, Yoshiro; Watanabe, Masatoshi; Inoue, Tamotsu

    1990-01-01

    New cell lines of small cell lung cancer (SCLC) were established from specimens of untreated primary tumors biopsied by diagnostic bronchofiberscopy. The advantage of this method was ease of obtaining specimens from lung tumors. Establishment of cell lines was successful with 4 of 13 specimens (30%). Clinical responses of the tumors showed considerable variation, but were well correlated with the in vitro sensitivity of the respective cell lines to chemotherapeutic drugs and irradiation. One of the cell lines was resistant to all drugs tested and irradiation, while another was sensitive to all of them. Although the acquired resistance of SCLC is the biggest problem in treatment, the natural resistance to therapy is another significant problem. Either acquired or natural, resistance mechanisms of SCLC may be elucidated by the use of such cell lines derived from untreated tumors. This method and these SCLC cell lines are expected to be useful for the serial study of biologic and genetic changes of untreated and pre-treated tumors, or primary and secondary tumors. (author)

  9. Prevention of lysosomal storage diseases and derivation of mutant stem cell lines by preimplantation genetic diagnosis.

    Science.gov (United States)

    Altarescu, Gheona; Beeri, Rachel; Eiges, Rachel; Epsztejn-Litman, Silvina; Eldar-Geva, Talia; Elstein, Deborah; Zimran, Ari; Margalioth, Ehud J; Levy-Lahad, Ephrat; Renbaum, Paul

    2012-01-01

    Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research.

  10. Prevention of Lysosomal Storage Diseases and Derivation of Mutant Stem Cell Lines by Preimplantation Genetic Diagnosis

    Science.gov (United States)

    Altarescu, Gheona; Beeri, Rachel; Eiges, Rachel; Epsztejn-Litman, Silvina; Eldar-Geva, Talia; Elstein, Deborah; Zimran, Ari; Margalioth, Ehud J.; Levy-Lahad, Ephrat; Renbaum, Paul

    2012-01-01

    Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research. PMID:23320174

  11. Tumorigenicity, invasion and metastasis of the small cell lung cancer cell line NCI-H69 and two derivative lines MOG-H69V and MOG-H69VZ.

    Science.gov (United States)

    Khan, M Z; McNicol, A M; Freshney, R I

    1996-01-01

    Two adherent cell lines MOG-H69V and MOG-H69VZ have been isolated from a continuous cell line, NCI-H69, derived from human small cell lung cancer by Carney et al, [1987]. They have been established and characterised morphologically, biochemically, and for growth characteristics in vitro Khan et al (19). In the present study both the parental and the derivative lines have been investigated for invasiveness in vitro and in vivo. The parental line showed an early invasiveness compared with both the derivative cell lines. All cell lines formed tumours in nude mice with 100% take rate. Xenograft histology of all the cell lines revealed pleomorphic tumours, however the derivative lines showed areas of focal, large, spindle cells containing both acidic and neutral mucin, and spaces between the cells were found filled with alcianophilic, amorphous material. The parental line was invasive and metastatic. Tumours of both the derivative lines were non-metastatic under similar conditions. They were also investigated for neuroendocrine-cell marker expression. These data show that while the behaviour of the parental line was compatible with small cell lung cancer, that of the derivative lines was more indicative of non-small cell lung cancer, both in vitro and in vivo. As previous data suggested a common origin of the parental and the derivative lines, probably from a stem cell subpopulation present in the parental line, these lines represent a useful model for the study of phenotypic changes in lung cancer.

  12. Characterization of HGF/Met Signaling in Cell Lines Derived From Urothelial Carcinoma of the Bladder

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young H. [Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Apolo, Andrea B. [Genitourinary Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Agarwal, Piyush K.; Bottaro, Donald P., E-mail: dbottaro@helix.nih.gov [Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

    2014-11-25

    There is mounting evidence of oncogenic hepatocyte growth factor (HGF)/Met signaling in urothelial carcinoma (UC) of the bladder. The effects of three kinase inhibitors, cabozantinib, crizotinib and EMD1214063, on HGF-driven signaling and cell growth, invasion and tumorigenicity were analyzed in cultured UC cell lines. SW780 xenograft growth in SCID and human HGF knock-in SCID (hHGF/SCID) mice treated with cabozantinib or vehicle, as well as tumor levels of Met and pMet, were also determined. Met content was robust in most UC-derived cell lines. Basal pMet content and effector activation state in quiescent cells were low, but significantly enhanced by added HGF, as were cell invasion, proliferation and anchorage independent growth. These HGF-driven effects were reversed by Met inhibitor treatment. Tumor xenograft growth was significantly higher in hHGF/SCID mice vs. SCID mice and significantly inhibited by cabozantinib, as was tumor phospho-Met content. These studies indicate the prevalence and functionality of the HGF/Met signaling pathway in UC cells, suggest that paracrine HGF may contribute to UC tumor growth and progression, and that support further preclinical investigation of Met inhibitors for the treatment of UC is warranted.

  13. Glial Cell Line-Derived Neurotrophic Factor-Transfected Placenta-Derived Versus Bone Marrow-Derived Mesenchymal Cells for Treating Spinal Cord Injury.

    Science.gov (United States)

    Lu, Yao; Gao, Hui; Zhang, Man; Chen, Bing; Yang, Huilin

    2017-04-14

    BACKGROUND Placenta-derived mesenchymal stem cells (PMSCs) were isolated from placenta and had differentiation and self-renewal potential. We transfected PMSCs with glial cell line-derived neurotrophic factor (GDNF) and compared their effect for repairing spinal cord injury (SCI) with that of GDNF-transfected bone marrow-derived mesenchymal stem cell (BMSC). MATERIAL AND METHODS The PMSCs were isolated from Sprague-Dawley rat placenta; BMSCs were isolated from Sprague-Dawley rat thigh bone marrow. Primary cultured BMSCs and PMSCs were uniformly spindle-shaped. Flow cytometry indicated that both cell types were CD29- and CD90-positive and CD34- and CD45-negative, confirming that they were MSCs. The PMSCs and BMSCs were transfected with recombinant lentivirus containing the GDNF gene in vitro. PMSC and BMSC viability was increased after transfection, and GDNF expression was increased until 10 d after transfection. SCI was created in the rats (n=64) and was repaired using transfected PMSCs and BMSCs or untransfected PMSCs and BMSCs. RESULTS The transfected PMSCs and BMSCs repaired the SCI. Flow cytometry, histology, immunohistochemical, kinesiology properties, and Basso-Beattie-Bresnahan locomotion score measurements determined no significant difference between transfected PMSCs and BMSCs at 7, 14, and 21 d post-transplantation (P>0.05); the injury healed better in transfected PMSCs and BMSCs than in untransfected PMSCs and BMSCs (P<0.05). CONCLUSIONS MSCs have similar biology characteristics and capacity for SCI repair to BMSCs and can be used as a new resource for treating SCI.

  14. X-radiation-induced transformation in a C3H mouse embryo-derived cell line

    International Nuclear Information System (INIS)

    Terzaghi, M.; Little, J.B.

    1976-01-01

    Reproducible x-ray-induced oncogenic transformation has been demonstrated in an established cell line of mouse embryo fibroblasts. Cells derived from transformed foci formed malignant tumors when injected into syngeneic hosts. An exponential increase in the number of transformants per viable cell occurred with doses of up to 400 rads of x-radiation. The transformation frequency in exponentially growing cultures remained constant at 2.3 x 10 -3 following doses of 400 to 1500 rads. There was little change in survival following x-ray doses up to 300 rads. Doses greater than 300 rads were associated with an exponential decline in survival; the D 0 for the survival curve was 175 rads. Transformation frequency varied with changes in the number of viable cells seeded per dish. There was about a 10-fold decline in the transformation frequency when the number of cells was increased from 400 to 1000 viable cells/100-mm Petri dish. Below this density range there was little change in transformation frequency. The presence of lethally preirradiated cells was not associated with an enhancement of transformation in irradiated cells or with the induction of transformation in unirradiated cell cultures. Amphotericin B (Fungizone) inhibited the appearance of transformants when added to the culture medium within 2 to 3 weeks after initiation of the experiment

  15. Stromal cell-associated hematopoiesis: immortalization and characterization of a primate bone marrow-derived stromal cell line.

    Science.gov (United States)

    Paul, S R; Yang, Y C; Donahue, R E; Goldring, S; Williams, D A

    1991-04-15

    An elucidation of the interaction between the bone marrow microenvironment and hematopoietic stem cells is critical to the understanding of the molecular basis of stem cell self renewal and differentiation. This interaction is dependent, at least in part, on direct cell to cell contact or cellular adhesion to extracellular matrix proteins. Long-term bone marrow cultures (LTMC) provide an appropriate microenvironment for maintenance of primitive hematopoietic stem cells and a means of analyzing this stem cell-stromal cell interaction in vitro. Although LTMC have been successfully generated from murine and human bone marrow, only limited success has been reported in a primate system. In addition, few permanent stromal cell lines are available from nonmurine bone marrow. Because the primate has become a useful model for large animal bone marrow transplant studies and, more specifically, retroviral-mediated gene transfer analysis, we have generated immortalized bone marrow stromal cell lines from primate bone marrow using gene transfer of the Simian virus large T (SV40 LT) antigen. At least one stromal cell line has demonstrated the capacity to maintain early hematopoietic cells in long-term cultures for up to 4 weeks as measured by in vitro progenitor assays. Studies were undertaken to characterize the products of extracellular matrix biosynthesis and growth factor synthesis of this cell line, designated PU-34. In contrast to most murine bone marrow-derived stromal cell lines capable of supporting hematopoiesis in vitro that have been examined, the extracellular matrix produced by this primate cell line includes collagen types I, laminin. Growth factor production analyzed through RNA blot analysis, bone marrow cell culture data, and factor-dependent cell line proliferation assays includes interleukin-6 (IL-6), IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, leukemia inhibitory factor, and a novel cytokine designated IL-11. This

  16. Exposure to cobalt causes transcriptomic and proteomic changes in two rat liver derived cell lines.

    Directory of Open Access Journals (Sweden)

    Matthew G Permenter

    Full Text Available Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies.

  17. Exposure to Cobalt Causes Transcriptomic and Proteomic Changes in Two Rat Liver Derived Cell Lines

    Science.gov (United States)

    Permenter, Matthew G.; Dennis, William E.; Sutto, Thomas E.; Jackson, David A.; Lewis, John A.; Stallings, Jonathan D.

    2013-01-01

    Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies. PMID:24386269

  18. Mapping of the secretome of primary isolates of mammalian cells, stem cells and derived cell lines

    Czech Academy of Sciences Publication Activity Database

    Skalníková, Helena; Motlík, Jan; Gadher, S. J.; Kovářová, Hana

    2011-01-01

    Roč. 11, - (2011), s. 691-708 ISSN 1615-9853 R&D Projects: GA MŠk 1M0538; GA MŠk(CZ) ME10044 Institutional research plan: CEZ:AV0Z50450515 Keywords : cell biology * conditioned media * cytokine Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.505, year: 2011

  19. Natural bizbenzoquinoline derivatives protect zebrafish lateral line sensory hair cells from aminoglycoside toxicity

    Directory of Open Access Journals (Sweden)

    Matthew eKruger

    2016-03-01

    Full Text Available Moderate to severe hearing loss affects 360 million people worldwide and most often results from damage to sensory hair cells. Hair cell damage can result from aging, genetic mutations, excess noise exposure, and certain medications including aminoglycoside antibiotics. Aminoglycosides are effective at treating infections associated with cystic fibrosis and other life-threatening conditions such as sepsis, but cause hearing loss in 20-30% of patients. It is therefore imperative to develop new therapies to combat hearing loss and allow safe use of these potent antibiotics. We approach this drug discovery question using the larval zebrafish lateral line because zebrafish hair cells are structurally and functionally similar to mammalian inner ear hair cells and respond similarly to toxins. We screened a library of 502 natural compounds in order to identify novel hair cell protectants. Our screen identified four bisbenzylisoquinoline derivatives: berbamine, E6 berbamine, hernandezine, and isotetrandrine, each of which robustly protected hair cells from aminoglycoside-induced damage. Using fluorescence microscopy and electrophysiology, we demonstrated that the natural compounds confer protection by reducing antibiotic uptake into hair cells and showed that hair cells remain functional during and after incubation in E6 berbamine. We also determined that these natural compounds do not reduce antibiotic efficacy. Together, these natural compounds represent a novel source of possible otoprotective drugs that may offer therapeutic options for patients receiving aminoglycoside treatment.

  20. Novel pancreatic cancer cell lines derived from genetically engineered mouse models of spontaneous pancreatic adenocarcinoma: applications in diagnosis and therapy.

    Directory of Open Access Journals (Sweden)

    María P Torres

    Full Text Available Pancreatic cancer (PC remains one of the most lethal human malignancies with poor prognosis. Despite all advances in preclinical research, there have not been significant translation of novel therapies into the clinics. The development of genetically engineered mouse (GEM models that produce spontaneous pancreatic adenocarcinoma (PDAC have increased our understanding of the pathogenesis of the disease. Although these PDAC mouse models are ideal for studying potential therapies and specific genetic mutations, there is a need for developing syngeneic cell lines from these models. In this study, we describe the successful establishment and characterization of three cell lines derived from two (PDAC mouse models. The cell line UN-KC-6141 was derived from a pancreatic tumor of a Kras(G12D;Pdx1-Cre (KC mouse at 50 weeks of age, whereas UN-KPC-960 and UN-KPC-961 cell lines were derived from pancreatic tumors of Kras(G12D;Trp53(R172H;Pdx1-Cre (KPC mice at 17 weeks of age. The cancer mutations of these parent mice carried over to the daughter cell lines (i.e. Kras(G12D mutation was observed in all three cell lines while Trp53 mutation was observed only in KPC cell lines. The cell lines showed typical cobblestone epithelial morphology in culture, and unlike the previously established mouse PDAC cell line Panc02, expressed the ductal marker CK19. Furthermore, these cell lines expressed the epithelial-mesenchymal markers E-cadherin and N-cadherin, and also, Muc1 and Muc4 mucins. In addition, these cell lines were resistant to the chemotherapeutic drug Gemcitabine. Their implantation in vivo produced subcutaneous as well as tumors in the pancreas (orthotopic. The genetic mutations in these cell lines mimic the genetic compendium of human PDAC, which make them valuable models with a high potential of translational relevance for examining diagnostic markers and therapeutic drugs.

  1. Effect of glial cell line-derived neurotrophic factor on retinal function after experimental branch retinal vein occlusion

    DEFF Research Database (Denmark)

    Ejstrup, Rasmus; Dornonville de la Cour, Morten; Kyhn, Maria Voss

    2012-01-01

    The objective of the study was to investigate the effect of glial cell line-derived neurotrophic factor (GDNF) on the multifocal electroretinogram (mfERG) following an induced branch retinal vein occlusion (BRVO) in pigs.......The objective of the study was to investigate the effect of glial cell line-derived neurotrophic factor (GDNF) on the multifocal electroretinogram (mfERG) following an induced branch retinal vein occlusion (BRVO) in pigs....

  2. Design, Synthesis and Evaluation of N13-Substituted Evodiamine Derivatives against Human Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Senchuan Song

    2013-12-01

    Full Text Available Attempting to improve the anticancer activity and solubility of evodiamine in simulated gastric fluid (SGF and simulated intestinal fluid (SIF solutions, thirty-eight N13-substituted evodiamine derivatives were designed, synthesized and tested for antitumor activities against six kinds of human cancer cell lines, namely prostate cancer (DU-145 and PC-3, lung cancer (H460, breast cancer (MCF-7, colon cancer (HCT-5 and glioblastoma (SF-268. The solubility of these compounds in SGF and SIF solutions was evaluated, and apoptosis induced by 2-2, 2-3, 2-16 and 3-2 was determined. The results showed: (1 among all compounds examined, 2-16 showed the highest antitumor activity and a broader spectrum of activity, with IC50 values ranging from 1–2 µM; (2 their solubility was obviously improved; (3 2-3, 2-16 and 3-2 had a significant impact inducing apoptosis in some cancer cell lines. The preliminary structure-activity relationships of these derivatives were discussed.

  3. Multiple breast cancer cell-lines derived from a single tumor differ in their molecular characteristics and tumorigenic potential.

    Directory of Open Access Journals (Sweden)

    Goar Mosoyan

    Full Text Available BACKGROUND: Breast cancer cell lines are widely used tools to investigate breast cancer biology and to develop new therapies. Breast cancer tissue contains molecularly heterogeneous cell populations. Thus, it is important to understand which cell lines best represent the primary tumor and have similarly diverse phenotype. Here, we describe the development of five breast cancer cell lines from a single patient's breast cancer tissue. We characterize the molecular profiles, tumorigenicity and metastatic ability in vivo of all five cell lines and compare their responsiveness to 4-hydroxytamoxifen (4-OHT treatment. METHODS: Five breast cancer cell lines were derived from a single patient's primary breast cancer tissue. Expression of different antigens including HER2, estrogen receptor (ER, CK8/18, CD44 and CD24 was determined by flow cytometry, western blotting and immunohistochemistry (IHC. In addition, a Fluorescent In Situ Hybridization (FISH assay for HER2 gene amplification and p53 genotyping was performed on all cell lines. A xenograft model in nude mice was utilized to assess the tumorigenic and metastatic abilities of the breast cancer cells. RESULTS: We have isolated, cloned and established five new breast cancer cell lines with different tumorigenicity and metastatic abilities from a single primary breast cancer. Although all the cell lines expressed low levels of ER, their growth was estrogen-independent and all had high-levels of expression of mutated non-functional p53. The HER2 gene was rearranged in all cell lines. Low doses of 4-OHT induced proliferation of these breast cancer cell lines. CONCLUSIONS: All five breast cancer cell lines have different antigenic expression profiles, tumorigenicity and organ specific metastatic abilities although they derive from a single tumor. None of the studied markers correlated with tumorigenic potential. These new cell lines could serve as a model for detailed genomic and proteomic analyses to

  4. The inhibitory influence of adipose tissue-derived mesenchymal stem cell environment and Wnt antagonism on breast tumour cell lines.

    Science.gov (United States)

    Visweswaran, Malini; Arfuso, Frank; Dilley, Rodney J; Newsholme, Philip; Dharmarajan, Arun

    2018-02-01

    Tumours exhibit a heterogeneous mix of cell types that reciprocally regulate their growth in the tumour stroma, considerably affecting the progression of the disease. Both adipose-derived mesenchymal stem cells and Wnt signalling pathway are vital in driving breast tumour growth. Hence, we examined the effect of secreted factors released by adipose-derived mesenchymal stem cells, and further explored the anti-tumour property of the Wnt antagonist secreted frizzled-related protein 4 (sFRP4) on MCF-7 and MDA-MB-231 breast tumour cells. We observed that conditioned medium and extracellular matrix derived from adipose-derived mesenchymal stem cells inhibited tumour viability. The inhibitory effect of the conditioned medium was retained within its low molecular weight and non-protein component. The conditioned medium also induced apoptosis accompanied by a decrease in the mitochondrial membrane potential in tumour cells, Furthermore, it downregulated the protein expression of active β-catenin and Cyclin D1, which are major target proteins of the Wnt signalling pathway, and reduced the expression of anti-apoptotic protein Bcl-xL. The combination of conditioned medium and sFRP4 further potentiated the effects, depending on the tumour cell line and experimental assay. We conclude that factors derived from conditioned medium of adipose-derived mesenchymal stem cells and sFRP4 significantly decreased the tumour cell viability and migration rates (MCF-7), accompanied with an enhanced apoptotic activity through inhibition of canonical Wnt signalling. Besides giving an insight to possible paracrine interactions and influence of signalling pathways, reflective of a breast tumour microenvironment, this study emphasises the utilization of cell free-secreted factors and Wnt antagonists to improve conventional anti-cancer strategies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. The niche-derived glial cell line-derived neurotrophic factor (GDNF induces migration of mouse spermatogonial stem/progenitor cells.

    Directory of Open Access Journals (Sweden)

    Lisa Dovere

    Full Text Available In mammals, the biological activity of the stem/progenitor compartment sustains production of mature gametes through spermatogenesis. Spermatogonial stem cells and their progeny belong to the class of undifferentiated spermatogonia, a germ cell population found on the basal membrane of the seminiferous tubules. A large body of evidence has demonstrated that glial cell line-derived neurotrophic factor (GDNF, a Sertoli-derived factor, is essential for in vivo and in vitro stem cell self-renewal. However, the mechanisms underlying this activity are not completely understood. In this study, we show that GDNF induces dose-dependent directional migration of freshly selected undifferentiated spermatogonia, as well as germline stem cells in culture, using a Boyden chamber assay. GDNF-induced migration is dependent on the expression of the GDNF co-receptor GFRA1, as shown by migration assays performed on parental and GFRA1-transduced GC-1 spermatogonial cell lines. We found that the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP is specifically expressed in undifferentiated spermatogonia. VASP belongs to the ENA/VASP family of proteins implicated in actin-dependent processes, such as fibroblast migration, axon guidance, and cell adhesion. In intact seminiferous tubules and germline stem cell cultures, GDNF treatment up-regulates VASP in a dose-dependent fashion. These data identify a novel role for the niche-derived factor GDNF, and they suggest that GDNF may impinge on the stem/progenitor compartment, affecting the actin cytoskeleton and cell migration.

  6. Serum glial cell line-derived neurotrophic factor levels and postoperative cognitive dysfunction after surgery for rheumatic heart disease.

    Science.gov (United States)

    Duan, Xiaoxia; Zhu, Tao; Chen, Chan; Zhang, Guanpeng; Zhang, Junhui; Wang, Lin; Zhang, Luye; Wang, Maohua; Wang, Xiaobin

    2018-03-01

    Postoperative cognitive dysfunction is an important complication of cardiac surgery with poor outcomes. Serum glial cell line-derived neurotrophic factor levels are decreased in patients with Alzheimer's disease, but the association between glial cell line-derived neurotrophic factor levels and postoperative cognitive dysfunction is poorly understood. The present study aimed to investigate the prognostic value of postoperative serum glial cell line-derived neurotrophic factor levels to predict postoperative cognitive dysfunction in patients with rheumatic heart disease undergoing heart valve replacement. This was a prospective observational study of 80 patients undergoing elective heart valve replacement surgery from June 2015 to June 2016 at the Affiliated Hospital of Southeast Medical University. Cognitive functions were assessed 1 day before and 7 days after surgery. Serum glial cell line-derived neurotrophic factor levels were measured by an enzyme-linked immunosorbent assay before (T1) and 1 (T2), 2 (T3), and 7 (T4) days after surgery. Perioperative parameters were evaluated to assess the relationship between glial cell line-derived neurotrophic factors and postoperative cognitive dysfunction. Postoperative cognitive dysfunction was identified in 38 patients (47.5%) 7 days after surgery. Average glial cell line-derived neurotrophic factor levels at 2 and 7 days after surgery in the postoperative cognitive dysfunction group were lower than in the nonpostoperative cognitive dysfunction group at the same time points (P derived neurotrophic factor (T1-T3) and Δglial cell line-derived neurotrophic factor (T1-T4) were identified as good predictors of postoperative cognitive dysfunction with threshold for postoperative cognitive dysfunction detection of 49.10 and 60.90, respectively. The perioperative glial cell line-derived neurotrophic factor levels in patients with postoperative cognitive dysfunction were lower than in patients without postoperative

  7. Synthesis and cytotoxic activity of certain benzothiazole derivatives against human MCF-7 cancer cell line.

    Science.gov (United States)

    Mohamed, Lamia W; Taher, Azza T; Rady, Ghada S; Ali, Mamdouh M; Mahmoud, Abeer E

    2017-04-01

    A new series of benzothiazole has been synthesized as cytotoxic agents. The new derivatives were tested for their cytotoxic activity toward the human breast cancer MCF-7 cell line against cisplatin as the reference drug. Many derivatives revealed good cytotoxic effect, whereas four of them, 4, 5c, 5d, and 6b, were more potent than cisplatin, with IC 50 values being 8.64, 7.39, 7.56, and 5.15 μm compared to 13.33 μm of cisplatin. The four derivatives' cytotoxic activity was accompanied by regulating free radicals production, by increasing the activity of superoxide dismutase and depletion of intracellular reduced glutathione, catalase, and glutathione peroxidase activities, accordingly, the high production of hydrogen peroxide, nitric oxide, and other free radicals causing tumor cell death as monitored by reduction in the synthesis of protein and nucleic acids. Most of the tested compounds showed potent to moderate growth inhibitory activity; in particular, compound 6b exhibited the highest activity suggesting it is a lead compound in cytotoxic activity. © 2016 John Wiley & Sons A/S.

  8. Establishment and characterization of a unique 1 {mu}m diameter liver-derived progenitor cell line

    Energy Technology Data Exchange (ETDEWEB)

    Aravalli, Rajagopal N., E-mail: arava001@umn.edu [Department of Radiology, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Behnan Sahin, M. [Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Cressman, Erik N.K. [Department of Radiology, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Steer, Clifford J., E-mail: steer001@umn.edu [Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, MN 55455 (United States)

    2010-01-01

    Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 {mu}m in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers.

  9. A dibenzoylmethane derivative inhibits lipopolysaccharide-induced NO production in mouse microglial cell line BV-2.

    Science.gov (United States)

    Takano, Katsura; Ishida, Natsumi; Kawabe, Kenji; Moriyama, Mitsuaki; Hibino, Satoshi; Choshi, Tominari; Hori, Osamu; Nakamura, Yoichi

    2017-04-05

    Microglial activation has been suggested to play important roles in various neurodegenerative diseases by phagocytosis and producing various factors such as nitric oxide (NO), proinflammatory cytokines. Excessive production of NO, as a consequence of increased inducible nitric oxide synthase (iNOS) in microglia, contributes to the neurodegeneration. During a search for compounds that regulate endoplasmic reticulum (ER) stress, a dibenzoylmethane derivative, 2,2'-dimethoxydibenzoylmethane (DBM 14-26) was identified as a novel neuroprotective agent (Takano et al., Am. J. Physiol. Cell Physiol. 293, C1884-1894, 2007). We previously reported in cultured astrocytes that DBM 14-26 protected hydrogen peroxide-induced cell death and inhibited lipopolysaccharide (LPS)-induced NO production (Takano et al., J. Neurosci. Res. 89, 955-965, 2011). In the present study, we assessed the effects of DBM 14-26 on microglia using the mouse cell line BV-2 and found that DBM 14-26 inhibited LPS-induced iNOS expression and NO production also in microglia. DBM 14-26 also suppressed LPS-induced IL-1β expression. Conditioned medium of BV-2 cells stimulated by LPS significantly decreased cell viability of neuron (human neuroblastoma SH-SY5Y cells) compared with the absence of LPS. Conditioned medium of BV-2 cells stimulated by LPS in the presence of DBM 14-26 did not significantly decreased cell viability of neuron. These results indicate that microglial activation by LPS causes neuronal cell death and DBM 14-26 protect neuron through the inhibition of microglial activation. Functional regulation of microglia by DBM 14-26 could be a therapeutic candidate for the treatment of neurodegenerative diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Comparison of radiosensitivity between human hematopoietic cell lines derived from patients with Down's syndrome and from normal persons

    International Nuclear Information System (INIS)

    Huang, C.C.; Banerjee, A.; Tan, J.C.; Hou, Y.

    1977-01-01

    Seven hematopoietic cell lines, four derived from the peripheral blood of patients with Down's syndrome (DS) and three from normal persons, were irradiated with 100, 150, 300, and 500 rads from a 60 Co source and harvested for cell count and chromosome aberration studies every 12 hours for 72 hours post irradiation. Cell growth inhibition and an increase in chromosome aberration were observed in all the cell lines at each dose level and time interval. No significant difference was observed in the effects between DS and normal cell lines. The most common types of aberrations in the 12-hour samples were chromosome and/or chromatid breaks. In the later samples, chromatid exchanges were predominant. The results of the variance analyses on the induced chromosome aberrations in six lines (three DS and three normal lines) showed radiation dosage to be the largest component of total variance, following postirradiation duration and cell lines. The samples harvested 24 and 36 hours post irradiation generally showed greater effects than the samples of other harvest durations. The cell line variance could only be attributed to the differences among and between individual cell lines rather than the difference between DS and normal cell lines

  11. Concentration of Glial Cell Line-Derived Neurotrophic Factor Positively Correlates with Symptoms in Functional Dyspepsia.

    Science.gov (United States)

    Tanaka, Fumio; Tominaga, Kazunari; Fujikawa, Yoshiko; Nagami, Yasuaki; Kamata, Noriko; Yamagami, Hirokazu; Tanigawa, Tetsuya; Shiba, Masatsugu; Watanabe, Toshio; Fujiwara, Yasuhiro; Arakawa, Tetsuo

    2016-12-01

    In patients with functional dyspepsia (FD), mild duodenal inflammation correlates with increased mucosal permeability. Enteric glial cells can produce glial cell line-derived neurotrophic factor (GDNF) to repair disrupted epithelial barrier function. We examined the role of duodenal GDNF in FD pathophysiology and its association with dyspeptic symptoms. Duodenal biopsies taken from FD patients and control subjects were used for analysis. GDNF protein expression and localization were examined. Cellular infiltration of eosinophils and mast cells was measured. We also examined the intercellular space between the adjacent epithelial cells at the apical junction complex using transmission electron microscopy. In FD patients, expression of GDNF protein was significantly increased compared with controls, 107.3 (95.3-136.7) versus 49.3 (38.0-72.6) pg/mg protein (median (interquartile range), p = 0.006), respectively. GDNF was localized in enteric glial cells, eosinophils, and epithelial cells. The number of eosinophils was significantly greater in FD patients than in controls, 1039 (923-1181) versus 553 (479-598) cells/mm 2 (p = 0.021), respectively. The intercellular space was dilated at the adherent junction in FD patients compared to control patients, 32.4 (29.8-34.8) versus 22.0 (19.9-26.1) nm (p = 0.002), respectively. Intercellular distance positively correlated with the frequency of postprandial fullness and early satiation (p = 0.001, r = 0.837 and p = 0.009, r = 0.693, respectively). Expression of GDNF correlated with epigastric burning (p = 0.041, r = 0.552). Increased expression of duodenal GDNF might be involved in FD pathophysiology and symptom perception.

  12. Cabergoline decreases alcohol drinking and seeking behaviors via glial cell line-derived neurotrophic factor.

    Science.gov (United States)

    Carnicella, Sebastien; Ahmadiantehrani, Somayeh; He, Dao-Yao; Nielsen, Carsten K; Bartlett, Selena E; Janak, Patricia H; Ron, Dorit

    2009-07-15

    Cabergoline is an ergotamine derivative that increases the expression of glial cell line-derived neurotrophic factor (GDNF) in vitro. We recently showed that GDNF in the ventral tegmental area (VTA) reduces the motivation to consume alcohol. We therefore set out to determine whether cabergoline administration decreases alcohol-drinking and -seeking behaviors via GDNF. Reverse transcription polymerase chain reaction (RT-PCR) and Enzyme-Linked ImmunoSorbent Assay (ELISA) were used to measure GDNF levels. Western blot analysis was used for phosphorylation experiments. Operant self-administration in rats and a two-bottle choice procedure in mice were used to assess alcohol-drinking behaviors. Instrumental performance tested during extinction was used to measure alcohol-seeking behavior. The [35S]GTPgammaS binding assay was used to assess the expression and function of the dopamine D2 receptor (D2R). We found that treatment of the dopaminergic-like cell line SH-SY5Y with cabergoline and systemic administration of cabergoline in rats resulted in an increase in GDNF level and in the activation of the GDNF pathway. Cabergoline treatment decreased alcohol-drinking and -seeking behaviors including relapse, and its action to reduce alcohol consumption was localized to the VTA. Finally, the increase in GDNF expression and the decrease in alcohol consumption by cabergoline were abolished in GDNF heterozygous knockout mice. Together, these findings suggest that cabergoline-mediated upregulation of the GDNF pathway attenuates alcohol-drinking behaviors and relapse. Alcohol abuse and addiction are devastating and costly problems worldwide. This study puts forward the possibility that cabergoline might be an effective treatment for these disorders.

  13. Probing the importance of clonality: Single cell subcloning of clonally derived CHO cell lines yields widely diverse clones differing in growth, productivity, and product quality.

    Science.gov (United States)

    Ko, Peggy; Misaghi, Shahram; Hu, Zhilan; Zhan, Dejin; Tsukuda, Joni; Yim, Mandy; Sanford, Mark; Shaw, David; Shiratori, Masaru; Snedecor, Brad; Laird, Michael; Shen, Amy

    2017-12-11

    In the past few decades, a large variety of therapeutic antibodies and proteins have been expressed in Chinese hamster ovary (CHO) cells. This mammalian expression system is robust, scalable, relatively inexpensive, and importantly allows for post-translational modifications that are important for some therapeutic proteins. Historically, CHO cell lines were derived from colonies of cells grown in semi-solid or liquid plates using either serum-containing or serum-free media. Current advancements in cell sorting and imaging technologies have allowed for isolating and imaging single cell progenitors at the seeding step, significantly increasing the probability of isolating clonally derived cell lines. However, it is debatable how much population heterogeneity can be eliminated when clonally derived cell lines, originated from a single cell progenitor, are scaled up. To further investigate this phenomenon, we subcloned two different clonally derived (day 0 imaged and visually inspected) cell lines expressing antibody-X. The results showed that when six randomly chosen subclones of each line were evaluated in a production assay, these subclones displayed a range of variation in titer, specific productivity, growth, and product quality attributes. Some subclones displayed variations in transgene copy numbers. Additionally, clonal derivation did not assure stability of the derived cell lines. Our findings show that cell heterogeneity exists in a population even when derived from a single cell progenitor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

  14. RET-mediated glial cell line-derived neurotrophic factor signaling inhibits mouse prostate development.

    Science.gov (United States)

    Park, Hyun-Jung; Bolton, Eric C

    2017-06-15

    In humans and rodents, the prostate gland develops from the embryonic urogenital sinus (UGS). The androgen receptor (AR) is thought to control the expression of morphogenetic genes in inductive UGS mesenchyme, which promotes proliferation and cytodifferentiation of the prostatic epithelium. However, the nature of the AR-regulated morphogenetic genes and the mechanisms whereby AR controls prostate development are not understood. Glial cell line-derived neurotrophic factor (GDNF) binds GDNF family receptor α1 (GFRα1) and signals through activation of RET tyrosine kinase. Gene disruption studies in mice have revealed essential roles for GDNF signaling in development; however, its role in prostate development is unexplored. Here, we establish novel roles of GDNF signaling in mouse prostate development. Using an organ culture system for prostate development and Ret mutant mice, we demonstrate that RET-mediated GDNF signaling in UGS increases proliferation of mesenchyme cells and suppresses androgen-induced proliferation and differentiation of prostate epithelial cells, inhibiting prostate development. We also identify Ar as a GDNF-repressed gene and Gdnf and Gfrα1 as androgen-repressed genes in UGS, thus establishing reciprocal regulatory crosstalk between AR and GDNF signaling in prostate development. © 2017. Published by The Company of Biologists Ltd.

  15. SYNTHESIS AND CYTOTOXIC ACTIVITY OF CHALCONE DERIVATIVES ON HUMAN BREAST CANCER CELL LINES

    Directory of Open Access Journals (Sweden)

    Nuraini Harmastuti

    2012-12-01

    Full Text Available Chalcone, an α,β-unsaturated ketone, has been shown have many biological activities such as anticancer and antifungi. This research was conducted to synthesize the chalcone derivatives and to obtain their cytotoxic activity on human cervix cancer cell lines. Synthesis of chalcone and its derivatives, 4II-methylchalcone, 4II-methoxychalcone, and 3II,4II-dichlorochalcone was carried out using starting materials of benzaldehide and acetofenon, p-methylacetophenone, p-methoxyacetophenone, as well as m,p-dichloroacetophenone through Claisen Schmidt condensation catalized by NaOH in ethanol at 15 °C. The purity of synthesized compounds were analyzed by thin layer chromatography, melting range, and gas chromatography. Structure elucidations were conducted by UV spectrophotometer, IR spectrometer, 1H-NMR spectrometer, as well as mass spectrometer. Cytotoxic activities were determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT microculture tetrazolium viability assay. The results showed that chalcone and derivatives compounds have been able to be synthesized and purified and had the same structure as a predicted structure. Chalcone had highest cytotoxic activity compared to that of its derivatives, with the IC50 values of chalcone, 4II-methylchalcone, 4II-methoxychalcone, and 3II,4II-dichlorochalcone were 9.49, 14.79, 11.48, and 24.26 µg/mL respectively. It was concluded that methyl, methoxy as well as chlorine substitution at 3 II and 4II position decrease the cytotoxic activity of chalcone.

  16. Effect of Mesenchymal Stem Cells and a Novel Curcumin Derivative on Notch1 Signaling in Hepatoma Cell Line

    Directory of Open Access Journals (Sweden)

    Mohamed Talaat Abdel Aziz

    2013-01-01

    Full Text Available This study was conducted to evaluate the effect of mesenchymal stem cells (MSCs and a novel curcumin derivative (NCD on HepG2 cells (hepatoma cell line and to investigate their effect on Notch1 signaling pathway target genes. HepG2 cells were divided into HepG2 control group, HepG2 cells treated with MSC conditioned medium (MSCs CM, HepG2 cells treated with a NCD, HepG2 cells treated with MSCs CM and NCD, and HepG2 cells treated with MSCs CM (CM of MSCs pretreated with a NCD. Expression of Notch1, Hes1, VEGF, and cyclin D1 was assessed by real-time, reverse transcription-polymerase chain reaction (RT-PCR in HepG2 cells. In addition, HepG2 proliferation assay was performed in all groups. Notch1 and its target genes (Hes1 and cyclin D1 were downregulated in all treated groups with more suppressive effect in the groups treated with both MSCs and NCD. Also, treated HepG2 cells showed significant decrease in cell proliferation rate. These data suggest that modulation of Notch1 signaling pathway by MSCs and/or NCD can be considered as a therapeutic target in HCC.

  17. Analysis of STAT4 expression in cutaneous T-cell lymphoma (CTCL) patients and patient-derived cell lines

    DEFF Research Database (Denmark)

    Litvinov, Ivan V; Cordeiro, Brendan; Fredholm, Simon Mayland

    2014-01-01

    Deregulation of STAT signaling has been implicated in the pathogenesis for a variety of cancers, including CTCL. Recent reports indicate that loss of STAT4 expression is an important prognostic marker for CTCL progression and is associated with the acquisition of T helper 2 cell phenotype......R-155 leads to upregulation in STAT4 expression in MyLa cells. In summary, our results suggest that loss of STAT4 expression and associated switch to Th2 phenotype during Mycosis Fungoides progression may be driven via aberrant histone acetylation and/or upregulation of oncogenic miR-155 microRNA....... by malignant cells. However, little is known about the molecular mechanism behind the downregulation of STAT4 in this cancer. In the current work we test the expression of STAT4 and STAT6 via RT-PCR and/or Western Blot in CTCL lesional skin samples and in immortalized patient-derived cell lines...

  18. Experimental infection of Leishmania (L. chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae

    Directory of Open Access Journals (Sweden)

    Felio J Bello

    2005-10-01

    Full Text Available The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5 cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6% and on day 4 in the J774 cells (51%. This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

  19. Establishment and characterization of an adherent pure epithelial cell line derived from the bovine oviduct.

    Science.gov (United States)

    Schoen, J; Bondzio, A; Topp, K; Einspanier, R

    2008-03-15

    The oviduct in vivo has to perform various tasks: maturation and transport of the gametes, milieu preparation for fertilization and embryonic development, and transport of the embryo. The complex arrangement of endocrine and paracrine signals being exchanged between the early embryo and the inner cell layers of the oviduct is barely understood. Therefore, a reproducible, well-characterized oviduct epithelial cell line as well as an optimized transfection protocol for DNA vectors and siRNA for this cell line has been established. A bovine oviduct primary cell culture system has been optimized using a selection medium permitting the survival of only epithelial cells. From this we established an adherent bovine oviduct pure epithelial cell line (aBOPEC-1). This cell line maintains some important characteristics of the primary cells such as the expression of estrogen receptors and p450 aromatase but it lacks some characteristics due to the selection and dedifferentiation processes (cilia, expression of progesterone receptor and oviduct specific glycoprotein-1). Optimization of the transfection protocols finally revealed a suitable DNA-transfection procedure yielding transfection efficiencies of over 50%. Additionally, siRNA transfection efficiency reached more than 90%. This new cell line builds an essential basis especially for future functional studies in the oviduct epithelium using distinct knock down experiments.

  20. Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts

    DEFF Research Database (Denmark)

    Jakobsen, Jannik E.; Li, Juan; Moldt, Brian

    2011-01-01

    We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon-based do......We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon...... transfer. PFV cells supported Flp mediated cassette exchange for transgene substitution of eGFP with dsRED, and the dsRED transgenic PFV cells generated blastocysts with transgene expression. Hence, the PFV cell line constitutes a valuable pig equivalent to transformed cell lines from other mammalian...

  1. Protein kinase C stimulation of phospholipid synthesis in a type II pneumocyte derived cell line

    International Nuclear Information System (INIS)

    Brandes, M.E.; Finkelstein, J.N.

    1986-01-01

    In the Type II pneumocyte-derived cell line, A549, addition of 50 nM 12-O-tetradecanoyl phorbol-13-acetate (TPA) and phorbol-12, 13-dibutyrate more than doubled the rate of de novo synthesis of phophatidylcholine (PC). A similar increase was observed with the addition of the exogenous diacylglycerol, 1-oleoyl-2-acetylglycerol. These results suggest a role for the activation of protein kinase C(PKC) in the stimulation of PC biosynthesis. The modulation of CTP:phosphocholine cytidyltransferase activity was examined as the locus for TPA mediated effects. Pulse chase experiments showed TPA caused a significant increase in the rate of utilization of phosphocholine. The observed effect of TPA on CT activity may be due to its nonspecific promotion of binding of the enzyme to the endoplasmic reticulum or a specific consequence of PKC activation and phosphorylation. Preincubation of cells with Compound 48/80, an inhibitor of PKC, reduced TPA stimulation of PC synthesis by 40-45%. Incubation with 4α-phorbol 12,13 didecanoate (PDD) which does not activate PKC had little stimulatory effect. Treatment of intact cells with TPA for 60 min increased CT from 0.401 nmol/min/mg to 0.787 nmol/min/mg. Preincubation with 48/80 prior to TPA reduced the increase in CT activity over 50%. Incubation with PDD only increased CT by 17%. These results strongly suggest that the phorbol ester stimulation of phospholipid synthesis is a specific effect due to its ability to activate PKC

  2. Incorporation study of 18FDG through its uptake into tumour-derived cell lines

    International Nuclear Information System (INIS)

    Costa, Flavia M.; Nascimento, Leonardo T.C.; Santos, Raquel G.

    2013-01-01

    In vitro tests of radiopharmaceuticals are an important instrument to study their mechanisms of action, binding and incorporation. 18 FDG is the most used radiopharmaceutical for diagnostics positron emission tomography (PET) on oncology, on the basis of accelerated rates of absorption of glucose in cell malignancies. This radiotracer has been routinely produced at CDTN; and therefore, it was selected for preliminary assays due to its availability. Nowadays, UPPR at CDTN produces routinely 18 FDG for the local PET Centers but others PET radiopharmaceuticals are in development such as 18 F-Fluorocholine and 18 F-Fluorothymidine. According to the Brazilian Health Regulatory Agency (ANVISA) it is necessary to validate and register these new radiopharmaceuticals in order to get the approval for their commercialization. Targeting efficacy is one of the important issues to be evaluated during radiopharmaceutical validation. The aim of this study was to develop a standard protocol to determine tumor targeting efficacy of PET radiopharmaceuticals in vitro. Therefore it was developed a protocol based on the incorporation og 18 FDG through the uptake in different tumor-derived cell lines. Three variables were investigated for the standardization of the test: the number of cells to be seeded in 96-well plates, the time of incubation with the radiopharmaceutical and the radiotracer concentration. The standardized protocol was considered suitable for 18 FDG incorporation assay and showed reproductive results. The protocol developed in this work will pave the way for the in vitro studies of incorporation of the new PET radiopharmaceuticals to be produced at UPPR-CDTN, such as: 18 F-Fluorocholine and 18 F-Fluorothymidine. (author)

  3. Incorporation study of {sup 18}FDG through its uptake into tumour-derived cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Costa, Flavia M.; Nascimento, Leonardo T.C.; Santos, Raquel G., E-mail: fnc@cdtn.br, E-mail: raqueou@uol.com.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN-CNEN/MG), Belo Horizonte, MG (Brazil)

    2013-07-01

    In vitro tests of radiopharmaceuticals are an important instrument to study their mechanisms of action, binding and incorporation. {sup 18}FDG is the most used radiopharmaceutical for diagnostics positron emission tomography (PET) on oncology, on the basis of accelerated rates of absorption of glucose in cell malignancies. This radiotracer has been routinely produced at CDTN; and therefore, it was selected for preliminary assays due to its availability. Nowadays, UPPR at CDTN produces routinely {sup 18}FDG for the local PET Centers but others PET radiopharmaceuticals are in development such as {sup 18}F-Fluorocholine and {sup 18}F-Fluorothymidine. According to the Brazilian Health Regulatory Agency (ANVISA) it is necessary to validate and register these new radiopharmaceuticals in order to get the approval for their commercialization. Targeting efficacy is one of the important issues to be evaluated during radiopharmaceutical validation. The aim of this study was to develop a standard protocol to determine tumor targeting efficacy of PET radiopharmaceuticals in vitro. Therefore it was developed a protocol based on the incorporation og {sup 18} FDG through the uptake in different tumor-derived cell lines. Three variables were investigated for the standardization of the test: the number of cells to be seeded in 96-well plates, the time of incubation with the radiopharmaceutical and the radiotracer concentration. The standardized protocol was considered suitable for {sup 18}FDG incorporation assay and showed reproductive results. The protocol developed in this work will pave the way for the in vitro studies of incorporation of the new PET radiopharmaceuticals to be produced at UPPR-CDTN, such as: {sup 18}F-Fluorocholine and {sup 18}F-Fluorothymidine. (author)

  4. Cytotoxic effect of ICD-85 (venom-derived peptides on MDA-MB-231 cell line

    Directory of Open Access Journals (Sweden)

    A Zare Mirakabadi

    2008-01-01

    Full Text Available Since 1987, when chemopreventive testing programs began, more than 1,000 agents and agent combinations have been selected and evaluated in preclinical studies of chemopreventive activity against various types of cancers. In the present study we aimed to provide quantitative and qualitative characterization of biological and pharmacological activities of ICD-85 on MDA-MB-231 cell line (a highly invasive breast cancer cell line in order to gain a better understanding of the cytotoxic and apoptotic effects of this compound. For this study, the MDA-MB-231 cell line was used and the effect of ICD-85 was assayed by measuring the activity of the cytosolic enzyme lactate dehydrogenase (LDH, released into the culture medium after membrane damage. Morphological alterations of cells were investigated in the control group and cells incubated with ICD-85 as cytotoxic agent. Results showed, in the test group, that cells incubated with 16 µg/mL of ICD-85 had decreased cytoplasmic branching. Some cells were had ruptured and lost the continuity of their surrounding membranes while some had shrunk. Cells incubated with higher doses (above16 µg/mL showed similar changes towards cellular normality with more severity. Results obtained from the ICD-85 stability test reveal that the effect of ICD-85 on MDA-MB-231 cell line in culture medium is stable throughout the incubation time period (24 hours. It appears that ICD-85 at higher concentrations acts at the membrane level, which allows the passage of ions down the concentration gradient, resulting in osmotic changes in organelles followed by several unidentified mechanisms leading to cell death. At lower concentrations, it appears that ICD-85 can prevent cell growth by another mechanism, which may be one of the causes for apoptosis in the cell line.

  5. A cell line resource derived from honey bee (Apis mellifera) embryonic tissues.

    Science.gov (United States)

    Goblirsch, Michael J; Spivak, Marla S; Kurtti, Timothy J

    2013-01-01

    A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz's L15 medium and incubated at 32(°)C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10-14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.

  6. A cell line resource derived from honey bee (Apis mellifera embryonic tissues.

    Directory of Open Access Journals (Sweden)

    Michael J Goblirsch

    Full Text Available A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz's L15 medium and incubated at 32(°C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711 has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32 and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1 gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10-14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.

  7. Cytotoxicity of Silver Nanoparticles in Human Embryonic Stem Cell-Derived Fibroblasts and an L-929 Cell Line

    Directory of Open Access Journals (Sweden)

    Hui Peng

    2012-01-01

    Full Text Available Consensus about the toxicity of silver nanoparticles (Ag-NPs has not been reached, even though extensive attention has been paid to this issue. This confusion may be due to physicochemical factors of Ag-NPs and the cell model used for biological safety evaluation. In the present study, human embryonic stem cell-derived fibroblasts (EBFs, which have been considered a closer representative of the in vivo response, were used as a novel cell model to assess the cytotoxicity of Ag-NPs (~20 nm and ~100 nm in comparison with L-929 fibroblast cell line. Cell proliferation, cell cycle, apoptosis, p53 expression, and cellular uptake were examined. Results showed that Ag-NPs presented higher cytotoxicity to EBF than to L-929. EBF demonstrated a stronger capacity to ingest Ag-NPs, a higher G2/M arrest, and more upgraduated p53 expression after exposed to Ag-NPs for 48 h when compared with L-929. It could be concluded that EBF exhibited a more sensitive response to Ag-NPs compared with L-929 cells, indicating that EBF may be a valid candidate for cytotoxicity screening assays of nanoparticles.

  8. Differentiation of Umbilical Cord Lining Membrane-Derived Mesenchymal Stem Cells into Endothelial-Like Cells

    Science.gov (United States)

    Chung Doan, Chinh; Long Le, Thanh; Son Hoang, Nghia; Trung Doan, Ngoc; Dong Le, Van; Si Do, Minh

    2014-01-01

    Background: Stem cell therapy for the treatment of vascular-related diseases through functional revascularization is one of the most important research areas in tissue engineering. The aim of this study was to investigate the in vitro differentiation of umbilical CL-MSC into endothelial lineage cells. Methods: In this study, isolated cells were characterized for expression of MSC-specific markers and osteogenic and adipogenic differentiation. They were induced to differentiate into endothelial-like cells and then examined for expression of the endothelial-specific markers, karyotype, and functional behavior of cells. Results: Isolated cells expressed MSC-specific markers and differentiated into adipocytes and osteoblasts. After endothelial differentiation, they expressed CD31, vWF, VE-cadherin, VEGFR1, and VEGFR2 at both mRNA and protein level, but their morphological changes were not apparent when compared with those of undifferentiated cells. There were no significant changes in karyotype of differentiated cells. Furthermore, angiogenesis assay and LDL uptake assay showed that differentiated cells were able to form the capillary-like structures and uptake LDL, respectively. Conclusion: The results indicated that umbilical CL-MSC could differentiate into functional endothelial-like cells. Also, they are suitable for basic and clinical studies to cure several vascular-related diseases. PMID:24518546

  9. Axon guidance of sympathetic neurons to cardiomyocytes by glial cell line-derived neurotrophic factor (GDNF.

    Directory of Open Access Journals (Sweden)

    Keiko Miwa

    Full Text Available Molecular signaling of cardiac autonomic innervation is an unresolved issue. Here, we show that glial cell line-derived neurotrophic factor (GDNF promotes cardiac sympathetic innervation in vitro and in vivo. In vitro, ventricular myocytes (VMs and sympathetic neurons (SNs isolated from neonatal rat ventricles and superior cervical ganglia were cultured at a close distance. Then, morphological and functional coupling between SNs and VMs was assessed in response to GDNF (10 ng/ml or nerve growth factor (50 ng/ml. As a result, fractions of neurofilament-M-positive axons and synapsin-I-positive area over the surface of VMs were markedly increased with GDNF by 9-fold and 25-fold, respectively, compared to control without neurotrophic factors. Pre- and post-synaptic stimulation of β1-adrenergic receptors (BAR with nicotine and noradrenaline, respectively, resulted in an increase of the spontaneous beating rate of VMs co-cultured with SNs in the presence of GDNF. GDNF overexpressing VMs by adenovirus vector (AdGDNF-VMs attracted more axons from SNs compared with mock-transfected VMs. In vivo, axon outgrowth toward the denervated myocardium in adult rat hearts after cryoinjury was also enhanced significantly by adenovirus-mediated GDNF overexpression. GDNF acts as a potent chemoattractant for sympathetic innervation of ventricular myocytes, and is a promising molecular target for regulation of cardiac function in diseased hearts.

  10. Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow.

    Directory of Open Access Journals (Sweden)

    Atsushi Nagai

    Full Text Available Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs and mesenchymal stem cells (MSCs. MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10, was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1, neurons (neurofilament protein, synapsin and MAP2, astrocytes (glial fibrillary acidic protein, GFAP and oligodendrocytes (myelin basic protein, MBP as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells, neurofilament protein and beta-tubulin III (neurons GFAP (astrocytes, and galactocerebroside (oligodendrocytes. Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders.

  11. Solutions for the Cell Cycle in Cell Lines Derived from Human Tumors

    Directory of Open Access Journals (Sweden)

    B. Zubik-Kowal

    2006-01-01

    Full Text Available The goal of the paper is to compute efficiently solutions for model equations that have the potential to describe the growth of human tumor cells and their responses to radiotherapy or chemotherapy. The mathematical model involves four unknown functions of two independent variables: the time variable t and dimensionless relative DNA content x. The unknown functions can be thought of as the number density of cells and are solutions of a system of four partial differential equations. We construct solutions of the system, which allow us to observe the number density of cells for different t and x values. We present results of our experiments which simulate population kinetics of human cancer cells in vitro. Our results show a correspondence between predicted and experimental data.

  12. Polyketide Derivatives from Annona muricata Linn Leaves as Potencial Anticancer Material by Combination Treatment With Doxorubicin on Hela Cell Line

    Science.gov (United States)

    Artanti, A. N.; Astirin, O. P.; Prayito, A.; Widiyaningsih, R. F.; Prihapsara, F.

    2017-02-01

    One of the compounds found effication as an anticancer agent on cervical cancer is acetogenin, a polyketide compound that is abundant in Annona muricata L. leaves. This study has been done to examine polyketide derivatives was isolated from Annona muricata L. which has potency to induce apoptosis by p53 expression on hela cell line. An approach recently develop to overcome side effect of chemoterapeutic agent is used of combined chemoterapeutic agent, i.e doxorubicin. The determination of cytotoxic combination activity from polyketide derivative and doxorubicin was evaluated using MTT assay to obtain the value of CI (combination index). The expression of p53 profile was evaluated by immunohistochemistry on hela cell line. Data analysis showed that combination of polyketide derivative from Annona muricata L. (38,5 µg/ml) and doxorubicin with all of concentration performed synergistic effect on hela cell line with CI value from 0,33 - 0,65. The analysis on immucytochemistry showed that polyketide derivative from Annona muricata L. leaves could enhance p53 pathway significantly on hela cell line.

  13. Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line.

    Science.gov (United States)

    Schemmerer, Mathias; Apelt, Silke; Trojnar, Eva; Ulrich, Rainer G; Wenzel, Jürgen J; Johne, Reimar

    2016-09-29

    Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Huh-7 Lunet BLR and MRC-5 cells only weakly supported virus replication. Inoculation of A549-derived subclone cell lines resulted in most cases in reduced HEV replication. However, the subclone A549/D3 was susceptible to lower virus concentrations and resulted in higher virus yields as compared to parental A549 cells. Transcriptome analysis indicated a downregulation of genes for carcinoembryonic antigen-related cell adhesion molecules ( CEACAM ) 5 and 6 , and an upregulation of the syndecan 2 ( SDC2 ) gene in A549/D3 cells compared to A549 cells. However, treatment of A549/D3 cells or A549 cells with CEACAM- or syndecan 2-specific antisera did not influence HEV replication. The results show that cells supporting more efficient HEV replication can be selected from the A549 cell line. The specific mechanisms responsible for the enhanced replication remain unknown.

  14. Establishment and cryopreservation of a fibroblast cell line derived from Bengal tiger (Panthera tigris tigris).

    Science.gov (United States)

    Guan, W J; Liu, C Q; Li, C Y; Liu, D; Zhang, W X; Ma, Y H

    2010-01-01

    The Bengal tiger ear marginal tissue fibroblasts cell line (BTF22), containing 157 tubes of frozen cells, was successfully established by using primary explants technique and cell cryoconservation technology. Biological analysis showed that the population doubling time (PDT) for revival cells was approximately 28 h. Measurement of LDH and MDH isoenzymes showed no cross-contamination among the cells. Karyotyping showed that the frequency of cells with chromosome number 2n = 38 was 90.6-92.2%. Tests for bacteria, fungi, viruses and mycoplasma were negative. Plasmids encoding the fluorescent proteins pEGFP-N3, pEGFP-C1, pECFP-N1, pECFP-mito, pDsRed1-N1, and pEYFP-N1 were transfected into cells to study exogenous gene expression in the cells. The plasmid transfection efficiency was between 4.4% and 31.9%. Every index of the BTF22 cell line meets all the standard quality controls of American type Culture Collection (ATCC). Not only has the germline of this important Bengal tiger species been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somacloning research. Moreover, the establishment of this technical platform would provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.

  15. Longitudinal Claudin Gene Expression Analyses in Canine Mammary Tissues and Thereof Derived Primary Cultures and Cell Lines

    Directory of Open Access Journals (Sweden)

    Susanne C. Hammer

    2016-09-01

    Full Text Available Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies.

  16. Synthesis of 2,3-diyne-1,4-naphthoquinone derivatives and evaluation of cytotoxic activity against tumor cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Mauro G.; Camara, Celso A.; Silva, Tania M.S., E-mail: ccelso@dcm.ufrpe.br [Universidade Federal Rural de Pernambuco (LSCB/UFRPE), Recife, PE (Brazil). Dept. de Ciencias Moleculares. Lab. de Sintese de Compostos Bioativos; Feitosa, Anderson C.S.; Meira, Assuero S.; Pessoa, Claudia [Universidade Federal do Ceara (LOE/UFC), Fortaleza, CE (Brazil). Dept. de Fisiologia e Farmacologia. Lab. de Oncologia Experimental

    2013-09-15

    A series of 2,3-diyne-1,4-naphthoquinone derivatives was synthesized from 2,3-dibromo- 1,4-naphthoquinone and various functionalized terminal alkynes using palladium-catalyzed Sonogashira cross-coupling reaction. The diynes were evaluated as potential cytotoxic agents against three tumor cell lines: human ovarian adenocarcinoma (OVCAR-8), human metastatic prostate cancer (PC-3M) and human bronchoalveolar lung carcinoma (NCI-H358M), presenting, in general, satisfactory results for inhibition of cell growth. (author)

  17. Synthesis of 2,3-diyne-1,4-naphthoquinone derivatives and evaluation of cytotoxic activity against tumor cell lines

    International Nuclear Information System (INIS)

    Silva, Mauro G.; Camara, Celso A.; Silva, Tania M.S.; Feitosa, Anderson C.S.; Meira, Assuero S.; Pessoa, Claudia

    2013-01-01

    A series of 2,3-diyne-1,4-naphthoquinone derivatives was synthesized from 2,3-dibromo- 1,4-naphthoquinone and various functionalized terminal alkynes using palladium-catalyzed Sonogashira cross-coupling reaction. The diynes were evaluated as potential cytotoxic agents against three tumor cell lines: human ovarian adenocarcinoma (OVCAR-8), human metastatic prostate cancer (PC-3M) and human bronchoalveolar lung carcinoma (NCI-H358M), presenting, in general, satisfactory results for inhibition of cell growth. (author)

  18. Prevention of Lysosomal Storage Diseases and Derivation of Mutant Stem Cell Lines by Preimplantation Genetic Diagnosis

    OpenAIRE

    Altarescu, Gheona; Beeri, Rachel; Eiges, Rachel; Epsztejn-Litman, Silvina; Eldar-Geva, Talia; Elstein, Deborah; Zimran, Ari; Margalioth, Ehud J.; Levy-Lahad, Ephrat; Renbaum, Paul

    2012-01-01

    Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative ma...

  19. Characterization of a novel breast cancer cell line derived from a metastatic bone lesion of a breast cancer patient.

    Science.gov (United States)

    Johnson, Julie; Bessette, Darrell C; Saunus, Jodi M; Smart, Chanel E; Song, Sarah; Johnston, Rebecca L; Cocciardi, Sibylle; Rozali, Esdy N; Johnstone, Cameron N; Vargas, Ana Christina; Kazakoff, Stephen H; BioBank, Victorian Cancer; Khanna, Kum Kum; Lakhani, Sunil R; Chenevix-Trench, Georgia; Simpson, Peter T; Nones, Katia; Waddell, Nicola; Al-Ejeh, Fares

    2018-02-21

    We aimed to generate and characterize a novel cell line from a breast cancer bone metastasis to better study the progression of the disease. The cell line, P7731, was derived from a metastatic bone lesion of a breast cancer patient and assessed for marker expression. P7731 was analyzed for DNA copy number variation, somatic mutations, and gene expression and was compared with the primary tumor. P7731 cells are negative for estrogen receptor alpha (ERα), progesterone receptor (PR), and HER2 (triple-negative); strongly express vimentin (100% of cells positive) and also express cytokeratins 8/18 and 19 but at lower frequencies. Flow cytometry indicates P7731 cells are predominantly CD44 + /CD49f + /EpCAM - , consistent with a primitive, mesenchymal-like phenotype. The cell line is tumorigenic in immunocompromised mice. Exome sequencing identified a total of 45 and 76 somatic mutations in the primary tumor and cell line, respectively, of which 32 were identified in both samples and included mutations in known driver genes PIK3CA, TP53, and ARID1A. P7731 retains the DNA copy number alterations present in the matching primary tumor. Homozygous deletions detected in the cell line and in the primary tumor were found in regions containing three known (CDKN2A, CDKN2B, and CDKN1B) and 23 putative tumor suppressor genes. Cell line-specific gene amplification coupled with mRNA expression analysis revealed genes and pathways with potential pro-metastatic functions. This novel human breast cancer-bone metastasis cell line will be a useful model to study aspects of breast cancer biology, particularly metastasis-related changes from breast to bone.

  20. Diatom-derived polyunsaturated aldehydes activate cell death in human cancer cell lines but not normal cells.

    Directory of Open Access Journals (Sweden)

    Clementina Sansone

    Full Text Available Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD, 2-trans,4-trans-octadienal (OD and 2-trans,4-trans-heptadienal (HD on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1 and Fas Associated Death Domain (FADD leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP. The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms.

  1. Determination of volatile organic compounds exhaled by cell lines derived from hematological malignancies.

    Science.gov (United States)

    Tang, Hongxia; Lu, Yan; Zhang, Lulu; Wu, Zhonghui; Hou, Xiaofang; Xia, Hailong

    2017-06-30

    Background : The gas human exhaled contains many volatile organic compounds (VOCs), which is related to the health status of body. Analysis of VOCs has been proposed as a noninvasive diagnostic tool for certain cancers. Detailed research on the VOCs in gas exhaled by cell can characterize cell type specific metabolites and may be helpful to detect the cancer markers in clinical practice.Methods : Solid-phase microextraction (SPME) gas chromatography-mass spectrometry was used to detect VOCs in the headspace of tissue culture flask in non-Hodgkin's lymphoma (NHL) cell line JEKO and acute mononuclear leukemia cell line SHI-1, to elaborate the characteristic gaseous biomarkers of hematological malignancies. While macrophage cells and lymphocytic cells were acted as control. The blank group was only the RPMI 1640 medium containing 10% fetal calf serum that without cells.Results : Comparing with control group, the concentration of dimethyl sulfide, 2,4-dimethylheptane, methylbenzene, o -xylene, dodecane, and 1,3-di-tert-butylbenzene in JEKO cells was relatively higher, while the concentration of ethanol, hexanal, and benzaldehyde was lower. In SHI-1 cells, the levels of 2,4-dimethylheptane, benzene, 4-methyldecane, chloroform, 3,7-dimethyl dodecane, and hexadecane were significantly elevated, but the levels of hexanol and cyclohexanol were distinctly reduced. This pilot study revealed that the malignant hematological cells could change the components of VOCs in the cell culture flask in a cell type-specific pattern. The traits of VOCs in our setting offered new strategy for hematological malignancies tracing, and would act as potential biomarkers in diagnosis of malignant hematological diseases. © 2017 The Author(s).

  2. Evaluation of anticancer effects and enhanced doxorubicin cytotoxicity of xanthine derivatives using canine hemangiosarcoma cell lines.

    Science.gov (United States)

    Motegi, Tomoki; Katayama, Masaaki; Uzuka, Yuji; Okamura, Yasuhiko

    2013-10-01

    Methylxanthine derivatives increase cAMP and are known to have diuretic, cardiac, and central nervous system stimulatory effects. Moreover, caffeine inhibits the development of tumors induced by various carcinogens. The aim of this work was to elucidate the anticancer effects on apoptosis of xanthine derivatives alone and with doxorubicin in canine hemangiosarcoma cells. Xanthine derivatives with or without doxorubicin were administered to cells, and the effects were investigated by measuring tumor cell proliferation, cell death (cytotoxicity) induction, and apoptosis by the expression of annexin V or caspase 3/7. Both caffeine and theophylline induced apoptosis, and the treated cells expressed annexin V and caspase 3/7. Both drugs enhanced doxorubicin-induced cytotoxicity; however, hypoxanthine showed no effect. These results indicate that theophylline is similar to caffeine; both drugs may enhance doxorubicin-induced cytotoxicity by inhibiting ATM/ATR kinases. Our data suggest that caffeine and theophylline have anticancer effects and can improve the treatment effect in canine hemangiosarcoma patients. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Differential responses to radiation and hyperthermia of cloned cell lines derived from a single human melanoma xenograft

    International Nuclear Information System (INIS)

    Rofstad, E.K.; Brustad, T.

    1984-01-01

    One uncloned and five cloned cell lines were derived from a single human melanoma xenograft. Cells from passages 7-12 were exposed to either radiation or hyperthermia (42.5 0 C, pH = 7.4) under aerobic conditions and the colony forming ability of the cells was assayed in soft agar. The five cloned lines showed individual and characteristic responses to radiation as well as to hyperthermia. The variation in the response to radiation was mainly reflected in the size of the shoulders of the survival curves rather than in the D 0 -values. The variation in the response to hyperthermia was mainly reflected in the terminal slopes of the survival curves. The survival curve of cells from the uncloned line, both when exposed to radiation and hyperthermia, was positioned in the midst of those of the cloned lines. The response of the cloned lines to radiation did not correlate with the response to hyperthermia, indicating that tumor cell subpopulations which are resistant to radiation may respond well to hyperthermia

  4. Irradiation With Carbon Ion Beams Induces Apoptosis, Autophagy, and Cellular Senescence in a Human Glioma-Derived Cell Line

    International Nuclear Information System (INIS)

    Jinno-Oue, Atsushi; Shimizu, Nobuaki; Hamada, Nobuyuki; Wada, Seiichi; Tanaka, Atsushi; Shinagawa, Masahiko; Ohtsuki, Takahiro; Mori, Takahisa; Saha, Manujendra N.; Hoque, Ariful S.; Islam, Salequl; Kogure, Kimitaka; Funayama, Tomoo; Kobayashi, Yasuhiko

    2010-01-01

    Purpose: We examined biological responses of human glioma cells to irradiation with carbon ion beams (C-ions). Methods and Materials: A human glioma-derived cell line, NP-2, was irradiated with C-ions. Apoptotic cell nuclei were stained with Hoechst 33342. Induction of autophagy was examined either by staining cells with monodansylcadaverine (MDC) or by Western blotting to detect conversion of microtuble-associated protein light chain 3 (MAP-LC3) (LC3-I) to the membrane-bound form (LC3-II). Cellular senescence markers including induction of senescence-associated β-galactosidase (SA-β-gal) were examined. The mean telomere length of irradiated cells was determined by Southern blot hybridization. Expression of tumor suppressor p53 and cyclin/cyclin-dependent kinase inhibitor p21 WAF1/CIP1 in the irradiated cells was analyzed by Western blotting. Results: When NP-2 cells were irradiated with C-ions at 6 Gy, the major population of the cells died of apoptosis and autophagy. The residual fraction of attached cells ( WAF1/CIP1 was induced in NP-2 cells after irradiation. Furthermore, we found that irradiation with C-ions induced cellular senescence in a human glioma cell line lacking functional p53. Conclusions: Irradiation with C-ions induced apoptosis, autophagy, and cellular senescence in human glioma cells.

  5. Cancer stemness and metastatic potential of the novel tumor cell line K3: an inner mutated cell of bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Qian, Hui; Ding, Xiaoqing; Zhang, Jiao; Mao, Fei; Sun, Zixuan; Jia, Haoyuan; Yin, Lei; Wang, Mei; Zhang, Xu; Zhang, Bin; Yan, Yongmin; Zhu, Wei; Xu, Wenrong

    2017-06-13

    Mesenchymal stem cells (MSCs) transplantation has been used for therapeutic applications in various diseases. Here we report MSCs can malignantly transform in vivo. The novel neoplasm was found on the tail of female rat after injection with male rat bone marrow-derived MSCs (rBM-MSCs) and the new tumor cell line, K3, was isolated from the neoplasm. The K3 cells expressed surface antigens and pluripotent genes similar to those of rBM-MSCs and presented tumor cell features. Moreover, the K3 cells contained side population cells (SP) like cancer stem cells (CSCs), which might contribute to K3 heterogeneity and tumorigenic capacity. To investigate the metastatic potential of K3 cells, we established the nude mouse models of liver and lung metastases and isolated the corresponding metastatic cell lines K3-F4 and K3-B6. Both K3-F4 and K3-B6 cell lines with higher metastatic potential acquired more mesenchymal and stemness-related features. Epithelial-mesenchymal transition is a potential mechanism of K3-F4 and K3-B6 formation.

  6. Establishment and characterisation of a new cell line derived from Culex quinquefasciatus (Diptera: Culicidae

    Directory of Open Access Journals (Sweden)

    Nidya A Segura

    2012-02-01

    Full Text Available Insect cell cultures are an important biotechnological tool for basic and applied studies. The objective of this work was to establish and characterise a new cell line from Culex quinquefasciatus embryonic tissues. Embryonated eggs were taken as a source of tissue to make explants that were seeded in L-15, Grace's, Grace's/L-15, MM/VP12, Schneider's and DMEM culture media with a pH range from 6.7-6.9 and incubated at 28ºC. The morphological, cytogenetic, biochemical and molecular characteristics of the cell cultures were examined by observing the cell shapes, obtaining the karyotypes, using a cellulose-acetate electrophoretic system and performing random amplified polymorphic DNA-polymerase chain reaction analysis, respectively. The Grace's/L-15 medium provided the optimal nutritional conditions for cell adhesion and proliferation. Approximately 40-60 days following the explant procedure, a confluent monolayer was formed. Cellular morphology in the primary cultures and the subcultures was heterogeneous, but in the monolayer the epithelioid morphology type predominated. A karyotype with a diploid number of six chromosomes (2n = 6 was observed. Isoenzymatic and molecular patterns of the mosquito cell cultures matched those obtained from the immature and adult forms of the same species. Eighteen subcultures were generated. These cell cultures potentially constitute a useful tool for use in biomedical applications.

  7. Effects of acetylcholine and electrical stimulation on glial cell line-derived neurotrophic factor production in skeletal muscle cells.

    Science.gov (United States)

    Vianney, John-Mary; Miller, Damon A; Spitsbergen, John M

    2014-11-07

    Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor required for survival of neurons in the central and peripheral nervous system. Specifically, GDNF has been characterized as a survival factor for spinal motor neurons. GDNF is synthesized and secreted by neuronal target tissues, including skeletal muscle in the peripheral nervous system; however, the mechanisms by which GDNF is synthesized and released by skeletal muscle are not fully understood. Previous results suggested that cholinergic neurons regulate secretion of GDNF by skeletal muscle. In the current study, GDNF production by skeletal muscle myotubes following treatment with acetylcholine was examined. Acetylcholine receptors on myotubes were identified with labeled alpha-bungarotoxin and were blocked using unlabeled alpha-bungarotoxin. The question of whether electrical stimulation has a similar effect to that of acetylcholine was also investigated. Cells were stimulated with voltage pulses; at 1 and 5 Hz frequencies for times ranging from 30 min to 48 h. GDNF content in myotubes and GDNF in conditioned culture medium were quantified by enzyme-linked immunosorbant assay. Results suggest that acetylcholine and short-term electrical stimulation reduce GDNF secretion, while treatment with carbachol or long-term electrical stimulation enhances GDNF production by skeletal muscle. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Oxidative Stress Triggered by Apigenin Induces Apoptosis in a Comprehensive Panel of Human Cervical Cancer-Derived Cell Lines

    Directory of Open Access Journals (Sweden)

    Raquel P. Souza

    2017-01-01

    Full Text Available Recently, the cytotoxic effects of apigenin (4′,5,7-trihydroxyflavone, particularly its marked inhibition of cancer cell viability both in vitro and in vivo, have attracted the attention of the anticancer drug discovery field. Despite this, there are few studies of apigenin in cervical cancer, and these studies have mostly been conducted using HeLa cells. To evaluate the possibility of apigenin as a new therapeutic candidate for cervical cancer, we evaluated its cytotoxic effects in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive, SiHa (HPV 16-positive, CaSki (HPV 16 and HPV 18-positive, and C33A (HPV-negative cells in comparison to a nontumorigenic spontaneously immortalized human epithelial cell line (HaCaT. Our results demonstrated that apigenin had a selective cytotoxic effect and could induce apoptosis in all cervical cancer cell lines which were positively marked with Annexin V, but not in HaCaT (control cells. Additionally, apigenin was able to induce mitochondrial redox impairment, once it increased ROS levels and H2O2, decreased the Δψm, and increased LPO. Still, apigenin was able to inhibit migration and invasion of cancer cells. Thus, apigenin appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV genotypes.

  9. Modulation of visceral hypersensitivity by glial cell line-derived neurotrophic factor family receptor α-3 in colorectal afferents

    OpenAIRE

    Tanaka, T.; Shinoda, M.; Feng, B.; Albers, K. M.; Gebhart, G. F.

    2010-01-01

    Irritable bowel syndrome is characterized by colorectal hypersensitivity and contributed to by sensitized mechanosensitive primary afferents and recruitment of mechanoinsensitive (silent) afferents. Neurotrophic factors are well known to orchestrate dynamic changes in the properties of sensory neurons. Although pain modulation by proteins in the glial cell line-derived neurotrophic factor (GDNF) family has been documented in various pathophysiological states, their role in colorectal hypersen...

  10. Canine distemper virus induces apoptosis in cervical tumor derived cell lines

    Directory of Open Access Journals (Sweden)

    Rajão Daniela S

    2011-06-01

    Full Text Available Abstract Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi, by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control.

  11. The effect of the two epipodophyllotoxin derivatives etoposide (VP-16) and teniposide (VM-26) on cell lines established from patients with small cell carcinoma of the lung

    DEFF Research Database (Denmark)

    Roed, H; Vindeløv, Lars; Christensen, I J

    1987-01-01

    To determine whether there is any difference between the two epipodophyllotoxin derivatives etoposide and teniposide in their therapeutic effect in small cell carcinoma of the lung (SCCL), they were compared against five human SCCL cell lines in vitro. When the two were compared at equimolar conc...

  12. Synthesis of Icaritin and β-anhydroicaritin Mannich Base Derivatives and Their Cytotoxic Activities on Three Human Cancer Cell Lines.

    Science.gov (United States)

    Nguyen, Van-Son; Shi, Ling; Wang, Sheng-Chun; Wang, Qiu-An

    2017-01-01

    Prenyl flavonoid icaritin (1) and β-anhydroicaritin (2) are two natural products with important biological and pharmacological effects. such as antiosteoporosis, estrogen regulation and antitumor properties. The present study investigates the synthesis and cytotoxic activities on three Human cancer cell lines (Hela, HCC1954 and SK-OV-3) of icaritin and β-anhydroicaritin Mannich base derivatives in vitro models. Preylated flavonoid icaritin (1) upon treatment with formic acid under microwave assistance gave another natural product β-anhydroicaritin (2) in good yield (89%). Based on Mannich reaction of 1 or 2 with various secondary amines and formaldehyde, two series eighteen new 6-aminomethylated flavonoids Mannich base derivatives 3-11 and 12-20 were synthesized. Their cytotoxic potential against three human cancer cell lines (Hela, HCC1954 and SK-OV-3) were evaluated by the standard MTT method with cis-Platin and Paclitaxel as positive control. Our research showed that most of these flavonoid Mannich base derivatives displayed equal or higher (lower IC50 values) cytotoxic activities than the positive control cis-Platin. Some compounds possess the IC50 value below 10µM. Compounds 6-(diisopropylamino)methyl- and 6-morpholinylmethyl substituted β-anhydroicaritin (15 and 19) showed selective cytotoxicity against HCC1954 cells (IC50 12.688 µM) and Hela cells (IC50 6.543 µM) respectively. Our finding most of icaritin and β-anhydroicaritin Mannich base derivatives possessing moderate to potent cytotoxicity against these three cancer cells (Hela, HCC1954 and SK-OV-3). Compound 15 and 19 showed selective cytotoxicity against HCC1954 cells and Hela cells respectively, they are potential and selective anticancer agent and worthy of further development.

  13. Enhanced expression of unique gangliosides with GM2-determinant in human uterine cervical carcinoma-derived cell lines.

    Science.gov (United States)

    Tanaka, Kyoko; Miyazawa, Masaki; Mikami, Mikio; Aoki, Daisuke; Kiguchi, Kazushige; Iwamori, Masao

    2016-10-01

    Monoclonal antibody YHD-06 generated by immunization with GM2 reacted with gangliosides with GM2-determinant, i.e., GM2, GalNAc-GM1b and GalNAc-GD1a, among which GalNAc-GD1a was characterized as an antigen of autoimmune peripheral neuropathies including Guillain-Barré syndrome. When glycolipids were examined by TLC-immunostaining with YHD-06 in seven human cervical carcinoma-derived cell lines, GM2 was found in all cell lines, amounting to 15.5 % to 57.5 % of total gangliosides. Whereas GalNAc-GD1a was present in three cell lines, amounting to 5.4-17.5 % of total gangliosides, and GalNAc-GM1b in four cell lines in amounts of less than 2 %. The elevated amounts of gangliosides with GM2 determinant were closely correlated with the relative intensities of gene expression of GalNAc transferase, this being characteristic of cervical carcinoma-derived cells. However, in tissues from patients with several histological types of cervical carcinomas, GM3 was ubiquitously expressed in amounts of more than 66 % of total gangliosides, GM2 was expressed in only five of 15 tissues, and both GalNAc-GM1b and GalNAc-GD1a were not even detected in trace amounts. Since GM1 was detected in all tissues in amounts of less than 0.06 μg/mg dried tissue, all cervical carcinoma tissues were revealed to exhibit GM2 synthesis, indicating that enhanced synthesis of gangliosides with GM2 determinant is a characteristic of cultivated cells in vitro. Similarly, although I(3)SO3-GalCer was not present in the squamous cell carcinoma (SCC) tissues, SCC-derived cells selectively expressed II(3)SO3-LacCer. Since enhanced synthesis of GM2 has been reported in SV-40 virus-transfected fibroblasts, papilloma virus might be involved in the expression of GM2 in cervical carcinoma-derived cells.

  14. Cell line provenance.

    Science.gov (United States)

    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.

  15. Suppression of the maturation and activation of the dendritic cell line DC2.4 by melanoma-derived factors.

    Science.gov (United States)

    Hargadon, Kristian M; Forrest, Osric A; Reddy, Pranay R

    2012-01-01

    Dendritic cells play critical roles in both innate and adaptive immunity, and their numerous functions are tightly linked to their maturation and activation status. Here, we characterize the murine dendritic cell line DC2.4 as a model for studying dendritic cell maturation and activation, and we evaluate the influence of melanoma tumor cells on these processes. Exposure of DC2.4 cells to the Toll-like receptor ligand lipopolysaccharide induces both maturation and activation of these cells, characterized by upregulation of costimulatory molecule expression and proinflammatory cytokine/chemokine production. This maturation and activation is suppressed by soluble factors derived from both the highly tumorigenic B16-F1 and the poorly tumorigenic D5.1G4 murine melanoma cell lines. Interestingly, the extent of DC2.4 immunosuppression by these melanomas correlates with their tumorigenicity, suggesting a potentially vital role for dendritic cell/tumor cell interactions in the regulation of anti-tumor immunity and tumor outgrowth. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Establishment and characterization of novel epithelial-like cell lines derived from human periodontal ligament tissue in vitro.

    Science.gov (United States)

    Tansriratanawong, Kallapat; Ishikawa, Hiroshi; Toyomura, Junko; Sato, Soh

    2017-10-01

    In this study, novel human-derived epithelial-like cells (hEPLCs) lines were established from periodontal ligament (PDL) tissues, which were composed of a variety of cell types and exhibited complex cellular activities. To elucidate the putative features distinguishing these from epithelial rest of Malassez (ERM), we characterized hEPLCs based on cell lineage markers and tight junction protein expression. The aim of this study was, therefore, to establish and characterize hEPLCs lines from PDL tissues. The hEPLCs were isolated from PDL of third molar teeth. Cellular morphology and cell organelles were observed thoroughly. The characteristics of epithelial-endothelial-mesenchymal-like cells were compared in several markers by gene expression and immunofluorescence, to ERM and human umbilical-vein endothelial cells (HUVECs). The resistance between cellular junctions was assessed by transepithelial electron resistance, and inflammatory cytokines were detected by ELISA after infecting hEPLCs with periodontopathic bacteria. The hEPLCs developed into small epithelial-like cells in pavement appearance similar to ERM. However, gene expression patterns and immunofluorescence results were different from ERM and HUVECs, especially in tight junction markers (Claudin, ZO-1, and Occludins), and endothelial markers (vWF, CD34). The transepithelial electron resistance indicated higher resistance in hEPLCs, as compared to ERM. Periodontopathic bacteria were phagocytosed with upregulation of inflammatory cytokine secretion within 24 h. In conclusion, hEPLCs that were derived using the single cell isolation method formed tight multilayers colonies, as well as strongly expressed tight junction markers in gene expression and immunofluorescence. Novel hEPLCs lines exhibited differently from ERM, which might provide some specific functions such as metabolic exchange and defense mechanism against bacterial invasion in periodontal tissue.

  17. Construction and characterization of yeast two-hybrid cDNA library derived from LFBK cell line.

    Science.gov (United States)

    Mahajan, Sonalika; Sharma, Gaurav Kumar; Matura, Rakesh; Subramaniam, Saravanan; Mohapatra, Jajati Keshari; Pattnaik, Bramhadev

    2015-05-01

    The cDNA libraries are indispensable and critical tools for performing protein-protein interaction studies. In this study, a high quality yeast two-hybrid cDNA library from the LFBK cell line was constructed and characterized. LFBK cell line was originally derived from the swine kidney cells and is highly susceptible to foot-and-mouth disease virus (FMDV) infection. The total RNA was extracted from the LFBK cells and the switching mechanism at the 5' end of RNA template (SMART) technique was employed for the cDNA synthesis. Subsequently, double stranded cDNA was amplified by long-distance PCR, purified and co-transformed with pGADT7-rec vector in yeast strain Y187. The quality parameters of the constructed library were evaluated to qualify the constructed library. Nucleotide sequencing of the randomly selected clones from the library confirmed the swine genotype of LFBK cell line. The LFBK cDNA library was mated with the 2C protein of FMDV in yeast two-hybrid (YTH) system and several putative interaction partners were identified in the preliminary screening. The LFBK library was observed to be of high quality and could potentially be applied to protein interaction studies between FMDV and the host cells using YTH system. Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  18. Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC

    Science.gov (United States)

    Besse, Benjamin; Charrier, Mélinda; Lapierre, Valérie; Dansin, Eric; Lantz, Olivier; Planchard, David; Le Chevalier, Thierry; Livartoski, Alain; Barlesi, Fabrice; Laplanche, Agnès; Ploix, Stéphanie; Vimond, Nadège; Peguillet, Isabelle; Théry, Clotilde; Lacroix, Ludovic; Zoernig, Inka; Dhodapkar, Kavita; Dhodapkar, Madhav; Viaud, Sophie; Soria, Jean-Charles; Reiners, Katrin S.; Pogge von Strandmann, Elke; Vély, Frédéric; Rusakiewicz, Sylvie; Eggermont, Alexander; Pitt, Jonathan M.; Zitvogel, Laurence; Chaput, Nathalie

    2016-01-01

    ABSTRACT Dendritic cell-derived exosomes (Dex) are small extracellular vesicles secreted by viable dendritic cells. In the two phase-I trials that we conducted using the first generation of Dex (IFN-γ-free) in end-stage cancer, we reported that Dex exerted natural killer (NK) cell effector functions in patients. A second generation of Dex (IFN-γ-Dex) was manufactured with the aim of boosting NK and T cell immune responses. We carried out a phase II clinical trial testing the clinical benefit of IFN-γ-Dex loaded with MHC class I- and class II-restricted cancer antigens as maintenance immunotherapy after induction chemotherapy in patients bearing inoperable non-small cell lung cancer (NSCLC) without tumor progression. The primary endpoint was to observe at least 50% of patients with progression-free survival (PFS) at 4 mo after chemotherapy cessation. Twenty-two patients received IFN-γ-Dex. One patient exhibited a grade three hepatotoxicity. The median time to progression was 2.2 mo and median overall survival (OS) was 15 mo. Seven patients (32%) experienced stabilization of >4 mo. The primary endpoint was not reached. An increase in NKp30-dependent NK cell functions were evidenced in a fraction of these NSCLC patients presenting with defective NKp30 expression. Importantly, MHC class II expression levels of the final IFN-γ-Dex product correlated with expression levels of the NKp30 ligand BAG6 on Dex, and with NKp30-dependent NK functions, the latter being associated with longer progression-free survival. This phase II trial confirmed the capacity of Dex to boost the NK cell arm of antitumor immunity in patients with advanced NSCLC. PMID:27141373

  19. Wharton’s Jelly-derived Mesenchymal Stem Cells can Differentiate into Hepatocyte-like Cells by HepG2 Cell Line Extract

    Directory of Open Access Journals (Sweden)

    Maryam Borhani-Haghighi

    2015-03-01

    Full Text Available Background: Wharton’s jelly is an unlimited source of stem cells that can be used in cell therapy and tissue engineering without any ethical concern. It has been revealed the cell-free extract could be effective to induce cell differentiation. The objective of this study was to induce Wharton’s jelly-derived mesenchymal stem cells (MSCs into hepatocyte-like cells by premeabilization of the cells in the presence of HepG2 cell line extract. Methods: MSCs were isolated from the umbilical cord, CD marker profile and their differentiation potential into adipogenic and osteogenic lineages were determined. The cells were then, permeabilized by streptolysin O in the presence of HepG cell extract. The treated cells were cultured for 17 days. The cell phenotype was evaluated and the hepatocyte specific markers were detected by immunofluorescence and immunocytochemistry. The Periodic Acid Schiff (PAS reaction and the cellular uptake of indocyanine green were performed to evaluate the functional behavior of the differentiated cells. Results: The phenotype of extract-treated MSCs changed into a round or polygonal cells with few short processes and they could express high level of albumin, cytokeratin 18 and 19. The MSCs also could store glycogen and uptake and release indocyanine green. Conclusion: We demonstrated for the first time that Wharton’s jelly-derived MSCs could differentiate into hepatocyte-like cells by premeabilization of them in the presence of HepG2 cell extract. This study suggests a feasible method to differentiate MSCs into functional hepatocyte-like cells.

  20. A Comparitive Assessement of Cytokine Expression in Human-Derived Cell Lines Exposed to Alpha Particles and X-Rays

    Directory of Open Access Journals (Sweden)

    Vinita Chauhan

    2012-01-01

    Full Text Available Alpha- (α- particle radiation exposure has been linked to the development of lung cancer and has been identified as a radiation type likely to be employed in radiological dispersal devices. Currently, there exists a knowledge gap concerning cytokine modulations associated with exposure to α-particles. Bio-plex technology was employed to investigate changes in proinflammatory cytokines in two human-derived cell lines. Cells were irradiated at a dose of 1.5 Gy to either α-particles or X-rays at equivalent dose rates. The two cell lines exhibited a unique pattern of cytokine expression and the response varied with radiation type. Of the 27 cytokines assessed, only vascular endothelin growth factor (VEGF was observed to be modulated in both cell lines solely after α-particle exposure, and the expression of VEGF was shown to be dose responsive. These results suggest that certain proinflammatory cytokines may be involved in the biological effects related to α- particle exposure and the responses are cell type and radiation type specific.

  1. The effects of ultraviolet light on aspects of DNA metabolism in cell lines derived from plodia interpunctella and Trichoplusia ni

    International Nuclear Information System (INIS)

    Styer, S.C.

    1991-01-01

    Insect cells are significantly more resistant to the lethal effects of 254 nm ultraviolet light (UV) than mammalian cells. The predominant photoproduct produced by UV is the (5-6) cyclobutyl pyrimidine dimer. There is controversy whether this lesion, or another, the pyrimidine (6-4) pyrimidone, is responsible for the biological effects of UV. Insect cells contain a photolyase which selectively removes the (5-6), but not the (6-4) lesion, so that the relative roles of these lesions can be studied. Insect cell lines derived from the cabbage looper and the Indian meal moth were exposed to UV and analyzed for their ability to incorporate 3 H-thymidine. After exposure, cells from the Indian meal moth exhibited a rapid and prolonged depression in the rate of thymidine incorporation, whereas cells from the cabbage looper showed only a slight drop in incorporation and a rapid recovery. The extent of depression in thymidine incorporation was not correlated to the amount of cell killing by UV in these cell lines. Blockage of fork progression was correlated to the depression in thymidine incorporation. Photoreactivation did not entirely relieve blockage, depression in thymidine incorporation or cell killing, indicating that although the (5-6) dimer appears to be the major lesion responsible for these effects, other lesions, such as the (6-4) photoproduct, may play a role. In addition, activation of alternative sites of replicon initiation appeared to correlate with the depression in thymidine incorporation and the excision capabilities in these cells. The resistance to UV in these insect cells compared to mammalian cells may be due to their ability to rapidly remove the (5-6) lesion, which is the critical lesion in these insect cells

  2. Development, characterization and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19

    Science.gov (United States)

    Totipotent embryonic stem cell lines have not been established from ungulates, however, we have developed several somatic cell lines from the in vitro culture of pig epiblast cells. One such cell line, PICM-19, was isolated via colony-cloning and was found to spontaneously differentiate into hepati...

  3. Cell Line Derived 5-FU and Irinotecan Drug-Sensitivity Profiles Evaluated in Adjuvant Colon Cancer Trial Data

    DEFF Research Database (Denmark)

    Buhl, Ida Kappel; Gerster, Sarah; Delorenzi, Mauro

    2016-01-01

    PURPOSE: This study evaluates whether gene signatures for chemosensitivity for irinotecan and 5-fluorouracil (5-FU) derived from in vitro grown cancer cell lines can predict clinical sensitivity to these drugs. METHODS: To test if an irinotecan signature and a SN-38 signature could identify...... patients who benefitted from the addition of irinotecan to 5-FU, we used gene expression profiles based on cell lines and clinical tumor material. These profiles were applied to expression data obtained from pretreatment formalin fixed paraffin embedded (FFPE) tumor tissue from 636 stage III colon cancer...... patients enrolled in the PETACC-3 prospective randomized clinical trial. A 5-FU profile developed similarly was assessed by comparing the PETACC-3 cohort with a cohort of 359 stage II colon cancer patients who underwent surgery but received no adjuvant therapy. RESULTS: There was no statistically...

  4. Renal carcinoma cell-derived exosomes induce human immortalized line of Jurkat T lymphocyte apoptosis in vitro.

    Science.gov (United States)

    Yang, Lin; Wu, Xiaohou; Wang, Dan; Luo, Chunli; Chen, Lixue

    2013-01-01

    Tumor-derived exosomes usually contain some molecules that can help immune evasion by tumors. This study is aimed at investigating the potential effect of exosomes from human kidney adenocarcinoma cells on a human immortalized line of Jurkat T lymphocytes in vitro. Exosomes were purified from human kidney adenocarcinoma ACHN cells by sequential centrifugations and ultrafiltrations, and characterized by transmission electron microscopy. The effects of exosomes on the proliferation, cytokine production and apoptosis of Jurkat T cells were determined using flow cytometry and enzyme-linked immunosorbent assay. The relative levels of pro- and anti-apoptotic molecules were determined by Western blotting. Exosomes were purified from ACHN cells and exhibited typical characteristics. Treatment with exosomes inhibited Jurkat T cell proliferation and induced Jurkat T cell apoptosis in a dose- and time-dependent manner. Treatment with exosomes reduced spontaneous interleukin-2 (IL-2), interferon-γ, IL-6 and IL-10 production by Jurkat T cells. Treatment with exosomes increased the relative levels of cleaved caspase-3, -8 and -9 as well as Bax, but reduced the levels of Bcl-2 in Jurkat T cells. The purified exosomes contained Fas ligand, and treatment with soluble Fas abrogated exosome-mediated Jurkat T cell apoptosis. Our data indicate that exosomes from kidney adenocarcinoma cells contain Fas ligand and trigger Jurkat T cell apoptosis, contributing to the immune evasion of tumors. Copyright © 2013 S. Karger AG, Basel.

  5. Foxa2-venus fusion reporter mouse line allows live-cell analysis of endoderm-derived organ formation.

    Science.gov (United States)

    Burtscher, Ingo; Barkey, Wenke; Lickert, Heiko

    2013-08-01

    The Foxa2-winged helix/forkhead box transcription factor (TF) is absolutely required for endoderm formation and organogenesis. Foxa2 plays essential roles during lung, liver, pancreas, and gastrointestinal tract development and regulates cell-type specific programs in the adult organism. To specifically address Foxa2 function during organ development and homeostasis, we generated a Foxa2-Venus fusion (FVF) reporter protein by gene targeting in embryonic stem (ES) cells. The FVF knock-in reporter is expressed under endogenous Foxa2 control and the fluorescent protein fusion does not interfere with TF function, as homozygous mice are viable and fertile. Moreover, the FVF protein localizes to the nucleus, associates with chromatin during mitosis, and reflects the endogenous Foxa2 protein distribution pattern in several tissues in heterozygous animals. Importantly, live-cell imaging on single-cell level of the FVF and Sox17-Cherry fusion double knock-in reporter ES cell line reveals the dynamics of endoderm TF accumulation during ES cell differentiation. The FVF reporter also allowed us to identify the endoderm progenitors during gastrulation and to visualize the different branching morphogenesis modes of the lung and pancreas epithelium in ex vivo embryo and organ cultures. In summary, the generation of the FVF reporter line adds an important new tool to visualize and analyse endoderm-derived organ development and homeostasis on the cellular and molecular level. Copyright © 2013 Wiley Periodicals, Inc.

  6. Synthesis of acetylcholine from choline derived from phosphatidylcholine in a human neuronal cell line

    International Nuclear Information System (INIS)

    Blusztajn, J.K.; Liscovitch, M.; Richardson, U.I.

    1987-01-01

    Cholinergic neurons are unique among cells since they alone utilize choline not only as a component of major membrane phospholipids, such as phosphatidylcholine (Ptd-Cho), but also as a precursor of their neurotransmitter acetylcholine (AcCho). It has been hypothesized that choline-phospholipids might serve as a storage pool of choline for AcCho synthesis. The selective vulnerability of cholinergic neurons in certain neurodegenerative diseases (e.g., Alzheimer disease, motor neuron disorders) might result from the abnormally accelerated liberation of choline (to be used a precursor of AcCho) from membrane phospholipids, resulting in altered membrane composition and function and compromised neuronal viability. However, the proposed metabolic link between membrane turnover and AcCho synthesis has been difficult to demonstrate because of the heterogeneity of the preparations used. Here the authors used a population of purely cholinergic cells (human neuroblastomas, LA-N-2), incubated in the presence of [methyl- 3 H]methionine to selectively label PtdCho synthesized by methylation of phosphatidylethanolamine, the only pathway of de novo choline synthesis. Three peaks of radioactive material that cochromatographed with authentic AcCho, choline, and phosphocholine were observed when the water-soluble metabolites of the [ 3 H]PtdCho were purified by high-performance liquid chromatography. The results demonstrate that AcCho can be synthesized from choline derived from the degradation of endogenous PtdCho formed de novo by methylation of phosphatidylethanolamine

  7. Establishment of an osteogenic cell line derived from adult mouse bone marrow stroma by use of a recombinant retrovirus.

    Science.gov (United States)

    Mathieu, E; Schoeters, G; vander Plaetse, F; Merregaert, J

    1992-04-01

    In order to characterize fibroblastic colony-forming units (CFU-F) from murine bone marrow in relation to osteogenesis, adherent cells of 7-day-old BALB/c mouse bone marrow cultures were infected with a recombinant retrovirus (N2/ delta fosB) containing the bacterial neomycin resistance gene. One of the G418-resistant clones, MN7, was selected for further analysis on the basis of its high expression of the bone-specific alkaline phosphatase. The cells have now been in culture for more than 1 year and maintain a stable phenotype. The osteogenic nature of the immortalized clone MN7 was demonstrated as follows: (1) Mineralization was detected by 85Sr uptake and with the Von Kossa staining method only after in vitro cultivation on a collagen type I matrix. (2) Osteoblastic phenotype markers, including the synthesis of type I collagen, osteonectin, and the bone-specific isoenzyme of alkaline phosphatase were expressed in vitro. (3) MN7 cells responded to bone effectors such as parathyroid hormone and 1,25-dihydroxyvitamin D3. (4) Intraperitoneal injection of MN7 cells into 1-day-old BALB/c mice produced typical osteosarcomas in all animals. We conclude that MN7, derived entirely in vitro from a stromal CFU-F colony, represents a stable murine osteosarcoma cell line expressing the osteoblastic phenotype and provides the first direct evidence needed to establish adult mouse marrow-derived, nonhematopoietic stromal cells as osteoprogenitors.

  8. Development of a xeno-free non-contact co-culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line.

    Science.gov (United States)

    Desai, Nina; Ludgin, Jennifer; Goldberg, Jeffrey; Falcone, Tommaso

    2013-06-01

    Mouse embryonic fibroblast feeder layers (MEF) have conventionally been used to culture and maintain the pluripotency of embryonic stem cells (ESC). This study explores the potential of using a novel human endometrial cell line to develop a non-xeno, non-contact co-culture system for ESC propagation and derivation. Such xeno-free systems may prove essential for the establishment of clinical grade human ESC lines suitable for therapeutic application. A novel line of human endometrial cells were seeded in a 6-well dish. Filter inserts containing mouse ESCs were placed on these wells and passaged 2-3 times per week. Inner cell masses derived from mouse blastocysts were also cultured on transwells in the presence of the feeder layer. In both cases, staining for SSEA-1, SOX-2, OCT-4 and alkaline phosphatase were used to monitor the retention of stem cells. ESC colonies retained their stem cell morphology and attributes for over 120 days in culture and 44 passages to date. Inner cell mass derived ESC cultures were maintained in a pluripotent state for 45 days, through 6 passages with retention of all stem cell characteristics. The stem cell colonies expressed stem cell specific markers SSEA-1, Sox 2, Oct-4 and alkaline phosphatase. Upon removal of the human feeder layer, there was a distinct change in cell morphology within the colonies and evidence of ESC differentiation. Human feeder layers offer a simple path away from the use of MEF feeder cells or MEF conditioned medium for ESC culture. Furthermore, indirect co-culture using porous membranes to separate the two cell types can prevent contamination of stem cell preparations with feeder cells during passaging.

  9. Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines.

    Science.gov (United States)

    Kaewkhaw, Rossukon; Swaroop, Manju; Homma, Kohei; Nakamura, Jutaro; Brooks, Matthew; Kaya, Koray Dogan; Chaitankar, Vijender; Michael, Sam; Tawa, Gregory; Zou, Jizhong; Rao, Mahendra; Zheng, Wei; Cogliati, Tiziana; Swaroop, Anand

    2016-04-01

    We discuss the use of pluripotent stem cell lines carrying fluorescent reporters driven by retinal promoters to derive three-dimensional (3-D) retina in culture and how this system can be exploited for elucidating human retinal biology, creating disease models in a dish, and designing targeted drug screens for retinal and macular degeneration. Furthermore, we realize that stem cell investigations are labor-intensive and require extensive resources. To expedite scientific discovery by sharing of resources and to avoid duplication of efforts, we propose the formation of a Retinal Stem Cell Consortium. In the field of vision, such collaborative approaches have been enormously successful in elucidating genetic susceptibility associated with age-related macular degeneration.

  10. Investigation of Content, Stoichiometry and Transfer of miRNA from Human Neural Stem Cell Line Derived Exosomes.

    Directory of Open Access Journals (Sweden)

    Lara Stevanato

    Full Text Available Exosomes are small (30-100 nm membrane vesicles secreted by a variety of cell types and only recently have emerged as a new avenue for cell-to-cell communication. They are natural shuttles of RNA and protein cargo, making them attractive as potential therapeutic delivery vehicles. MicroRNAs (miRNAs are short non-coding RNAs which regulate biological processes and can be found in exosomes. Here we characterized the miRNA contents of exosomes derived from human neural stem cells (hNSCs. Our investigated hNSC line is a clonal, conditionally immortalized cell line, compliant with good manufacturing practice (GMP, and in clinical trials for stroke and critical limb ischemia in the UK (clinicaltrials.gov: NCT01151124, NCT02117635, and NCT01916369. By using next generation sequencing (NGS technology we identified the presence of a variety of miRNAs in both exosomal and cellular preparations. Many of these miRNAs were enriched in exosomes indicating that cells specifically sort them for extracellular release. Although exosomes have been proven to contain miRNAs, the copy number quantification per exosome of a given miRNA remains unclear. Herein we quantified by real-time PCR a highly shuttled exosomal miRNA subtype (hsa-miR-1246 in order to assess its stoichiometry per exosome. Furthermore, we utilized an in vitro system to confirm its functional transfer by measuring the reduction in luciferase expression using a 3' untranslated region dual luciferase reporter assay. In summary, NGS analysis allowed the identification of a unique set of hNSC derived exosomal miRNAs. Stoichiometry and functional transfer analysis of one of the most abundant identified miRNA, hsa-miR-1246, were measured to support biological relevance of exosomal miRNA delivery.

  11. Significance of CD133 positive cells in four novel HPV-16 positive cervical cancer-derived cell lines and biopsies of invasive cervical cancer.

    Science.gov (United States)

    Javed, Shifa; Sharma, Bal Krishan; Sood, Swati; Sharma, Sanjeev; Bagga, Rashmi; Bhattacharyya, Shalmoli; Rayat, Charan Singh; Dhaliwal, Lakhbir; Srinivasan, Radhika

    2018-04-02

    Cervical cancer is a major cause of cancer-related mortality in women in the developing world. Cancer Stem cells (CSC) have been implicated in treatment resistance and metastases development; hence understanding their significance is important. Primary culture from tissue biopsies of invasive cervical cancer and serial passaging was performed for establishing cell lines. Variable Number Tandem Repeat (VNTR) assay was performed for comparison of cell lines with their parental tissue. Tumorsphere and Aldefluor assays enabled isolation of cancer stem cells (CSC); immunofluorescence and flow cytometry were performed for their surface phenotypic expression in cell lines and in 28 tissue samples. Quantitative real-time PCR for stemness and epithelial-mesenchymal transition (EMT) markers, MTT cytotoxicity assay, cell cycle analysis and cell kinetic studies were performed. Four low-passage novel cell lines designated RSBS-9, - 14 and - 23 from squamous cell carcinoma and RSBS-43 from adenocarcinoma of the uterine cervix were established. All were HPV16+. VNTR assay confirmed their uniqueness and derivation from respective parental tissue. CSC isolated from these cell lines showed CD133 + phenotype. In tissue samples of untreated invasive cervical cancer, CD133 + CSCs ranged from 1.3-23% of the total population which increased 2.8-fold in radiation-resistant cases. Comparison of CD133 + with CD133 - bulk population cells revealed increased tumorsphere formation and upregulation of stemness and epithelial-mesenchymal transition (EMT) markers with no significant difference in cisplatin sensitivity. Low-passage cell lines developed would serve as models for studying tumor biology. Cancer Stem Cells in cervical cancer display CD133 + phenotype and are increased in relapsed cases and hence should be targeted for achieving remission.

  12. A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties

    International Nuclear Information System (INIS)

    Wang, Meng-Yu; Nestvold, Janne; Rekdal, Øystein; Kvalheim, Gunnar; Fodstad, Øystein

    2017-01-01

    Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cell marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo. - Highlights: • Spontaneously transformed rat MSCs (rTMSCs) share characteristics with normal MSCs. • rTMSCs possess a side population, enriched with tumorigenic cells. • rTMSCs model fibrosarcoma in vivo.

  13. A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Meng-Yu [Department of Cell Therapy, Institute for Cancer Research, Oslo University Hospital, Oslo (Norway); Nestvold, Janne, E-mail: j.m.nestvold@medisin.uio.no [Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo (Norway); Rekdal, Øystein [Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Tromsø (Norway); Kvalheim, Gunnar [Department of Cell Therapy, Institute for Cancer Research, Oslo University Hospital, Oslo (Norway); Fodstad, Øystein [Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital, Oslo (Norway)

    2017-03-15

    Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cell marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo. - Highlights: • Spontaneously transformed rat MSCs (rTMSCs) share characteristics with normal MSCs. • rTMSCs possess a side population, enriched with tumorigenic cells. • rTMSCs model fibrosarcoma in vivo.

  14. Glial cell line-derived neurotrophic factor up-regulates GTP-cyclohydrolase I activity and tetrahydrobiopterin levels in primary dopaminergic neurones

    DEFF Research Database (Denmark)

    Bauer, M; Suppmann, S; Meyer, M

    2002-01-01

    Glial cell line-derived neurotrophic factor (GDNF) protects dopaminergic neurones against toxic and physical damage. In addition, GDNF promotes differentiation and structural integrity of dopaminergic neurones. Here we show that GDNF can support the function of primary dopaminergic neurones...

  15. Conditioned Media from Human Adipose Tissue-Derived Mesenchymal Stem Cells and Umbilical Cord-Derived Mesenchymal Stem Cells Efficiently Induced the Apoptosis and Differentiation in Human Glioma Cell Lines In Vitro

    Directory of Open Access Journals (Sweden)

    Chao Yang

    2014-01-01

    Full Text Available Human mesenchymal stem cells (MSCs have an intrinsic property for homing towards tumor sites and can be used as tumor-tropic vectors for tumor therapy. But very limited studies investigated the antitumor properties of MSCs themselves. In this study we investigated the antiglioma properties of two easily accessible MSCs, namely, human adipose tissue-derived mesenchymal stem cells (ASCs and umbilical cord-derived mesenchymal stem cells (UC-MSCs. We found (1 MSC conditioned media can significantly inhibit the growth of human U251 glioma cell line; (2 MSC conditioned media can significantly induce apoptosis in human U251 cell line; (3 real-time PCR experiments showed significant upregulation of apoptotic genes of both caspase-3 and caspase-9 and significant downregulation of antiapoptotic genes such as survivin and XIAP after MSC conditioned media induction in U 251 cells; (4 furthermore, MSCs conditioned media culture induced rapid and complete differentiation in U251 cells. These results indicate MSCs can efficiently induce both apoptosis and differentiation in U251 human glioma cell line. Whereas UC-MSCs are more efficient for apoptosis induction than ASCs, their capability of differentiation induction is not distinguishable from each other. Our findings suggest MSCs themselves have favorable antitumor characteristics and should be further explored in future glioma therapy.

  16. Lipid-mediated glial cell line-derived neurotrophic factor gene transfer to cultured porcine ventral mesencephalic tissue

    DEFF Research Database (Denmark)

    Bauer, Matthias; Meyer, Morten; Brevig, Thomas

    2002-01-01

    Transplantation of dopaminergic ventral mesencephalic (VM) tissue into the basal ganglia of patients with Parkinson's disease (PD) shows at best moderate symptomatic relief in some of the treated cases. Experimental animal studies and clinical trials with allogenic and xenogenic pig-derived VM......-mediated transfer of the gene for human glial cell line-derived neurotrophic factor (GDNF) to embryonic (E27/28) porcine VM tissue kept as organotypic explant cultures. Treatment of the developing VM with two mitogens, basic fibroblast growth factor and epidermal growth factor, prior to transfection significantly...... increased transfection yields. Expression of human GDNF via an episomal vector could be detected by in situ hybridization and by the measuring of GDNF protein secreted into the culture medium. When compared to mock-transfected controls, VM tissue expressing recombinant GDNF contained significantly higher...

  17. Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Felix Hart

    2017-05-01

    Full Text Available IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich and hematological (CD20 cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.

  18. A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties.

    Science.gov (United States)

    Wang, Meng-Yu; Nestvold, Janne; Rekdal, Øystein; Kvalheim, Gunnar; Fodstad, Øystein

    2017-03-15

    Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cell marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Glial cell line-derived neurotrophic factor promotes the development of adrenergic neurons in mouse neural crest cultures

    Science.gov (United States)

    Maxwell, Gerald D.; Reid, Kate; Elefanty, Andrew; Bartlett, Perry F.; Murphy, Mark

    1996-01-01

    Growth of mouse neural crest cultures in the presence of glial cell line-derived neurotrophic factor (GDNF) resulted in a dramatic dose-dependent increase in the number of tyrosine hydroxylase (TH)-positive cells that developed when 5% chicken embryo extract was present in the medium. In contrast, growth in the presence of bone morphogenetic protein (BMP)-2, BMP-4, BMP-6, transforming growth factor (TGF) β1, TGF-β2, and TGF-β3 elicited no increase in the number of TH-positive cells. The TH-positive cells that developed in the presence of GDNF had neuronal morphology and contained the middle and low molecular weight neurofilament proteins. Numerous TH-negative cells with the morphology of neurons also were observed in GDNF-treated cultures. Analysis revealed that the period from 6 to 12 days in vitro was the critical time for exposure to GDNF to generate the increase in TH-positive cell number. The growth factors neurotrophin-3 and fibroblast growth factor-2 elicited increases in the number of TH-positive cells similar to that seen in response to GDNF. In contrast, nerve growth factor was unable to substitute for GDNF. These findings extend the previously reported biological activities of GDNF by showing that it can act on mouse neural crest cultures to promote the development of neurons. PMID:8917581

  20. Effects of hypoxia on expression of a panel of stem cell and chemosensitivity markers in glioblastoma cell line-derived spheroids

    DEFF Research Database (Denmark)

    Kolenda, Jesper; Jensen, Stine Skov; Aaberg-Jessen, Charlotte

    Glioblastomas are the most frequent and malignant primary brain tumor. Tumor stem cells in these tumors have recently been suggested to possess innate resistance mechanisms against radiation and chemotherapy possibly explaining their high level of therapeutic resistance. Moreover tumor hypoxia...... of a panel of stem cell and chemosensitivity markers was therefore investigated using glioma spheroids derived from the conventional glioblastoma cell line U87. The glioma spheroids were derived at normoxic (21% O2) and hypoxic (1% O2) culturing conditions in serum-free medium with EGF and bFGF. The entire...... immunohistochemical panel included hypoxia (HIF-1α, HIF-2α), proliferation (Ki-67) and stem cell (CD133, nestin, podoplanin, Bmi-1, Sox-2) markers as well as markers related to chemosensitivity (MGMT, MDR-1, TIMP-1, Lamp-1). Since spheroids derived in hypoxia were smaller than in normoxia, a set of experiments...

  1. New cancer cachexia rat model generated by implantation of a peritoneal dissemination-derived human stomach cancer cell line.

    Science.gov (United States)

    Terawaki, Kiyoshi; Sawada, Yumi; Kashiwase, Yohei; Hashimoto, Hirofumi; Yoshimura, Mitsuhiro; Suzuki, Masami; Miyano, Kanako; Sudo, Yuka; Shiraishi, Seiji; Higami, Yoshikazu; Yanagihara, Kazuyoshi; Kase, Yoshio; Ueta, Yoichi; Uezono, Yasuhito

    2014-02-15

    Cancer cachexia (CC), a syndrome characterized by anorexia and body weight loss due to low fat-free mass levels, including reduced musculature, markedly worsens patient quality of life. Although stomach cancer patients have the highest incidence of cachexia, few experimental models for the study of stomach CC have been established. Herein, we developed stomach CC animal models using nude rats subcutaneously implanted with two novel cell lines, i.e., MKN45c185, established from the human stomach cancer cell line MKN-45, and 85As2, derived from peritoneal dissemination of orthotopically implanted MKN45c185 cells in mice. Both CC models showed marked weight loss, anorexia, reduced musculature and muscle strength, increased inflammatory markers, and low plasma albumin levels; however, CC developed earlier and was more severe in rats implanted with 85As2 than in those implanted with MKN45cl85. Moreover, human leukemia inhibitory factor (LIF), a known cachectic factor, and hypothalamic orexigenic peptide mRNA levels increased in the models, whereas hypothalamic anorexigenic peptide mRNA levels decreased. Surgical removal of the tumor not only abolished cachexia symptoms but also reduced plasma LIF levels to below detectable limits. Importantly, oral administration of rikkunshito, a traditional Japanese medicine, substantially ameliorated CC-related anorexia and body composition changes. In summary, our novel peritoneal dissemination-derived 85As2 rat model developed severe cachexia, possibly caused by LIF from cancer cells, that was ameliorated by rikkunshito. This model should provide a useful tool for further study into the mechanisms and treatment of stomach CC.

  2. Evidence for epithelial-mesenchymal transition in cancer stem-like cells derived from carcinoma cell lines of the cervix uteri.

    Science.gov (United States)

    Lin, Jiaying; Liu, Xishi; Ding, Ding

    2015-01-01

    The cancer stem cell (CSC) paradigm is one possible way to understand the genesis of cancer, and cervical cancer in particular. We quantified and enriched ALDH1(+) cells within cervical cancer cell lines and subsequently characterized their phenotypical and functional properties like invasion capacity and epithelial-mesenchymal transition (EMT). ALDH1 expression in spheroid-derived cells (SDC) and the parental monolayer-derived cell (MDC) line was compared by flow-cytometry. Invasion capability was evaluated by Matrigel assay and expression of EMT-related genes Twist 1, Twist 2, Snail 1, Snail 2, Vimentin and E-cadherin by real-time PCR. ALDH1 expression was significantly higher in SDC. ALDH1(+) cells showed increased colony-formation. SDC expressed lower levels of E-cadherin and elevated levels of Twist 1, Twist 2, Snail 1, Snail 2 and Vimentin compared to MDC. Cervical cancer cell lines harbor potential CSC, characterized by ALDH1 expression as well as properties like invasiveness, colony-forming ability, and EMT. CSC can be enriched by anchorage-independent culture techniques, which may be important for the investigation of their contribution to therapy resistance, tumor recurrence and metastasis.

  3. A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

    OpenAIRE

    Goblirsch, Michael J.; Spivak, Marla S.; Kurtti, Timothy J.

    2013-01-01

    A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to la...

  4. Cytotoxicity of nano-hydroxyapatite on human-derived oral epithelium cell line: an in vitro study

    Directory of Open Access Journals (Sweden)

    Farid Abassi

    2016-08-01

    Full Text Available Background: Hydroxyapatite nanoparticles have a more surface contact and solubility than conventional hydroxyapatite. Hydroxynanoparticles enhances the biological and mechanical properties of new regenerated tissues. The hydroxyapatite nanoparticles have received attention as a new and effective osseous graft for using as scaffolds in bone regeneration. The reports on hydroxyapatite nanoparticles biocompatibility are controversial. It has been shown that hydroxyapatite nanoparticles induces inflammatory reaction and apoptosis. The aim of the present study was to evaluate the cytotoxicity of nano-hydroxyapatite on the human epithelial cells. Methods: The study was experimental and completed in vitro. The study was carried out in department of Immonulogy, Faculty of Medicine, Shahid Beheshti University of Medical Sciences in November 2014. The human-derived oral epithelium cell line (KB obtained from Pasteur Institute, Tehran, Iran were exposed to hydroxyapatite nanoparticles at 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2.5 and 5 mg/ml concentrations in 24, 48 and 72 hours. Rod-shaped hydroxyapatite nanoparticles with 99% purity and maximum 100 nm sized particles were used. Methylthiazol tetrazolium bromide (MTT method was employed for cell vitality evaluation. Enzyme-linked immunosorbent assay (ELISA was used for assessing the viability of cells. Distilled water and fetal bovine serum (FBS were positive and negative controls. ANOVA and Duncan tests were used for statistical analysis. Results: The cytotoxicity of different concentrations of hydroxyapatite nanoparticles on human-derived oral epithelium cell line in 24 (P< 0.001, 48 (P< 0.001 and 72 hours (P< 0.001 was significantly different. The nano-hydroxyapatite particles at 0.5 to 1 mg/ml had the highest cytotoxicity effect on human-derived oral epithelium cells in 24, 48 and 72 hours. Lower concentrations than 0.05 mg/ml had the best biocompatibility properties in 24, 48 and 72 hours. Conclusion

  5. Understanding the role of keratins 8 and 18 in neoplastic potential of breast cancer derived cell lines.

    Directory of Open Access Journals (Sweden)

    Sapna V Iyer

    Full Text Available BACKGROUND: Breast cancer is a complex disease which cannot be defined merely by clinical parameters like lymph node involvement and histological grade, or by routinely used biomarkers like estrogen receptor (ER, progesterone receptor (PGR and epidermal growth factor receptor 2 (HER2 in diagnosis and prognosis. Breast cancer originates from the epithelial cells. Keratins (K are cytoplasmic intermediate filament proteins of epithelial cells and changes in the expression pattern of keratins have been seen during malignant transformation in the breast. Expression of the K8/18 pair is seen in the luminal cells of the breast epithelium, and its role in prognostication of breast cancer is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have modulated K8 expression to understand the role of the K8/18 pair in three different breast epithelium derived cell lines: non-transformed MCF10A, transformed but poorly invasive MDA MB 468 and highly invasive MDA MB 435. The up-regulation of K8 in the invasive MDA MB 435 cell line resulted in a significant decrease in proliferation, motility, in-vitro invasion, tumor volume and lung metastasis. The down-regulation of K8 in MDA MB 468 resulted in a significant increase in transformation potential, motility and invasion in-vitro, while MCF10A did not show any changes in cell transformation assays. CONCLUSIONS/SIGNIFICANCE: These results indicate the role of K8/18 in modulating invasion in breast cancer -its presence correlating with less invasive phenotype and absence correlating with highly invasive, dedifferentiated phenotype. These data may have important implications for prognostication of breast cancer.

  6. Establishment and molecular characterization of mesenchymal stem cell lines derived from human visceral & subcutaneous adipose tissues.

    Science.gov (United States)

    Potdar, Pd; Sutar, Jp

    2010-01-01

    Mesenchymal stem cells (MSCs), are multipotent stem cells that can differentiate into osteoblasts, chondrocytes, myocytes and adipocytes. We utilized adipose tissue as our primary source, since it is a rich source of MSCs as well as it can be harvested using a minimally invasive surgical procedure. Both visceral and subcutaneous adipose tissue (VSAT, SCAT respectively) samples were cultured using growth medium without using any substratum for their attachment. We observed growth of mesenchymal like cells within 15 days of culturing. In spite of the absence of any substratum, the cells adhered to the bottom of the petri dish, and spread out within 2 hours. Presently VSAT cells have reached at passage 10 whereas; SCAT cells have reached at passage 14. Morphologically MSCs obtained from visceral adipose tissue were larger in shape than subcutaneous adipose tissue. We checked these cells for presence or absence of specific stem cell molecular markers. We found that VSAT and SCAT cells confirmed their MSC phenotype by expression of specific MSC markers CD 105 and CD 13 and absence of CD34 and CD 45 markers which are specific for haematopoietic stem cells. These cells also expressed SOX2 gene confirming their ability of self-renewal as well as expressed OCT4, LIF and NANOG for their properties for pluripotency & plasticity. Overall, it was shown that adipose tissue is a good source of mesenchymal stem cells. It was also shown that MSCs, isolated from adipose tissue are multipotent stem cells that can differentiate into osteoblasts, chondrocytes, cardiomyocytes, adipocytes and liver cells which may open a new era for cell based regenerative therapies for bone, cardiac and liver disorders.

  7. Establishment and Molecular Characterization of Mesenchymal Stem Cell Lines Derived From Human Visceral & Subcutaneous Adipose Tissues

    Directory of Open Access Journals (Sweden)

    Jyoti Prakash Sutar

    2010-01-01

    Full Text Available Mesenchymal stem cells (MSCs, are multipotent stem cells that can differentiate into osteoblasts, chondrocytes, myocytes and adipocytes. We utilized adipose tissue as our primary source, since it is a rich source of MSCs as well as it can be harvested using a minimally invasive surgical procedure. Both visceral and subcutaneous adipose tissue (VSAT, SCAT respectively samples were cultured using growth medium without using any substratum for their attachment. We observed growth of mesenchymal like cells within 15 days of culturing. In spite of the absence of any substratum, the cells adhered to the bottom of the petri dish, and spread out within 2 hours. Presently VSAT cells have reached at passage 10 whereas; SCAT cells have reached at passage 14. Morphologically MSCs obtained from visceral adipose tissue were larger in shape than subcutaneous adipose tissue. We checked these cells for presence or absence of specific stem cell molecular markers. We found that VSAT and SCAT cells confirmed their MSC phenotype by expression of specific MSC markers CD 105 and CD13 and absence of CD34 and CD 45 markers which are specific for haematopoietic stem cells. These cells also expressed SOX2 gene confirming their ability of self-renewal as well as expressed OCT4, LIF and NANOG for their properties for pluripotency & plasticity. Overall, it was shown that adipose tissue is a good source of mesenchymal stem cells. It was also shown that MSCs, isolated from adipose tissue are multipotent stem cells that can differentiate into osteoblasts, chondrocytes, cardiomyocytes, adipocytes and liver cells which may open a new era for cell based regenerative therapies for bone, cardiac and liver disorders.

  8. Synthesis and Evaluation of New Pyrazoline Derivatives as Potential Anticancer Agents in HepG-2 Cell Line

    Directory of Open Access Journals (Sweden)

    Weijie Xu

    2017-03-01

    Full Text Available Cancer is a major public health concern worldwide. Adverse effects of cancer treatments still compromise patients’ quality of life. To identify new potential anticancer agents, a series of novel pyrazoline derivatives were synthesized and evaluated for cytotoxic effects on HepG-2 (human liver hepatocellular carcinoma cell line and primary hepatocytes. Compound structures were confirmed by 1H-NMR, mass spectrometry, and infrared imaging. An in vitro assay demonstrated that several compounds exerted cytotoxicity in the micromolar range. Benzo[b]thiophen-2-yl-[5-(4-hydroxy-3,5-dimethoxy-phenyl-3-(2-hydroxy-phenyl-4,5-dihydo-pyrazol-1-yl]-methanone (b17 was the most effective anticancer agent against HepG-2 cells owing to its notable inhibitory effect on HepG-2 with an IC50 value of 3.57 µM when compared with cisplatin (IC50 = 8.45 µM and low cytotoxicity against primary hepatocytes. Cell cycle analysis and apoptosis/necrosis evaluation using this compound revealed that b17 notably arrested HepG-2 cells in the G2/M phase and induced HepG-2 cells apoptosis. Our findings indicate that compound b17 may be a promising anticancer drug candidate.

  9. Precursor N-cadherin mediates glial cell line-derived neurotrophic factor-promoted human malignant glioma.

    Science.gov (United States)

    Xiong, Ye; Liu, Liyun; Zhu, Shuang; Zhang, Baole; Qin, Yuxia; Yao, Ruiqin; Zhou, Hao; Gao, Dian Shuai

    2017-04-11

    As the most prevalent primary brain tumor, gliomas are highly metastatic, invasive and are characteristic of high levels of glial cell-line derived neurotrophic factor (GDNF). GDNF is an important factor for invasive glioma cell growth; however, the underlying mechanism involved is unclear. In this study, we affirm a significantly higher expression of the precursor of N-cadherin (proN-cadherin) in most gliomas compared with normal brain tissues. Our findings reveal that GDNF interacts with the extracellular domain of proN-cadherin, which suggests that proN-cadherin mediates GDNF-induced glioma cell migration and invasion. We hypothesize that proN-cadherin might cause homotypic adhesion loss within neighboring cells and at the same time promote heterotypic adhesion within the extracellular matrix (ECM) through a certain mechanism. This study also demonstrates that the interaction between GDNF and proN-cadherin activates specific intracellular signaling pathways; furthermore, GDNF promoted the secretion of matrix metalloproteinase-9 (MMP-9), which degrades the ECM via proN-cadherin. To reach the future goal of developing novel therapies of glioma, this study, reveals a unique mechanism of glioma cell migration and invasion.

  10. Variations in Humanized and Defined Culture Conditions Supporting Derivation of New Human Embryonic Stem Cell Lines

    DEFF Research Database (Denmark)

    Fletcher, Judy M; Ferrier, Patricia M; Gardner, John O

    2006-01-01

    The evolution of "humanized" (i.e., free of animal sourced reagents) and ultimately chemically defined culture systems for human embryo stem cell (hESC) isolation and culture is of importance to improving their efficacy and safety in research and therapeutic applications. This can be achieved...... serum-free medium (SFM) containing only human sourced and recombinant protein. Further, outgrowth of embryonic cells from whole blastocysts in both media could be achieved for up to 1 week without reliance on feeder cells. All variant conditions sustained undifferentiated cell status, a stable karyotype......, with a transitional requirement for human feeder cells. This represents another sequential step in the generation of therapeutic grade stem cells with reduced risk of zoonotic pathogen transmission....

  11. Diterpenoid B derived from Plectranthus excisus inhibits the melanoma cell cycle in the B16 melanoma cell line.

    Science.gov (United States)

    Liu, Ya-Nan; Gu, Jun-Lian; Ma, Meng-Shi; Guo, Hua; Liu, Long; Guo, Li-Rong; Wang, Yue; Li, Yang

    2015-09-01

    Plectranthus excisus is widely distributed throughout northeast China. Its active ingredient, diterpenoids, exhibits significant antitumor effects. The present study examined the antitumor effects of diterpenoid B (DB), derived from Plectranthus excisus, and demonstrated that DB inhibited the proliferation of tumor cells by inhibiting the cell cycle. Reverse transcription‑quantitative polymerase chain reaction and western blot analysis were used to determine mRNA and protein expression levels, respectively. The results revealed that exposure to DB increased the expression levels of the transformation associated, protein 53, and cyclin‑dependent kinase inhibitor 1A, and decreased the expression of cyclin‑dependent kinase 2. The results of the present study demonstrated that DB can inhibit cell cycle progression and, therefore, offers potential as a beneficial antitumor drug.

  12. Generation of an isogenic, gene-corrected control cell line of the spinocerebellar ataxia type 2 patient-derived iPSC line H266

    DEFF Research Database (Denmark)

    Marthaler, Adele Gabriele; Tubsuwan, Alisa; Schmid, Benjamin

    2016-01-01

    (iPSC) lines of SCA2 patients in order to study a disease-specific phenotype. Here, we demonstrate the gene correction of the iPSC line H266 clone 10 where we have exchanged the expanded CAG repeat of the ATXN2 gene with the normal length found in healthy alleles. This gene corrected cell line...

  13. Generation of an isogenic, gene-corrected control cell line of the spinocerebellar ataxia type 2 patient-derived iPSC line H196

    DEFF Research Database (Denmark)

    Marthaler, Adele Gabriele; Schmid, Benjamin; Tubsuwan, Alisa

    2016-01-01

    (iPSC) lines of SCA2 patients in order to study a disease-specific phenotype. Here, we demonstrate the gene correction of the iPSC line H196 clone 7 where we have exchanged the expanded CAG repeat of the ATXN2 gene with the normal length found in healthy alleles. This gene corrected cell line...

  14. Generation of an isogenic, gene-corrected control cell line of the spinocerebellar ataxia type 2 patient-derived iPSC line H271

    DEFF Research Database (Denmark)

    Marthaler, Adele Gabriele; Schmid, Benjamin; Tubsuwan, Alisa

    2016-01-01

    (iPSC) lines of SCA2 patients in order to study a disease-specific phenotype. Here, we demonstrate the gene correction of the iPSC line H271 clone 1 where we have exchanged the expanded CAG repeat of the ATXN2 gene with the normal length found in healthy alleles. This gene corrected cell line...

  15. Amiodarone increases the accumulation of DEA in a human alveolar epithelium-derived cell line.

    Science.gov (United States)

    Seki, Satoru; Itagaki, Shirou; Kobayashi, Masaki; Hirano, Takeshi; Iseki, Ken

    2008-07-01

    Amiodarone (AMD)-induced pulmonary toxicity (AIPT) is the most life-threatening side-effect of AMD treatment. N-Monodesethylamiodarone (DEA), an active metabolite of AMD, also exhibits cytotoxicity and tends to accumulate in the lung more intensively than AMD. In this study, we characterized the mechanism of DEA accumulation using A549 cells as a model of the alveolar epithelium. Typical ATP-depletion compounds caused an approximately 30% increase in the accumulation of DEA in A549 cells, although these effects were less than those in Caco-2 cells. Triiodothyronine (T(3)), which exhibited an inhibitory effect on DEA efflux in Caco-2 cells, did not affect the accumulation of DEA in A549 cells. On the other hand, 100 microM AMD caused an approximately 200% increase in DEA content in A549 cells, although AMD accumulation was not affected by 100 microM DEA. Since the reducing effect of AMD on cellular ATP levels and that of FCCP were similar, the mechanism by which DEA accumulation is increased by AMD might be different from the ATP-dependent DEA efflux mechanism. The decrease in cell viability by DEA in the presence of AMD (IC(50) value of DEA for A549 cell viability: 25.4+/-2.4 microM) was more pronounced than that by DEA alone (IC(50) value: 11.5+/-3.0 microM). This further DEA accumulation by AMD might be a factor responsible for the greater accumulation of DEA than that of AMD in the lung in long-term AMD-treated patients.

  16. Characterization of a new insect cell line that is derived from the neonate larvae of Papilio xuthus (Lepidoptera: Papilionidae) and its susceptibility to AcNPV.

    Science.gov (United States)

    Zhang, Xin; Feng, Ying; Ding, Wei-Feng; Chen, Xiao-Ming; Wang, Cheng-Ye; Ma, Tao

    2012-06-01

    The cell line RIRI-PX1 was established from neonate larval tissues of Papilio xuthus by performing primary cultures in the modified Grace medium that was supplemented with 20% fetal bovine serum (FBS). The cell line primarily consisted of spindle-shaped and spherical cells which attached themselves to the flask. The population-doubling times (PDTs) at the 50th and 60th passage were 42.5 h and 42.1 h respectively. The average chromosome numbers of RIRI-PX1 cell line from passage 5 to passage 50 ranged from 103 to 199. It was confirmed that RIRI-PX1 cell line was derived from P. xuthus by comparing the mitochondrial cytochrome c oxidase subunit I gene (COI) of RIRI-PX1 cells and P. xuthus eggs. This cell line was susceptible to the Autographa californica nucleopolyhedrovirus (AcNPV) and produced high yield of polyhedral occlusion bodies (43.9OBs/cell) after 10 days of infection by AcNPV. The virus titer of AcNPV infected RIRI-PX1 cells was 3.25×10⁷ TCID₅₀/ml. We concluded that the RIRI-PX1 cell line is established from the neonate larvae tissues successfully and the cells of the cell line are sensitive to AcNPV. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Establishment, characterization, chemosensitivity, and radiosensitivity of two different cell lines derived from a human breast cancer biopsy

    International Nuclear Information System (INIS)

    Gioanni, J.; Courdi, A.; Lalanne, C.M.; Fischel, J.L.; Zanghellini, E.; Lambert, J.C.; Ettore, F.; Namer, M.

    1985-01-01

    In vitro culture of a human breast cancer biopsy fragment gave rise to two permanent cell lines, CAL 18 A and CAL 18 B, which were differentiated by both morphological and ultrastructural analysis. The karyotypic and growth properties of these two cell lines also differed, providing further evidence of cell heterogeneity within a given tumor. Both cell lines lost their hormone receptors in vitro. CAL 18 A cells grew in agar and were tumorigenic after inoculation into nude mice; neither of these properties was observed in CAL 18 B cells. The chemosensitivity of 12 antineoplastic drugs was assessed by a short-term assay, using inhibition of tritiated thymidine incorporation by the cells after contact with the drugs as the end point. Only a few drugs were active at moderate concentrations. The overall responses of both cell lines were similar. The cell survival curves, established by the colony method following a single dose of radiation, were also very similar, despite the greater heterogeneity of CAL 18 B cells. The two cell lines appear to be interrelated, since CAL 18 B cells were occasionally observed to emerge from CAL 18 A clones, suggesting that malignant cell redifferentiation may occur spontaneously in vitro

  18. Zirconium oxide ceramic foam: a promising supporting biomaterial for massive production of glial cell line-derived neurotrophic factor.

    Science.gov (United States)

    Liu, Zhong-wei; Li, Wen-qiang; Wang, Jun-kui; Ma, Xian-cang; Liang, Chen; Liu, Peng; Chu, Zheng; Dang, Yong-hui

    2014-12-01

    This study investigated the potential application of a zirconium oxide (ZrO2) ceramic foam culturing system to the production of glial cell line-derived neurotrophic factor (GDNF). Three sets of ZrO2 ceramic foams with different pore densities of 10, 20, and 30 pores per linear inch (PPI) were prepared to support a 3D culturing system. After primary astrocytes were cultured in these systems, production yields of GDNF were evaluated. The biomaterial biocompatibility, cell proliferation and activation of cellular signaling pathways in GDNF synthesis and secretion in the culturing systems were also assessed and compared with a conventional culturing system. In this study, we found that the ZrO2 ceramic foam culturing system was biocompatible, using which the GDNF yields were elevated and sustained by stimulated cell proliferation and activation of signaling pathways in astrocytes cultured in the system. In conclusion, the ZrO2 ceramic foam is promising for the development of a GDNF mass production device for Parkinson's disease treatment.

  19. Exposure to 1800 MHz radiofrequency electromagnetic radiation induces oxidative DNA base damage in a mouse spermatocyte-derived cell line.

    Science.gov (United States)

    Liu, Chuan; Duan, Weixia; Xu, Shangcheng; Chen, Chunhai; He, Mindi; Zhang, Lei; Yu, Zhengping; Zhou, Zhou

    2013-03-27

    Whether exposure to radiofrequency electromagnetic radiation (RF-EMR) emitted from mobile phones can induce DNA damage in male germ cells remains unclear. In this study, we conducted a 24h intermittent exposure (5 min on and 10 min off) of a mouse spermatocyte-derived GC-2 cell line to 1800 MHz Global System for Mobile Communication (GSM) signals in GSM-Talk mode at specific absorption rates (SAR) of 1 W/kg, 2 W/kg or 4 W/kg. Subsequently, through the use of formamidopyrimidine DNA glycosylase (FPG) in a modified comet assay, we determined that the extent of DNA migration was significantly increased at a SAR of 4 W/kg. Flow cytometry analysis demonstrated that levels of the DNA adduct 8-oxoguanine (8-oxoG) were also increased at a SAR of 4 W/kg. These increases were concomitant with similar increases in the generation of reactive oxygen species (ROS); these phenomena were mitigated by co-treatment with the antioxidant α-tocopherol. However, no detectable DNA strand breakage was observed by the alkaline comet assay. Taking together, these findings may imply the novel possibility that RF-EMR with insufficient energy for the direct induction of DNA strand breaks may produce genotoxicity through oxidative DNA base damage in male germ cells. Crown Copyright © 2013. Published by Elsevier Ireland Ltd. All rights reserved.

  20. Characteristics and anti-proliferative activity of azelaic acid and its derivatives entrapped in bilayer vesicles in cancer cell lines.

    Science.gov (United States)

    Manosroi, Aranya; Panyosak, Atchara; Rojanasakul, Yon; Manosroi, Jiradej

    2007-06-01

    The hydrophilicity and lipophilicity of azelaic acid (AA) were modified to diethyl azelate (DA) which was synthesized by Fisher esterification reaction and identified by IR, MS and (1)H NMR and to azelaic acid-beta-cyclodextrin complex (AACD) which was prepared by inclusion complexation and identified by IR, DSC and XRD respectively. AA, DA and AACD were entrapped in liposomes and niosomes comprising of L-alpha-dipalmitoyl phosphatidylcholine (DPPC)/cholesterol at 7:3 molar ratio and Tween61/cholesterol at 1:1 molar ratio, respectively, using a thin-film hydration method with sonication. The size and morphology of these bilayer vesicles were determined by optical and transmission electron microscopy. The particle size was found to be in the range of 90-190 nm. The entrapment efficiency of AA, DA and AACD in all vesicular formulations was more than 80%, as analyzed by HPLC for AA and AACD, and GC for DA. Anti-proliferative activity of AA and its derivatives (DA and AACD) both entrapped and not entrapped in bilayer vesicles, using MTT assay in three cancer cell lines (HeLa, KB and B(16)F(10)) comparing with vincristine, were investigated. AACD showed the highest potency comparing to AA in HeLa, KB and B(16)F(10) of 1.48, 1.6 and 1.5 times, respectively. AA entrapped in liposomes was about 90 times more potent than the free AA, and about 1.5 times less potent than vincristine. When entrapped in bilayer vesicles, DA and AACD were more effective than AA in killing cancer cells. AACD entrapped in liposomes gave the highest anti-proliferation activity in HeLa cell lines with the IC(50) of 2.3 and 327 times more potent than vincristine and AA, respectively. DA in liposomes demonstrated the IC(50) of 0.03 times less potent than vincristine in KB cell lines, while in B(16)F(10) AACD in niosomes showed the IC(50) of 0.05 times less potent than vincristine. This study has suggested that the modification of AA by derivatization and complexation as well as the entrapment in

  1. Photophysical Properties of Pheophorbide-a Derivatives and Their Photodynamic Therapeutic Effects on a Tumor Cell Line In Vitro

    Directory of Open Access Journals (Sweden)

    Kang-Kyun Wang

    2014-01-01

    Full Text Available Pheophorbide-a derivatives have been reported to be potential photosensitizers for photodynamic therapy (PDT. In this study, photophysics of pheophorbide-a derivatives (PaDs were investigated along with their photodynamic tumoricidal effect in vitro. PaDs were modified by changing the coil length and/or making the hydroxyl group (–OH substitutions. Their photophysical properties were studied by steady-state and time-resolved spectroscopic methods. The photodynamic tumoricidal effect was evaluated in the mouse breast cancer cell line (EMT6. Lifetime and quantum yield of fluorescence and quantum yields of triplet state and singlet oxygen were studied to determine the dynamic energy flow. The coil length of the substituted alkyl group did not significantly affect the spectroscopic properties. However, the substitution with the hydroxyl group increased the quantum yields of the triplet state and the singlet oxygen due to the enhanced intersystem crossing. In order to check the application possibility as a photodynamic therapy agent, the PaDs with hydroxyl group were studied for the cellular affinity and the photodynamic tumoricidal effect of EMT6. The results showed that the cellular affinity and the photodynamic tumoricidal effect of PaDs with the hydroxyl group depended on the coil-length of the substituted alkyl group.

  2. A Sclerostin super-producer cell line derived from the human cell line SaOS-2: a new tool for the study of the molecular mechanisms driving Sclerostin expression.

    Science.gov (United States)

    Pérez-Campo, Flor M; Sañudo, Carolina; Delgado-Calle, Jesús; Arozamena, Jana; Zarrabeitia, María T; Riancho, José A

    2014-08-01

    Sclerostin, the product of the SOST gene, is a key regulator of bone homeostasis. Sclerostin interferes with the Wnt signalling pathway and, therefore, has a negative effect on bone formation. Although the importance of sclerostin in bone homeostasis is well established, many aspects of its biology are still unknown. Due to its restricted pattern of expression, in vitro studies of SOST gene regulation are technically challenging. Furthermore, a more profound investigation of the molecular mechanism controlling sclerostin expression has been hampered by the lack of a good human in vitro model. Here, we describe two cell lines derived from the human osteosarcoma cell line SaOS-2 that produce elevated levels of sclerostin. Analysis of the super-producer cell lines showed that sclerostin levels were still reduced in response to parathyroid hormone treatment or in response to mechanical loading, indicating that these regulatory mechanisms were not affected in the presented cell lines. In addition, we did not find differences between the promoter or ECR5 sequences of our clones and the SaOS-2 parental line. However, the methylation of the proximal CpG island located at the SOST promoter was lower in the super-producer clones, in agreement with a higher level of SOST transcription. Although the underlying biological causes of the elevated levels of sclerostin production in this cell line are not yet clear, we believe that it could be an extremely useful tool to study the molecular mechanisms driving sclerostin expression in humans.

  3. Characterization of Camptothecin-induced Genomic Changes in the Camptothecin-resistant T-ALL-derived Cell Line CPT-K5

    DEFF Research Database (Denmark)

    Kjeldsen, Eigil; Nielsen, Christine J F; Roy, Amit

    2018-01-01

    -K5 and its parental cell line. We identified copy number alterations affecting genes important for maintaining genome integrity and reducing CPT-induced DNA damage. We show for the first time that short tandem repeats are targets for TOP1 cleavage, that can be differentially stimulated by CPT.......Acquisition of resistance to topoisomerase I (TOP1)-targeting camptothecin (CPT) derivatives is a major clinical problem. Little is known about the underlying chromosomal and genomic mechanisms. We characterized the CPT-K5 cell line expressing mutant CPT-resistant TOP1 and its parental T......-cell derived acute lymphoblastic leukemia CPT-sensitive RPMI-8402 cell line by karyotyping and molecular genetic methods, including subtractive oligo-based array comparative genomic hybridization (soaCGH) analysis. Karyotyping revealed that CPT-K5 cells had acquired additional structural aberrations...

  4. Sericin, a protein derived from silkworms, accelerates the proliferation of several mammalian cell lines including a hybridoma

    OpenAIRE

    Terada, Satoshi; Nishimura, Taeko; Sasaki, Masahiro; Yamada, Hideyuki; Miki, Masao

    2002-01-01

    Sericin, a constituent of the silkworm cocoon, was added to the culture of four mammalian cell lines: murine hybridoma 2E3-O,human hepatoblastoma HepG2, human epithelial HeLa and human embryonal kidney 293 cells. The proliferation of all cell lineswas accelerated in the presence of sericin. The hybridoma cellline was further studied. The 2E3-O cell line was so well adapted to serum-free medium that both the proliferation rate and maximum cell density in serum-free ASF103 medium were higher th...

  5. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.

    Directory of Open Access Journals (Sweden)

    Jin Hee Kim

    Full Text Available When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i there are no publicly available cloning vectors harboring a 2A peptide gene and (ii comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.

  6. Lymphoblast-derived integration-free ISRM-CON9 iPS cell line from a 75 year old female

    Directory of Open Access Journals (Sweden)

    Soraia Martins

    2018-01-01

    Full Text Available Human lymphoblast cells were used to generate integration-free induced pluripotent stem cells (iPSCs employing episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and L-MYC. The derived iPSCs were defined as pluripotent based on (i expression of pluripotency-associated markers, (ii embryoid body-based differentiation into cell types representative of the three germ layers and (iii the similarity between the transcriptomes of the iPSC line and the human embryonic stem cell line H1 with a Pearson correlation of 0.95.

  7. Differences in the transcriptome signatures of two genetically related Entamoeba histolytica cell lines derived from the same isolate with different pathogenic properties

    Directory of Open Access Journals (Sweden)

    Stanley Samuel L

    2010-01-01

    Full Text Available Abstract Background The availability of two genetically very similar cell lines (A and B derived from the laboratory isolate Entamoeba histolytica HM-1:IMSS, which differ in their virulence properties, provides a powerful tool for identifying pathogenicity factors of the causative agent of human amoebiasis. Cell line A is incapable inducing liver abscesses in gerbils, whereas interaction with cell line B leads to considerable abscess formation. Phenotypic characterization of both cell lines revealed that trophozoites from the pathogenic cell line B have a larger cell size, an increased growth rate in vitro, an increased cysteine peptidase activity and higher resistance to nitric oxide stress. To find proteins that may serve as virulence factors, the proteomes of both cell lines were previously studied, resulting in the identification of a limited number of differentially synthesized proteins. This study aims to identify additional genes, serving as virulence factors, or virulence markers. Results To obtain a comprehensive picture of the differences between the cell lines, we compared their transcriptomes using an oligonucleotide-based microarray and confirmed findings with quantitative real-time PCR. Out of 6242 genes represented on the array, 87 are differentially transcribed (≥two-fold in the two cell lines. Approximately 50% code for hypothetical proteins. Interestingly, only 19 genes show a five-fold or higher differential expression. These include three rab7 GTPases, which were found with a higher abundance in the non-pathogenic cell line A. The aig1-like GTPasesare of special interest because the majority of them show higher levels of transcription in the pathogenic cell line B. Only two molecules were found to be differentially expressed between the two cell lines in both this study and our previous proteomic approach. Conclusions In this study we have identified a defined set of genes that are differentially transcribed between the non

  8. Production of offspring from a germline stem cell line derived from prepubertal ovaries of germline reporter mice.

    Science.gov (United States)

    Zhang, Chen; Wu, Ji

    2016-07-01

    We investigated whether DEAD-box polypeptide 4 (DDX4) positive cells from post-natal ovaries of germline lineage reporter mice can be isolated based on endogenously expressed fluorescent proteins and used to establish a cell line for producing offspring. DDX4-positive cells from post-natal ovaries of germline lineage reporter mice can be isolated and used to establish a cell line for producing offspring. In recent years, female germline stem cells (FGSCs) have been isolated from the ovaries of post-natal mice by magnetic-activated cell sorting or fluorescence-activated cell sorting (FACS) relying on an antibody against DDX4. However, whether DDX4-positive cells from post-natal ovaries of germline lineage reporter mice can be established without using an antibody, as well as a cell line established for producing offspring, remains unknown. To obtain the expected offspring (Ddx4-Cre;mT/mG mice), Ddx4-Cre mice were crossed with mT/mG mice. In the ovaries of Ddx4-Cre;mT/mG mice, germ cells were destined to express enhanced green fluorescent protein (EGFP) while somatic cells still express tandem dimer Tomato (tdTomato). Therefore, the germ cells could be clearly distinguished from somatic cells by fluorescent proteins. Then, we investigated the pattern of fluorescent cells in the ovaries of 21-day-old Ddx4-Cre;mT/mG mice under a fluorescent microscope. Germ cells were sorted by FACS without using antibody and used to establish a FGSC line. The FGSC line was analyzed by DDX4 immunostaining, Edu (5-ethynyl-2'-deoxyuridine) labeling, and RT-PCR for germ cell markers. Finally, the physiological function of the FGSC line was examined by transplanting FGSCs into the ovaries of sterilized recipients and subsequent mating. Firstly, we have successfully isolated FGSCs from the ovaries of 21-day-old Ddx4-Cre;mT/mG mice based on endogenously expressed fluorescent proteins. FACS was used to separate the cells and 2.3% of all viable cells was EGFP-positive germ cells. Subsequently

  9. Combination effects of epidermal growth factor and glial cell line-derived neurotrophic factor on the in vitro developmental potential of porcine oocytes

    DEFF Research Database (Denmark)

    Valleh, Mehdi Vafaye; Rasmussen, Mikkel Aabech; Hyttel, Poul

    2016-01-01

    of improving this issue, the single and combined effects of epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) on oocyte developmental competence were investigated. Porcine cumulus–oocyte cell complexes (COCs) were matured in serum-free medium supplemented with EGF (0, 10...... with the combination of EGF and GDNF was shown to significantly improve oocyte competence in terms of blastocyst formation, blastocyst cell number and blastocyst hatching rate (P

  10. Characterization of two novel cell lines with distinct heterogeneity derived from a single human bile duct carcinoma.

    Directory of Open Access Journals (Sweden)

    Jinghan Wang

    Full Text Available BACKGROUND: Intratumoral heterogeneity reflects subclonal diversity and accounts for a variety of clinically defined phenotypes including the development of drug resistance and recurrence. However, intratumoral heterogeneity of bile duct carcinoma (BDC is rarely studied. METHODS: Two highly heterogeneous cell lines named EH-CA1a and EH-CA1b were established from a primary tumor tissue of a pathologically proven BDC. Distinct heterogeneity and underlying mechanisms of two cell lines in karyotype, colony formation, tumorgenicity, and sensitivity to chemoradiotherapy were intensively studied. RESULTS: Both cell lines showed typical morphology of cancer cells. EH-CA1a cells grew as free-floating aggregates, while EH-CA1b cells grew adherently as a monolayer. EH-CA1a cells had higher cloning efficiencies and were able to keep proliferating under hypoxic condition. Coincidentally, hypoxia-induced factor-1α (HIF1α and vascular endothelial growth factor (VEGF mRNA were significantly higher in EH-CA1a cells than in EH-CA1b cells. Both cell lines were tumorigenic in nude mouse, however, EH-CA1a cells showed more aggressive characteristics. Most importantly, the EH-CA1a cells showed much more resistance against radiation and chemotherapy with gemcitabine. Metastasis-related genes including matrix metalloproteinase 2 (MMP-2, MMP-9, epithelial-mesenchymal transition (EMT markers such as Vimentin, Snail, and Twist, are more highly expressed in EH-CA1a cells than in EH-CA1b cells. Moreover, the percentage of cells expressing cancer stem cell-like marker, CD133, in EH-CA1a cells is much higher than that in EH-CA1b cells. Moreover, knockdown of CD133 in both EH-CA1a and EH-CA1b cells significantly reduced their invasive potential and increased their sensitivities to radiation and gemcitabine, suggesting the differential expression of CD133 protein may partially account for the difference in malignancy between these two cancer cells. CONCLUSION: Establishment

  11. Synthesis and Pharmacophore Modelling of 2,6,9-Trisubstituted Purine Derivatives and Their Potential Role as Apoptosis-Inducing Agents in Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Jeannette Calderón-Arancibia

    2015-04-01

    Full Text Available A series of 2,6,9-trisubstituted purine derivatives have been synthesized and investigated for their potential role as antitumor agents. Twelve compounds were obtained by a three step synthetic procedure using microwave irradiation in a pivotal step. All compounds were evaluated in vitro to determine their potential effect on cell toxicity by the MTT method and flow cytometry analysis on four cancer cells lines and Vero cells. Three out of twelve compounds were found to be promising agents compared to a known and effective anticancer drug, etoposide, in three out of four cancer cell lines assayed with considerable selectivity. Preliminary flow cytometry data suggests that compounds mentioned above induce apoptosis on these cells. The main structural requirements for their activity for each cancer cell line were characterized with a preliminary pharmacophore model, which identified aromatic centers, hydrogen acceptor/donor center and a hydrophobic area. These features were consistent with the cytotoxic activity of the assayed compounds.

  12. Organotypic culture of normal, dysplastic and squamous cell carcinoma-derived oral cell lines reveals loss of spatial regulation of CD44 and p75 NTR in malignancy.

    Science.gov (United States)

    Dalley, Andrew J; AbdulMajeed, Ahmad A; Upton, Zee; Farah, Camile S

    2013-01-01

    Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted, yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence, in this study, we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2), mild dysplasia (DOK), severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44, p75(NTR), CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75(NTR) was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44, p75(NTR), CD24 antigens and ALDH activity (ALDEFLUOR(®) assay), with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer, increased FOXA1 and decreased FOXA2 expression correlated with disease severity, but OCT3/4, Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells. © 2012 John Wiley & Sons A/S.

  13. Sympathetic Innervation Induced in Engrafted Engineered Cardiomyocyte Sheets by Glial Cell Line Derived Neurotrophic Factor In Vivo

    Directory of Open Access Journals (Sweden)

    Xian-ming Fu

    2013-01-01

    Full Text Available The aim of myocardial tissue engineering is to repair or regenerate damaged myocardium with engineered cardiac tissue. However, this strategy has been hampered by lack of functional integration of grafts with native myocardium. Autonomic innervation may be crucial for grafts to function properly with host myocardium. In this study, we explored the feasibility of in vivo induction of autonomic innervation to engineered myocardial tissue using genetic modulation by adenovirus encoding glial cell line derived neurotrophic factor (GDNF. GFP-transgene (control group or GDNF overexpressing (GDNF group engineered cardiomyocyte sheets were transplanted on cryoinjured hearts in rats. Nerve fibers in the grafts were examined by immunohistochemistry at 1, 2, and 4 weeks postoperatively. Growth associated protein-43 positive growing nerves and tyrosine hydroxylase positive sympathetic nerves were first detected in the grafts at 2 weeks postoperatively in control group and 1 week in GDNF group. The densities of growing nerve and sympathetic nerve in grafts were significantly increased in GDNF group. No choline acetyltransferase immunopositive parasympathetic nerves were observed in grafts. In conclusion, sympathetic innervation could be effectively induced into engrafted engineered cardiomyocyte sheets using GDNF.

  14. Efficient Skipping of Single Exon Duplications in DMD Patient-Derived Cell Lines Using an Antisense Oligonucleotide Approach.

    Science.gov (United States)

    Wein, Nicolas; Vulin, Adeline; Findlay, Andrew R; Gumienny, Felecia; Huang, Nianyuan; Wilton, Steve D; Flanigan, Kevin M

    2017-01-01

    Exon skipping strategies in Duchenne muscular dystrophy (DMD) have largely been directed toward altering splicing of exons flanking out-of-frame deletions, with the goal of restoring an open mRNA reading frame that leads to production of an internally deleted but partially functional dystrophin protein. We sought to apply exon skipping to duplication mutations, assuming that the inherently limited efficiency of antisense oligonucleotide-induced exon skipping would more frequently skip a single copy of a duplicated exon, rather than both and result in significant amounts of wild-type DMD mRNA. We tested this hypothesis in fibroblast cell lines derived from patients with a variety of single or multiple exon duplications that have been modified to allow transdifferentiation into a myogenic lineage. Using a variety of 2'O-methyl antisense oligonucleotides, significant skipping was induced for each duplication leading to a wild-type transcript as a major mRNA product. This study provides another proof of concept for the feasibility of therapeutic skipping in patients carrying exon duplications in order to express wild-type, full-length mRNA, although careful evaluation of the skipping efficiency should be performed as some exons are easier to skip than others. Such a personalized strategy is expected to be highly beneficial for this subset of DMD patients, compared to inducing expression of an internally-deleted dystrophin.

  15. Constitutive phosphorylation of the mTORC2/Akt/4E-BP1 pathway in newly derived canine hemangiosarcoma cell lines

    Directory of Open Access Journals (Sweden)

    Murai Atsuko

    2012-07-01

    Full Text Available Abstract Background Canine hemangiosarcoma (HSA is a malignant tumor with poor long-term prognosis due to development of metastasis despite aggressive treatment. The phosphatidyl-inositol-3 kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR pathway is involved in its endothelial pathologies; however, it remains unknown how this pathway plays a role in canine HSA. Here, we characterized new canine HSA cell lines derived from nude mice-xenografted canine HSAs and investigated the deregulation of the signaling pathways in these cell lines. Results Seven canine HSA cell lines were established from 3 xenograft canine HSAs and showed characteristics of endothelial cells (ECs, that is, uptake of acetylated low-density lipoprotein and expression of canine-specific CD31 mRNA. They showed varied morphologies and mRNA expression levels for VEGF-A, bFGF, HGF, IGF-I, EGF, PDGF-B, and their receptors. Cell proliferation was stimulated by these growth factors and fetal bovine serum (FBS in 1 cell line and by FBS alone in 3 cell lines. However, cell proliferation was not stimulated by growth factors and FBS in the remaining 3 cell lines. Phosphorylated p44/42 Erk1/2 was increased by FBS stimulation in 4 cell lines. In contrast, phosphorylation of Akt at Ser473, mTOR complex 1 (mTORC1 at Ser2448, and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1 at Ser65 was high in serum-starved condition and not altered by FBS stimulation in 6 cell lines, despite increased phosphorylation of these residues in normal canine ECs. This suggested that the mTORC2/Akt/4E-BP1 pathway was constitutively activated in these 6 canine HSA cell lines. After cell inoculation into nude mice, canine HSA tumors were formed from 4 cell lines and showed Akt and 4E-BP1 phosphorylation identical to the parental cell lines. Conclusions Our findings suggest that the present cell lines may be useful tools for investigating the role of the mTORC2/Akt/4E-BP1 pathway in

  16. Derivation of the human induced pluripotent stem cell line MUi017-A from a patient with homozygous Hemoglobin Constant Spring

    Directory of Open Access Journals (Sweden)

    Wasinee Wongkummool

    2017-04-01

    Full Text Available Hemoglobin Constant Spring (HbCS, HBA2: c.427T>C is a common nondeletional α-thalassemia resulting from a nucleotide substitution at the termination codon of the HBA2 gene. Homozygosity for HbCS is characterized with mild anemia, jaundice, and splenomegaly. In this study, the human induced pluripotent stem cell line MUi017-A was successfully generated from peripheral blood CD34+ hematopoietic progenitors of a 52 year old female with homozygous HbCS. The MUi017-A cell line exhibited embryonic stem cell characteristics with consistent expression of specific pluripotency markers and the capability of differentiating into the three germ layers. The cell line may be used for the disease modeling.

  17. Delayed administration of glial cell line-derived neurotrophic factor (GDNF) protects retinal ganglion cells in a pig model of acute retinal ischemia

    DEFF Research Database (Denmark)

    Kyhn, Maria Voss; Klassen, Henry; Johansson, Ulrica Englund

    2009-01-01

    This study investigates whether intravitreal administration of glial cell line-derived neurotrophic factor (GDNF) enhances survival of NeuN positive retinal cells in a porcine model of retinal ischemia. 16 pigs were subjected to an ischemic insult where intraocular pressure was maintained at 5 mm...... electroretinography (mfERG), quantification of NeuN positive cells and evaluation of the degree of retinal perivasculitis and inflammation 6 weeks after the insult. In the post-injection eyes (days 14, 28 and 42), the ratios of the iN1 and the iP2 amplitudes were 0.10 (95% CI: 0.05-0.15) and 0.09 (95% CI: 0.......04-0.16) in eyes treated with blank microspheres, and 0.24 (95% CI: 0.18-0.32) and 0.23 (95% CI: 0.15-0.33) in eyes treated with GDNF microspheres. These differences were statistically significant (P eyes...

  18. Cell lines derived from a Medaka radiation-sensitive mutant have defects in DNA double-strand break responses

    International Nuclear Information System (INIS)

    Hidaka, Masayuki; Oda, Shoji; Mitani, Hiroshi; Kuwahara, Yoshikazu; Fukumoto, Manabu

    2010-01-01

    It was reported that the radiation-sensitive Medaka mutant 'ric1' has a defect in the repair of DNA double-strand breaks (DSBs) induced by γ-rays during early embryogenesis. To study the cellular response of a ric1 mutant to ionizing radiation (IR), we established the mutant embryonic cell lines RIC1-e9, RIC1-e42, RIC1-e43. Following exposure to γ-irradiation, the DSBs in wild-type cells were repaired within 1 h, while those in RIC1 cells were not rejoined even after 2 h. Cell death was induced in the wild-type cells with cell fragmentation, but only a small proportion of the RIC1 cells underwent cell death, and without cell fragmentation. Although both wild-type and RIC1 cells showed mitotic inhibition immediately after γ-irradiation, cell division was much slower to resume in the wild-type cells (20 h versus 12 h). In both wild-type and RIC1 cells, Ser139 phosphorylated H2AX (γH2AX) foci were formed after γ-irradiation, however, the γH2AX foci disappeared more quickly in the RIC1 cell lines. These results suggest that the instability of γH2AX foci in RIC1 cells cause an aberration of the DNA damage response. As RIC1 cultured cells showed similar defective DNA repair as ric1 embryos and RIC1 cells revealed defective cell death and cell cycle checkpoint, they are useful for investigating DNA damage responses in vitro. (author)

  19. Establishment and characterization of murine small cell lung carcinoma cell lines derived from HPV-16 E6/E7 transgenic mice.

    Science.gov (United States)

    Carraresi, Laura; Martinelli, Rosanna; Vannoni, Alessandro; Riccio, Massimo; Dembic, Maja; Tripodi, Sergio; Cintorino, Marcella; Santi, Spartaco; Bigliardi, Elisa; Carmellini, Mario; Rossini, Mara

    2006-01-08

    We have established two murine cell lines derived from Small Cell Lung Carcinomas (SCLCs) developed by HPV-E6/E7 transgenic mice. These cells named PPAP-9 and PPAP-10 were isolated from mice bearing tumors, 9 and 10 months old, respectively. The cells, 5 microm in diameter, express HPV oncoproteins and sustain tumor formation after subcutaneous injection in syngenic mice. A detailed analysis indicated the epithelial origin and the neuroendocrine differentiation of these cells. We showed by confocal immunofluorescence the expression of the epithelial marker cytokeratin 5, whose gene promoter was used to direct the expression of HPV E6/E. Cells express several neuroendocrine markers such as CGRP, MAP-2, Ash1, CgrA, Scg2. The neuroendocrine differentiation of these cells was further confirmed by electron microscopy demonstrating neuropeptides secreting granules in their cytoplasm. Furthermore, in agreement with the altered expression observed in the majority of human SCLC we showed in these cells the absence of both p53 and pRB and a dramatic reduction in the expression of Caveolin-1.

  20. E-cadherin-downregulation and RECK-upregulation are coupled in the non-malignant epithelial cell line MCF10A but not in multiple carcinoma-derived cell lines.

    Science.gov (United States)

    Yuki, Kanako; Yoshida, Yoko; Inagaki, Ryosaku; Hiai, Hiroshi; Noda, Makoto

    2014-04-02

    Expression of a mesenchymal phenotype is often associated with invasive/metastatic behaviors of carcinoma cells. Acquisition of a mesenchymal phenotype by a carcinoma cell is known as epithelial-mesenchymal transition (EMT). The membrane-anchored matrix metalloproteinase-regulator RECK is abundant in normal mesenchymal cells. In aggressive carcinomas, however, RECK expression is often downregulated. This apparent paradox prompted us to clarify the relationship between EMT and RECK. We found that TGFβ-induced E-cadherin downregulation, a hallmark of EMT, is accompanied by RECK-upregulation in a non-tumorigenic epithelial cell line (MCF10A). In contrast, the loss of E-cadherin expression is uncoupled from RECK-upregulation in carcinoma-derived cell lines (MCF7, MDA-MB-231, and A549). When RECK was artificially expressed in A549 cells, it showed little effect on EMT but elevated the level of integrin α5 and attenuated cell proliferation and migration. These findings implicate RECK in the regulation of proliferation and migration of normal epithelial cells after EMT and suggest how the uncoupling between EMT and RECK-upregulation impacts on the fates and behaviors of carcinoma cells.

  1. Characterization of cells recovered from the xenotransplanted NG97 human-derived glioma cell line subcultured in a long-term in vitro

    Directory of Open Access Journals (Sweden)

    Heinrich Juliana K

    2008-10-01

    Full Text Available Abstract Background In order to elucidate tumoral progression and drug resistance, cultured cell lines are valuable tools applied on tumor related assays provided they are well established and characterized. Our laboratory settled the NG97 cell line derived from a human astrocytoma grade III, which started to develop and express important phenotypical characteristics of an astrocytoma grade IV after injection in the flank of nude mice. Astrocytomas are extremely aggressive malignancies of the Central Nervous System (CNS and account for 46% of all primary malignant brain tumors. Progression to worse prognosis occurs in 85% of the cases possibly due to changes in cell tumor microenvironment and through biological pathways that are still unclear. Methods This work focused on characterizing the NG97 cell line specifically after being recovered from the xenotransplant, who maintained their undifferentiated characteristics along the following 60th passages in vitro. These cells were subcultivated to evaluate the possible contribution of these undifferentiated characteristics to the malignant progression phenotype. These characteristics were the expression of molecules involved in the processes of migration, dedifferentiation and chromosomal instability. Results Results showed that NG97(ht had an decrease in doubling time through sub cultivation, which was characterized by a converse modulation between the expression of glial fibrillary acidic protein (GFAP and vimentin. In addition, β1 integrins were present in intermediate levels while α5 integrins had a high expression profile as well as fibronectin and laminin. Cytogenetic analysis of NG97(ht revealed several chromosomal abnormalities, 89% of the cells showed to be hyperdiploid and the modal number was assigned to be 63. Several acrocentric chromosomes were visualized and at least 30 figures were attributed to be murine. These findings suggest a possible fusion between the original NG97 cells

  2. Characterization of cells recovered from the xenotransplanted NG97 human-derived glioma cell line subcultured in a long-term in vitro

    International Nuclear Information System (INIS)

    Machado, Camila ML; Boetcher-Luiz, Fátima; Verinaud, Liana; Ikemori, Rafael Y; Zorzeto, Tatiana Q; Nogueira, Ana CMA; Barbosa, Suse DS; Savino, Wilson; Schenka, André A; Vassallo, José; Heinrich, Juliana K

    2008-01-01

    In order to elucidate tumoral progression and drug resistance, cultured cell lines are valuable tools applied on tumor related assays provided they are well established and characterized. Our laboratory settled the NG97 cell line derived from a human astrocytoma grade III, which started to develop and express important phenotypical characteristics of an astrocytoma grade IV after injection in the flank of nude mice. Astrocytomas are extremely aggressive malignancies of the Central Nervous System (CNS) and account for 46% of all primary malignant brain tumors. Progression to worse prognosis occurs in 85% of the cases possibly due to changes in cell tumor microenvironment and through biological pathways that are still unclear. This work focused on characterizing the NG97 cell line specifically after being recovered from the xenotransplant, who maintained their undifferentiated characteristics along the following 60 th passages in vitro. These cells were subcultivated to evaluate the possible contribution of these undifferentiated characteristics to the malignant progression phenotype. These characteristics were the expression of molecules involved in the processes of migration, dedifferentiation and chromosomal instability. Results showed that NG97(ht) had an decrease in doubling time through sub cultivation, which was characterized by a converse modulation between the expression of glial fibrillary acidic protein (GFAP) and vimentin. In addition, β1 integrins were present in intermediate levels while α5 integrins had a high expression profile as well as fibronectin and laminin. Cytogenetic analysis of NG97(ht) revealed several chromosomal abnormalities, 89% of the cells showed to be hyperdiploid and the modal number was assigned to be 63. Several acrocentric chromosomes were visualized and at least 30 figures were attributed to be murine. These findings suggest a possible fusion between the original NG97 cells with stromal murine cells in the xenotransplant. In

  3. Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line

    OpenAIRE

    Schemmerer, Mathias; Apelt, Silke; Trojnar, Eva; Ulrich, Rainer G.; Wenzel, J?rgen J.; Johne, Reimar

    2016-01-01

    Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Hu...

  4. The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture

    Science.gov (United States)

    Schatten, H.; Hedrick, J.; Chakrabarti, A.

    1998-01-01

    Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

  5. Broad in vitro efficacy of plant-derived betulinic acid against cell lines derived from the most prevalent human cancer types

    NARCIS (Netherlands)

    Kessler, Jan H.; Mullauer, Franziska B.; de Roo, Guido M.; Medema, Jan Paul

    2007-01-01

    Betulinic acid (BA) is a widely available plant-derived triterpene with reported activity against cancer cells of neuroectodermal origin and leukaemias. Treatment with BA was shown to protect mice against transplanted human melanoma and led to tumor regression. In contrast, cells from healthy

  6. MSLN gene silencing has an anti-malignant effect on cell lines overexpressing mesothelin deriving from malignant pleural mesothelioma.

    Directory of Open Access Journals (Sweden)

    Ombretta Melaiu

    Full Text Available Genes involved in the carcinogenetic mechanisms underlying malignant pleural mesothelioma (MPM are still poorly characterized. So far, mesothelin (MSLN has aroused the most interest. It encodes for a membrane glycoprotein, frequently over-expressed in various malignancies such as MPM, and ovarian and pancreatic cancers. It has been proposed as a diagnostic and immunotherapeutic target with promising results. However, an alternative therapeutic approach seems to rise, whereby synthetic molecules, such as antisense oligonucleotides, could be used to inhibit MSLN activity. To date, such a gene-level inhibition has been attempted in two studies only, both on pancreatic and ovarian carcinoma cell lines, with the use of silencing RNA approaches. With regard to MPM, only one cell line (H2373 has been employed to study the effects of MSLN depletion. Indeed, the knowledge on the role of MSLN in MPM needs expanding. Accordingly, we investigated the expression of MSLN in a panel of three MPM cell lines, i.e., NCI-H28, Mero-14, and IstMes2; one non-MPM cell line was used as reference (Met5A. MSLN knock-down experiments on MSLN-overexpressing cells were also performed through silencing RNA (siRNA to verify whether previous findings could be generalized to a different set of cell cultures. In agreement with previous studies, transient MSLN-silencing caused decreased proliferation rate and reduced invasive capacity and sphere formation in MSLN-overexpressing Mero-14 cells. Moreover, MSLN-siRNA combined with cisplatin, triggered a marked increase in apoptosis and a decrease in proliferation as compared to cells treated with each agent alone, thereby suggesting a sensitizing effect of siRNA towards cisplatin. In summary, our findings confirm that MSLN should be considered a key molecular target for novel gene-based targeted therapies of cancer.

  7. In vitro and in vivo characterizations of established human follicular carcinoma cell line derived from thyroid cancer

    International Nuclear Information System (INIS)

    Tsuda, Takatoshi

    1992-01-01

    A continuous cell line, named SMC R86 F1, was established from a surgically resected primary thyroid lesion. The cell grew as an adhering monolayer with a doubling time of about 25 hours in modified Eagle's medium supplemented with fetal bovine serum. When the cells were transplanted into athymic nude mice, tumors developed at the site of inoculation. The cells not only showed epithelial origin upon light and electron microscopic examination but also possessed a biosynthetic marker human thyroglobulin (hTg). In order to examine the iodide trapping ability of the xenografts, radioiodine at doses of 3.7 MBq was injected into the peritoneum of 131 I treated nude mice bearing xenografts at about 4 weeks after the cell inoculation. Judging from the results of scintigraphic, autoradiographic and biodistribution studies, viable tissue of the xenografts in the treated mice had the ability to trap radioiodine. Histological sections of the xenografts resected from the treated mice consisted of follicle-like and trabecular growing structures, and immunohistochemically the cytoplasm of the tissues was hTg positive. The cells possessed the ability to trap radioactive iodine in vitro under the control of TSH. In addition, the expression of iodinated 19S Tg in the cell cytoplasms in the monolayer cultures was revealed by immunoblotting and autoradiographic assays. These observations provide strong evidence that the SMC R86 F1 cell line possesses well-differentiated properties of the malignant thyroid follicular epithelial cells. (author)

  8. Skin fibroblast cell lines derived from non-Hodgkin's-lymphoma (NHL) patients show increased sensitivity to chronic gamma irradiation.

    Science.gov (United States)

    Hannan, M A; Greer, W; Smith, B P; Sigut, D; Ali, M A; Amer, M H

    1991-01-21

    Cultured fibroblast cells from 19 patients with non-Hodgkin's lymphoma (NHL), 3 patients with ataxia telangiectasia (AT), 3 AT heterozygotes and 11 (presumed) normal subjects were studied for impaired colony-forming ability after chronic gamma irradiation. Five cell lines from the NHL patients were also examined for the sensitivity to acute gamma irradiation, as compared with those of normal subjects. To ascertain the degree of radiosensitivity of different cell lines, a comparison was made of the D10 values (radiation dose resulting in 10% survival) for each cell line, estimated "by eye" from the actual survival curves, and also from the calculated curves fitted to a log-linear model. It was observed that the acute gamma irradiation failed to show any appreciable difference in the radiation response of the cell lines from NHL patients as compared with those of normal subjects. However, chronic irradiation demonstrated significantly increased radiosensitivity in at least 10-12 NHL patients with a p value of less than 0.05, when the D10 values of each patient's cell line were compared with the calculated composite values for the normals. When the D10 values of the NHL patients and the normal subjects were compared as 2 groups, the former appeared to be significantly more sensitive to chronic gamma irradiation (p less than 0.0001). The same level of significant difference in radiosensitivity was found between the 2 groups when their D37 values (radiation dose resulting in 37% survival) were compared. In general, the radiation response of the NHL patients was similar to that of the AT homozygotes and heterozygotes used as a positive control group. Our data thus show that increased radiosensitivity is associated with the NHL patients studied, indicating an underlying abnormality of their DNA repair.

  9. Derivation of mouse embryonic stem cell lines from tyrosine hydroxylase reporter mice crossed with a human SNCA transgenic mouse model of Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Margarita Chumarina

    2017-03-01

    Full Text Available Mouse embryonic stem cell (mESC lines were derived by crossing heterozygous transgenic (tg mice expressing green fluorescent protein (GFP under the control of the rat tyrosine hydroxylase (TH promoter, with homozygous alpha-synuclein (aSYN mice expressing human mutant SNCAA53T under the control of the mouse Prion promoter (MoPrP, or wildtype (WT mice. The expression of GFP and human aSYN was validated by immunocytochemistry in midbrain neuron cultures upon differentiation of mESC lines using stromal cell-derived inducing activity. These mESC lines can help to study the impact of human aSYN expression in neurons and oligodendrocytes, and also trace GFP-expressing midbrain neurons.

  10. Derivation of mouse embryonic stem cell lines from tyrosine hydroxylase reporter mice crossed with a human SNCA transgenic mouse model of Parkinson's disease.

    Science.gov (United States)

    Chumarina, Margarita; Azevedo, Carla; Bigarreau, Julie; Vignon, Clémentine; Kim, Kwang-Soo; Li, Jia-Yi; Roybon, Laurent

    2017-03-01

    Mouse embryonic stem cell (mESC) lines were derived by crossing heterozygous transgenic (tg) mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase (TH) promoter, with homozygous alpha-synuclein (aSYN) mice expressing human mutant SNCA A53T under the control of the mouse Prion promoter (MoPrP), or wildtype (WT) mice. The expression of GFP and human aSYN was validated by immunocytochemistry in midbrain neuron cultures upon differentiation of mESC lines using stromal cell-derived inducing activity. These mESC lines can help to study the impact of human aSYN expression in neurons and oligodendrocytes, and also trace GFP-expressing midbrain neurons. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Distribution of glial cell line-derived neurotrophic factor receptor alpha-1 in the brain of adult zebrafish.

    Science.gov (United States)

    Lucini, Carla; Carla, Lucini; Facello, Bruna; Bruna, Facello; Maruccio, Lucianna; Lucianna, Maruccio; Langellotto, Fernanda; Fernanda, Langellotto; Sordino, Paolo; Paolo, Sordino; Castaldo, Luciana; Luciana, Castaldo

    2010-08-01

    Glial cell line-derived neurotrophic factor (GDNF) is a potent trophic factor for several types of neurons in the central and peripheral nervous systems. The biological activity of GDNF is mediated by a multicomponent receptor complex that includes a common transmembrane signaling component (the rearranged during transfection (RET) proto-oncogene product, a tyrosine kinase receptor) as well as a GDNF family receptor alpha (GFRalpha) subunit, a high-affinity glycosyl phosphatidylinositol (GPI)-linked binding element. Among the four known GFRalpha subunits, GFRalpha1 preferentially binds to GDNF. In zebrafish (Danio rerio) embryos, the expression of the GFRalpha1a and GFRalpha1b genes has been shown in primary motor neurons, the kidney, and the enteric nervous system. To examine the activity of GFRalpha in the adult brain of a lower vertebrate, we have investigated the localization of GFRalpha1a and GFRalpha1b mRNA and the GFRalpha1 protein in zebrafish. GFRalpha1a and GFRalpha1b transcripts were observed in brain extracts by reverse transcription-polymerase chain reaction. Whole-mount in-situ hybridization experiments revealed a wide distribution of GFRalpha1a and GFRalpha1b mRNAs in various regions of the adult zebrafish brain. These included the olfactory bulbs, dorsal and ventral telencephalic area (telencephalon), preoptic area, dorsal and ventral thalamus, posterior tuberculum and hypothalamus (diencephalon), optic tectum (mesencephalon), cerebellum, and medulla oblongata (rhombencephalon). Finally, expression patterns of the GFRalpha1 protein, detected immunohistochemically, correlated well with the mRNA expression and provided further insights into translational activity at the neuroanatomical level. In conclusion, the current study demonstrated that the presence of GFRalpha1 persists beyond the embryonic development of the zebrafish brain and, together with the GDNF ligand, is probably implicated in the brain physiology of an adult teleost fish.

  12. Characterization of four clones derived from human adenocarcinoma cell line, HT29, and analysis of their response to sodium butyrate

    Czech Academy of Sciences Publication Activity Database

    Štokrová, Jitka; Šloncová, Eva; Sovová, Vlasta; Kučerová, Dana; Žíla, Vojtěch; Turečková, Jolana; Vojtěchová, Martina; Korb, Jan; Tuháčková, Zdena

    2006-01-01

    Roč. 28, č. 2 (2006), s. 559-565 ISSN 1019-6439 R&D Projects: GA AV ČR(CZ) KJB5052302 Institutional research plan: CEZ:AV0Z50520514 Keywords : adenocarcinoma * HT29 cell line * ultrastructure analysis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.556, year: 2006

  13. Derivation of HVR1, HVR2 and HVR3 human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis (PGD) for monogenic disorder

    OpenAIRE

    Abdelkrim Hmadcha; Yolanda Aguilera; Maria Dolores Lozano-Arana; Nuria Mellado; Javier Sánchez; Cristina Moya; Luis Sánchez-Palazón; Jose Palacios; Guillermo Antiñolo; Bernat Soria

    2016-01-01

    From 106 human blastocyts donate for research after in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for monogenetic disorder, 3 human embryonic stem cells (hESCs) HVR1, HVR2 and HVR3 were successfully derived. HVR1 was assumed to be genetically normal, HVR2 carrying Becker muscular dystrophy and HVR3 Hemophilia B. Despite the translocation t(9;15)(q34.3;q14) detected in HVR2, all the 3 cell lines were characterised in vitro and in vivo as normal hESCs lines and were r...

  14. Synthesis of novel 1,8-acridinediones derivatives: Investigation of MDR reversibility on breast cancer cell lines T47D and tamoxifen-resistant T47D.

    Science.gov (United States)

    Moallem, S A; Dehghani, N; Mehri, S; Shahsavand, Sh; Alibolandi, M; Hadizadeh, F

    2015-01-01

    Multi drug resistance (MDR) is a serious obstacle in the management of breast cancer. Therefore, overcoming MDR using novel anticancer agents is a top priority for medicinal chemists. It was found that dihydropyridines lacking calcium antagonistic activity (e.g acridinediones) possess MDR modifier potency. In this study, the capability of four novel acridine-1,8-diones derivatives 3a-d were evaluated as MDR reversing agents. In addition, the relationship between structural properties and biological effects of synthesized compounds was discussed. In vitro cytotoxicity of acridine-1,8-diones 3a-d derivatives in combination with doxorubicin (DOX) on T47D and tomoxifen-resistant T47D (TAMR-6) breast cancer cell lines were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. Drug resistant index (DRI), which is equal to the ratio of IC50 in drug-resistant cells over IC50 in drug-sensitive cells, was calculated for each substance. Flowcytometry experiments were also implemented to distinguish cells undergoing apoptosis from those undergoing necrosis. The results from MTT and flowcytometry experiments indicated that 1 nM 3c derivative along with DOX significantly (PT47D and TAMR-6 breast cancer cell lines. Synthesized compounds 3a and 3b also at concentrations of 1 nM with DOX significantly increased the cytotoxicity of DOX on T47D and TAMR-6 breast cancer cell lines. Meanwhile, 3d derivative with DOX did not exhibit good synergistic effect on cytotoxic activity of DOX, and slightly increased DOX cytotoxicity in both cell lines. Our results proposed that 3c may be an attractive lead compound for further development as a chemotherapeutic agent for MDR breast cancer therapy in combination with routine chemotherapeutic agents such as DOX.

  15. Apoptosis Activation in Human Lung Cancer Cell Lines by a Novel Synthetic Peptide Derived from Conus californicus Venom.

    Science.gov (United States)

    Oroz-Parra, Irasema; Navarro, Mario; Cervantes-Luevano, Karla E; Álvarez-Delgado, Carolina; Salvesen, Guy; Sanchez-Campos, Liliana N; Licea-Navarro, Alexei F

    2016-02-05

    Lung cancer is one of the most common types of cancer in men and women and a leading cause of death worldwide resulting in more than one million deaths per year. The venom of marine snails Conus contains up to 200 pharmacologically active compounds that target several receptors in the cell membrane. Due to their diversity and specific binding properties, Conus toxins hold great potential as source of new drugs against cancer. We analyzed the cytotoxic effect of a 17-amino acid synthetic peptide (s-cal14.1a) that is based on a native toxin (cal14.1a) isolated from the sea snail Conus californicus. Cytotoxicity studies in four lung cancer cell lines were complemented with measurement of gene expression of apoptosis-related proteins Bcl-2, BAX and the pro-survival proteins NFκB-1 and COX-2, as well as quantification of caspase activity. Our results showed that H1299 and H1437 cell lines treated with s-call4.1a had decreased cell viability, activated caspases, and reduced expression of the pro-survival protein NFκB-1. To our knowledge, this is the first report describing activation of apoptosis in human lung cancer cell lines by s-cal14.1a and we offer insight into the possible mechanism of action.

  16. Characterisation Of Forebrain Neurons Derived From Late-Onset Huntington’s Disease Human Embryonic Stem Cell Lines

    Directory of Open Access Journals (Sweden)

    Jonathan Christos Niclis

    2013-04-01

    Full Text Available Huntington's Disease (HD is an incurable neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the Huntingtin gene. Recently, induced pluripotent stem cell lines carrying atypical and aggressive (CAG60+ HD variants have been generated, and perplexingly exhibit disparate molecular pathologies. Here we investigate two human embryonic stem cell (hESC lines carrying CAG37 and CAG51 repeats to assess whether typical late-onset expansions exhibit HD pathologies. HD hESC properties were assessed in comparison to wildtype control lines at undifferentiated states and throughout forebrain neuronal differentiation. Pluripotent HD lines demonstrate growth, viability, pluripotent gene expression, mitochondrial activity and forebrain specification that is indistinguishable from control lines. Expression profiles of crucial genes known to be dysregulated in HD remain unperturbed in the presence of mutant protein and throughout differentiation; however, elevated glutamate responses were observed in HD CAG51 neurons. These findings suggest typical late-onset HD mutations do not alter pluripotent parameters or differentiation mechanics but that neuronal progeny may possess the capacity to recapitulate neuropathologies seen in human patients. Such HD models will help further our understanding of the cascade of pathological events leading to disease onset and progression, while simultaneously facilitating the identification of candidate HD therapeutics.

  17. Low or undetectable TPO receptor expression in malignant tissue and cell lines derived from breast, lung, and ovarian tumors

    Directory of Open Access Journals (Sweden)

    Erickson-Miller Connie L

    2012-09-01

    Full Text Available Abstract Background Numerous efficacious chemotherapy regimens may cause thrombocytopenia. Thrombopoietin receptor (TPO-R agonists, such as eltrombopag, represent a novel approach for the treatment of chemotherapy-induced thrombocytopenia. The TPO-R MPL is expressed on megakaryocytes and megakaryocyte precursors, although little is known about its expression on other tissues. Methods Breast, lung, and ovarian tumor samples were analyzed for MPL expression by microarray and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR, and for TPO-R protein expression by immunohistochemistry (IHC. Cell line proliferation assays were used to analyze the in vitro effect of eltrombopag on breast, lung, and ovarian tumor cell proliferation. The lung carcinoma cell lines were also analyzed for TPO-R protein expression by Western blot. Results MPL mRNA was not detectable in 118 breast tumors and was detectable at only very low levels in 48% of 29 lung tumors studied by microarray analysis. By qRT-PCR, low but detectable levels of MPL mRNA were detectable in some normal (14-43% and malignant (3-17% breast, lung, and ovarian tissues. A comparison of MPL to EPOR, ERBB2, and IGF1R mRNA demonstrates that MPL mRNA levels were far lower than those of EPOR and ERBB2 mRNA in the same tissues. IHC analysis showed negligible TPO-R protein expression in tumor tissues, confirming mRNA analysis. Culture of breast, lung, and ovarian carcinoma cell lines showed no increase, and in fact, showed a decrease in proliferation following incubation with eltrombopag. Western blot analyses revealed no detectable TPO-R protein expression in the lung carcinoma cell lines. Conclusions Multiple analyses of breast, lung, and ovarian tumor samples and/or cell lines show no evidence of MPL mRNA or TPO-R protein expression. Eltrombopag does not stimulate growth of breast, lung, or ovarian tumor cell lines at doses likely to exert their actions on megakaryocytes and

  18. Low or undetectable TPO receptor expression in malignant tissue and cell lines derived from breast, lung, and ovarian tumors.

    Science.gov (United States)

    Erickson-Miller, Connie L; Pillarisetti, Kodandaram; Kirchner, Jennifer; Figueroa, David J; Ottesen, Lone; Martin, Anne-Marie; Liu, Yuan; Kamel, Yasser Mostafa; Messam, Conrad

    2012-09-11

    Numerous efficacious chemotherapy regimens may cause thrombocytopenia. Thrombopoietin receptor (TPO-R) agonists, such as eltrombopag, represent a novel approach for the treatment of chemotherapy-induced thrombocytopenia. The TPO-R MPL is expressed on megakaryocytes and megakaryocyte precursors, although little is known about its expression on other tissues. Breast, lung, and ovarian tumor samples were analyzed for MPL expression by microarray and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and for TPO-R protein expression by immunohistochemistry (IHC). Cell line proliferation assays were used to analyze the in vitro effect of eltrombopag on breast, lung, and ovarian tumor cell proliferation. The lung carcinoma cell lines were also analyzed for TPO-R protein expression by Western blot. MPL mRNA was not detectable in 118 breast tumors and was detectable at only very low levels in 48% of 29 lung tumors studied by microarray analysis. By qRT-PCR, low but detectable levels of MPL mRNA were detectable in some normal (14-43%) and malignant (3-17%) breast, lung, and ovarian tissues. A comparison of MPL to EPOR, ERBB2, and IGF1R mRNA demonstrates that MPL mRNA levels were far lower than those of EPOR and ERBB2 mRNA in the same tissues. IHC analysis showed negligible TPO-R protein expression in tumor tissues, confirming mRNA analysis. Culture of breast, lung, and ovarian carcinoma cell lines showed no increase, and in fact, showed a decrease in proliferation following incubation with eltrombopag. Western blot analyses revealed no detectable TPO-R protein expression in the lung carcinoma cell lines. Multiple analyses of breast, lung, and ovarian tumor samples and/or cell lines show no evidence of MPL mRNA or TPO-R protein expression. Eltrombopag does not stimulate growth of breast, lung, or ovarian tumor cell lines at doses likely to exert their actions on megakaryocytes and megakaryocyte precursors.

  19. Generation, isolation, and maintenance of human mast cells and mast cell lines derived from peripheral blood or cord blood

    DEFF Research Database (Denmark)

    Rådinger, Madeleine; Jensen, Bettina M; Kuehn, Hye Sun

    2010-01-01

    Antigen-mediated mast cell activation is a pivotal step in the initiation of allergic disorders including anaphylaxis and atopy. To date, studies aimed at investigating the mechanisms regulating these responses, and studies designed to identify potential ways to prevent them, have primarily been...

  20. A novel cell line derived from pleomorphic adenoma expresses MMP2, MMP9, TIMP1, TIMP2, and shows numeric chromosomal anomalies.

    Directory of Open Access Journals (Sweden)

    Aline Semblano Carreira Falcão

    Full Text Available Pleomorphic adenoma is the most common salivary gland neoplasm, and it can be locally invasive, despite its slow growth. This study aimed to establish a novel cell line (AP-1 derived from a human pleomorphic adenoma sample to better understand local invasiveness of this tumor. AP-1 cell line was characterized by cell growth analysis, expression of epithelial and myoepithelial markers by immunofluorescence, electron microscopy, 3D cell culture assays, cytogenetic features and transcriptomic study. Expression of matrix metalloproteinases (MMPs and their tissue inhibitors (TIMPs was also analyzed by immunofluorescence and zymography. Furthermore, epithelial and myoepithelial markers, MMPs and TIMPs were studied in the tumor that originated the cell line. AP-1 cells showed neoplastic epithelial and myoepithelial markers, such as cytokeratins, vimentin, S100 protein and smooth-muscle actin. These molecules were also found in vivo, in the tumor that originated the cell line. MMPs and TIMPs were observed in vivo and in AP-1 cells. Growth curve showed that AP-1 exhibited a doubling time of 3.342 days. AP-1 cells grown inside Matrigel recapitulated tumor architecture. Different numerical and structural chromosomal anomalies were visualized in cytogenetic analysis. Transcriptomic analysis addressed expression of 7 target genes (VIM, TIMP2, MMP2, MMP9, TIMP1, ACTA2 e PLAG1. Results were compared to transcriptomic profile of non-neoplastic salivary gland cells (HSG. Only MMP9 was not expressed in both libraries, and VIM was expressed solely in AP-1 library. The major difference regarding gene expression level between AP-1 and HSG samples occurred for MMP2. This gene was 184 times more expressed in AP-1 cells. Our findings suggest that AP-1 cell line could be a useful model for further studies on pleomorphic adenoma biology.

  1. Synthesis of an anthraquinone derivative (DHAQC) and its effect on induction of G2/M arrest and apoptosis in breast cancer MCF-7 cell line.

    Science.gov (United States)

    Yeap, SweeKeong; Akhtar, Muhammad Nadeem; Lim, Kian Lam; Abu, Nadiah; Ho, Wan Yong; Zareen, Seema; Roohani, Kiarash; Ky, Huynh; Tan, Sheau Wei; Lajis, Nordin; Alitheen, Noorjahan Banu

    2015-01-01

    Anthraquinones are an important class of naturally occurring biologically active compounds. In this study, anthraquinone derivative 1,3-dihydroxy-9,10-anthraquinone-2- carboxylic acid (DHAQC) (2) was synthesized with 32% yield through the Friedel-Crafts condensation reaction. The mechanisms of cytotoxicity of DHAQC (2) in human breast cancer MCF-7 cells were further investigated. Results from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that DHAQC (2) exhibited potential cytotoxicity and selectivity in the MCF-7 cell line, comparable with the naturally occurring anthraquinone damnacanthal. DHAQC (2) showed a slightly higher IC50 (inhibitory concentration with 50% cell viability) value in the MCF-7 cell line compared to damnacanthal, but it is more selective in terms of the ratio of IC50 on MCF-7 cells and normal MCF-10A cells. (selective index for DHAQC (2) was 2.3 and 1.7 for damnacanthal). The flow cytometry cell cycle analysis on the MCF-7 cell line treated with the IC50 dose of DHAQC (2) for 48 hours showed that DHAQC (2) arrested MCF-7 cell line at the G2/M phase in association with an inhibited expression of PLK1 genes. Western blot analysis also indicated that the DHAQC (2) increased BAX, p53, and cytochrome c levels in MCF-7 cells, which subsequently activated apoptosis as observed in annexin V/propidium iodide and cell cycle analyses. These results indicate that DHAQC (2) is a synthetic, cytotoxic, and selective anthraquinone, which is less toxic than the natural product damnacanthal, and which demonstrates potential in the induction of apoptosis in the breast cancer MCF-7 cell line.

  2. The production of glial cell line-derived neurotrophic factor by human sertoli cells is substantially reduced in sertoli cell-only testes.

    Science.gov (United States)

    Singh, D; Paduch, D A; Schlegel, P N; Orwig, K E; Mielnik, A; Bolyakov, A; Wright, W W

    2017-05-01

    Do human Sertoli cells in testes that exhibit the Sertoli cell-only (SCO) phenotype produce substantially less glial cell line-derived neurotrophic factor (GDNF) than Sertoli cells in normal testes? In human SCO testes, both the amounts of GDNF mRNA per testis and the concentration of GDNF protein per Sertoli cell are markedly reduced as compared to normal testes. In vivo, GDNF is required to sustain the numbers and function of mouse spermatogonial stem cells (SSCs) and their immediate progeny, transit-amplifying progenitor spermatogonia. GDNF is expressed in the human testis, and the ligand-binding domain of the GDNF receptor, GFRA1, has been detected on human SSCs. The numbers and/or function of these stem cells are markedly reduced in some infertile men, resulting in the SCO histological phenotype. We determined the numbers of human spermatogonia per mm2 of seminiferous tubule surface that express GFRA1 and/or UCHL1, another marker of human SSCs. We measured GFRA1 mRNA expression in order to document the reduced numbers and/or function of SSCs in SCO testes. We quantified GDNF mRNA in testes of humans and mice, a species with GDNF-dependent SSCs. We also compared GDNF mRNA expression in human testes with normal spermatogenesis to that in testes exhibiting the SCO phenotype. As controls, we also measured transcripts encoding two other Sertoli cell products, kit ligand (KITL) and clusterin (CLU). Finally, we compared the amounts of GDNF per Sertoli cell in normal and SCO testes. Normal human testes were obtained from beating heart organ donors. Biopsies of testes from men who were infertile due to maturation arrest or the SCO phenotype were obtained as part of standard care during micro-testicular surgical sperm extraction. Cells expressing GFRA1, UCHL1 or both on whole mounts of normal human seminiferous tubules were identified by immunohistochemistry and confocal microscopy and their numbers were determined by image analysis. Human GDNF mRNA and GFRA1 mRNA were

  3. Apoptotic induction by pinobanksin and some of its ester derivatives from Sonoran propolis in a B-cell lymphoma cell line.

    Science.gov (United States)

    Alday, Efrain; Valencia, Dora; Carreño, Ana Laura; Picerno, Patrizia; Piccinelli, Anna Lisa; Rastrelli, Luca; Robles-Zepeda, Ramon; Hernandez, Javier; Velazquez, Carlos

    2015-12-05

    Propolis is a resinous substance produced by honeybees (Apis mellifera) from the selective collection of exudates and bud secretions from several plants. In previous works, we reported the antiproliferative activity of Sonoran propolis (SP) on cancer cells; in addition we suggested the induction of apoptosis after treatment with SP due to the presence of morphological changes and a characteristic DNA fragmentation pattern. Herein, in this study we demonstrated that the antiproliferative effect of SP is induced through apoptosis in a B-cell lymphoma cancer cell line, M12.C3.F6, by an annexin V-FITC/Propidium iodide double labeling. This apoptotic effect of SP resulted to be mediated by modulations in the loss of mitochondrial membrane potential (ΔΨm) and through activation of caspases signaling pathway (3, 8 and 9). Afterward, in order to characterize the chemical constituents of SP that induce apoptosis in cancer cells, an HPLC-PDA-ESI-MS/MS method followed by a preparative isolation procedure and NMR spectroscopy analysis have been used. Eighteen flavonoids, commonly described in propolis from temperate regions, were characterized. Chrysin, pinocembrin, pinobanksin and its ester derivatives are the main constituents of SP and some of them have never been reported in SP. In addition, two esters of pinobanksin (8 and 13) are described by first time in propolis samples in general. The antiproliferative activity on M12.C3.F6 cells through apoptosis induction was exhibited by pinobanksin (4), pinobanksin-3-O-propanoate (14), pinobanksin-3-O-butyrate (16), pinobanksin-3-O-pentanoate (17), and the already reported galangin (11), chrysin (9) and CAPE. To our knowledge this is the first report of bioactivity of pinobanksin and some of its ester derivatives as apoptosis inducers. Further studies are needed to advance in the understanding of the molecular basis of apoptosis induction by SP and its constituents, as well as the structure-activity relationship of them

  4. Differences in cell cycle regulation after platinum derivatives treatment in sensitive and cisplatin resistant ovarian cancer cell lines

    Czech Academy of Sciences Publication Activity Database

    Horváth, Viktor; Souček, Karel; Šindlerová, Lenka; Hofmanová, Jiřina; Sova, P.; Kozubík, Alois

    2006-01-01

    Roč. 100, č. 5 (2006), s. 383-384 ISSN 0009-2770. [Mezioborové setkání mladých biologů, biochemiků a chemiků /6./. 14.06.2006-17.06.2006, Milovy] R&D Projects: GA AV ČR(CZ) 1QS500040507; GA MPO(CZ) PZ-Z2/29 Institutional research plan: CEZ:AV0Z50040507 Keywords : ovarian cancer * cell cycle * cisplatin Subject RIV: BO - Biophysics

  5. The Actin Cytoskeleton Is Involved in Glial Cell Line-Derived Neurotrophic Factor (GDNF)-Induced Ret Translocation into Lipid Rafts in Dopaminergic Neuronal Cells.

    Science.gov (United States)

    Li, Li; Song, Haijing; Mu, Peipei; Xu, Ming; Liu, Chaoxia; Wang, Ying; Qin, Yingsong; Sun, Shen; Gao, Jin; Wang, Ting; Gao, Dianshuai

    2017-09-07

    Glial cell line-derived neurotrophic factor (GDNF), a potential therapeutic factor for Parkinson's disease (PD), exerts its biological effects through the Ret receptor tyrosine kinase. The redistribution of Ret into lipid rafts substantially influences Ret signaling, but the mechanisms underlying Ret translocation remain unclear. The purpose of our study was to further explore the signaling mechanisms of GDNF and to determine whether the actin cytoskeleton is involved in the GDNF-induced Ret translocation into lipid rafts. In MN9D dopaminergic neuronal cells, we used density gradient centrifugation and immunofluorescence confocal microscopy to separate and visualize lipid rafts, co-immunoprecipitation to analyze protein-protein interactions, and latrunculin B (Lat B) and jasplakinolide (Jas) to disrupt and enhance the polymerization of the actin cytoskeleton, respectively. The results showed that Ret translocated into lipid rafts and coimmunoprecipitated with actin in response to GDNF treatment. After Lat B or Jas treatment, the Ret-F-actin association induced by GDNF was impaired or enhanced respectively and then the levels of Ret translocated into lipid rafts were correspondingly inhibited or promoted. These data indicate that actin polymerization and cytoskeletal remodeling are integral to GDNF-induced cell signaling in dopaminergic cells and define a new role of the actin cytoskeleton in promoting Ret redistribution into lipid rafts.

  6. The Actin Cytoskeleton Is Involved in Glial Cell Line-Derived Neurotrophic Factor (GDNF-Induced Ret Translocation into Lipid Rafts in Dopaminergic Neuronal Cells

    Directory of Open Access Journals (Sweden)

    Li Li

    2017-09-01

    Full Text Available Glial cell line-derived neurotrophic factor (GDNF, a potential therapeutic factor for Parkinson’s disease (PD, exerts its biological effects through the Ret receptor tyrosine kinase. The redistribution of Ret into lipid rafts substantially influences Ret signaling, but the mechanisms underlying Ret translocation remain unclear. The purpose of our study was to further explore the signaling mechanisms of GDNF and to determine whether the actin cytoskeleton is involved in the GDNF-induced Ret translocation into lipid rafts. In MN9D dopaminergic neuronal cells, we used density gradient centrifugation and immunofluorescence confocal microscopy to separate and visualize lipid rafts, co-immunoprecipitation to analyze protein-protein interactions, and latrunculin B (Lat B and jasplakinolide (Jas to disrupt and enhance the polymerization of the actin cytoskeleton, respectively. The results showed that Ret translocated into lipid rafts and coimmunoprecipitated with actin in response to GDNF treatment. After Lat B or Jas treatment, the Ret–F-actin association induced by GDNF was impaired or enhanced respectively and then the levels of Ret translocated into lipid rafts were correspondingly inhibited or promoted. These data indicate that actin polymerization and cytoskeletal remodeling are integral to GDNF-induced cell signaling in dopaminergic cells and define a new role of the actin cytoskeleton in promoting Ret redistribution into lipid rafts.

  7. Growth inhibitory activity for cancer cell lines of lapachol and its natural and semi-synthetic derivatives.

    Science.gov (United States)

    Fiorito, Serena; Epifano, Francesco; Bruyère, Céline; Mathieu, Véronique; Kiss, Robert; Genovese, Salvatore

    2014-01-15

    A series of 17 selected natural and semisynthetic 1,4-naphthoquinones were synthesized, and their growth inhibitory activity was evaluated in vitro. The compounds were tested on six human cancer cell lines using the MTT colorimetric assay. The data revealed that of the chemicals under study only lapachol, its acetate and 3-geranyllawsone displayed the highest activity, recording mean IC50 values ranging from 15 to 22 μM. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Establishment of a normal-derived estrogen receptor-positive cell line comparable to the prevailing human breast cancer subtype

    DEFF Research Database (Denmark)

    Hopkinson, Branden Michael; Klitgaard, Marie Christine; Petersen, Ole William

    2017-01-01

    Understanding human cancer increasingly relies on insight gained from subtype specific comparisons between malignant and non-malignant cells. The most frequent subtype in breast cancer is the luminal. By far the most frequently used model for luminal breast cancer is the iconic estrogen receptor......-positive (ERpos) MCF7 cell line. However, luminal specific comparisons have suffered from the lack of a relevant non-malignant counterpart. Our previous work has shown that transforming growth factor-β receptor (TGFβR) inhibition suffices to propagate prospectively isolated ERpos human breast luminal cells from...... reduction mammoplasties (HBEC). Here we demonstrate that transduction of these cells with hTERT/shp16 renders them immortal while remaining true to the luminal lineage including expression of functional ER (iHBECERpos). Under identical culture conditions a major difference between MCF7 and normal...

  9. Synthesis and Anticancer Activities of 4-[(Halophenyldiazenyl]phenol and 4-[(Halophenyldiazenyl]phenyl Aspirinate Derivatives against Nasopharyngeal Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Boon Kui Ho

    2017-01-01

    Full Text Available Aspirin and azo derivatives have been widely studied and have drawn considerable attention due to diverse biological activities. In this study, a series of 4-[(halophenyldiazenyl]phenyl aspirinate derivatives were synthesized from the reaction of aspirin with 4-[(halophenyldiazenyl]phenol via esterification, in the presence of DCC/DMAP in DCM with overall yield of 45–54%. 4-[(Halophenyldiazenyl]phenol was prepared prior to esterification from coupling reaction of aniline derivatives and phenol in basic solution. All compounds were characterized using elemental analysis, FTIR, and 1H and 13C NMR spectroscopies. All compounds were screened for their anticancer activities against nasopharyngeal cancer (NPC HK-1 cell lines and the viability of cultured cells was determined by MTS [3-(4,5-dimethylthiazol-2-yl-5-(3-carboxylmethoxylphenyl-2-(4-sulfophenyl-2H-tetrazolium]-based colorimetric assay. 4-[(E-(Fluorophenyldiazenyl]phenol showed the highest anticancer activity against NPC HK-1 cell lines compared to other synthesized compounds. 4-[(Halophenyldiazenyl]phenyl aspirinate showed low cytotoxicity against NPC HK-1 cell lines compared to 4-[(halophenyldiazenyl]phenol but better anticancer activity than aspirin alone.

  10. Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF

    Directory of Open Access Journals (Sweden)

    Coppock Donald L

    2008-12-01

    Full Text Available Abstract Background Multiple physiologic impairments are responsible for chronic wounds. A cell line grown which retains its phenotype from patient wounds would provide means of testing new therapies. Clinical information on patients from whom cells were grown can provide insights into mechanisms of specific disease such as diabetes or biological processes such as aging. The objective of this study was 1 To culture human cells derived from patients with chronic wounds and to test the effects of putative therapies, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF on these cells. 2 To describe a methodology to create fibroblast cell lines from patients with chronic wounds. Methods Patient biopsies were obtained from 3 distinct locations on venous ulcers. Fibroblasts derived from different wound locations were tested for their migration capacities without stimulators and in response to GM-CSF. Another portion of the patient biopsy was used to develop primary fibroblast cultures after rigorous passage and antimicrobial testing. Results Fibroblasts from the non-healing edge had almost no migration capacity, wound base fibroblasts were intermediate, and fibroblasts derived from the healing edge had a capacity to migrate similar to healthy, normal, primary dermal fibroblasts. Non-healing edge fibroblasts did not respond to GM-CSF. Six fibroblast cell lines are currently available at the National Institute on Aging (NIA Cell Repository. Conclusion We conclude that primary cells from chronic ulcers can be established in culture and that they maintain their in vivo phenotype. These cells can be utilized for evaluating the effects of wound healing stimulators in vitro.

  11. Selective toxicity of a quinoxaline 1,4-di-N-oxide derivative in human tumour cell lines.

    Science.gov (United States)

    Amaia, Azquetaa; Pachón, Gisela; Cascante, Marta; Creppy, Edmond E; Monge, Antonio; López de Cerain, Adela

    2005-01-01

    The presence of hypoxic cells in human solid tumours is one of the causes of tumour resistance to conventional therapy, and is also associated with processes that promote the tumour progression. Different chemical agents have been designed in order to take advantage of the particular metabolic characteristics of hypoxic regions. These drugs, called bioreductive agents, are activated inside the hypoxic cells to give active species that, in the presence of oxygen, are oxidised back to the non-toxic parent compound. Several quinoxaline 1,4-di-N-oxides have been described as potential bioreductive agents, and among them, 7-cloro-3-[[(N,N-dimethylamino)propy]amino]-2-quinoxalinecarbonitrile 1,4-di-N-oxide hydrocloride (Q-85 HCl) appeared to be the most promising one. In the present work, the selective cytotoxicity of Q-85 HCl was studied in several human tumour cell lines of different origin (Caco2, MCF-7, HT-29 and Tk-10). Cell viability was calculated after 2 h treatment under hypoxic and well-oxygenated conditions. The potency (the concentration that gives 1% of cell survival) in hypoxia and hypoxia cytotoxicity ratio (HCR = potency in oxygenated conditions/potency in hypoxia) were calculated after a 14-day clonogenic assay. Q-85 HCl was more toxic in hypoxia than in well-oxygenated cells in all the tumour cell lines. The best profile of potency in hypoxia (0.4 micromol/L) and selectivity (HCR=155) was found in CaCo-2 cells. Altogether, these results suggest an in vitro biological profile for Q-85 HCl that makes it an interesting candidate for the development as a bioreductive agent.

  12. Soy Promotes Juvenile Granulosa Cell Tumor Development in Mice and in the Human Granulosa Cell Tumor-Derived COV434 Cell Line1

    Science.gov (United States)

    Mansouri-Attia, Nadéra; James, Rebecca; Ligon, Alysse; Li, Xiaohui; Pangas, Stephanie A.

    2014-01-01

    ABSTRACT Soy attracts attention for its health benefits, such as lowering cholesterol or preventing breast and colon cancer. Soybeans contain isoflavones, which act as phytoestrogens. Even though isoflavones have beneficial health effects, a role for isoflavones in the initiation and progression of diseases including cancer is becoming increasingly recognized. While data from rodent studies suggest that neonatal exposure to genistein (the predominant isoflavone in soy) disrupts normal reproductive function, its role in ovarian cancers, particularly granulosa cell tumors (GCT), is largely unknown. Our study aimed to define the contribution of a soy diet in GCT development using a genetically modified mouse model for juvenile GCTs (JGCT; Smad1 Smad5 conditional double knockout mice) as well as a human JGCT cell line (COV434). While dietary soy cannot initiate JGCT development in mice, we show that it has dramatic effects on GCT growth and tumor progression compared to a soy-free diet. Loss of Smad1 and Smad5 alters estrogen receptor alpha (Esr1) expression in granulosa cells, perhaps sensitizing the cells to the effects of genistein. In addition, we found that genistein modulates estrogen receptor expression in the human JGCT cell line and positively promotes cell growth in part by suppressing caspase-dependent apoptosis. Combined, our work suggests that dietary soy consumption has deleterious effects on GCT development. PMID:25165122

  13. Uptake of Ga-curcumin derivatives in different cancer cell lines: Toward the development of new potential 68Ga-labelled curcuminoids-based radiotracers for tumour imaging.

    Science.gov (United States)

    Rubagotti, Sara; Croci, Stefania; Ferrari, Erika; Orteca, Giulia; Iori, Michele; Capponi, Pier C; Versari, Annibale; Asti, Mattia

    2017-08-01

    Thanks to the ability to suppress the proliferation and to kill tumour cells, several studies have shown the anti-cancer effects of curcumin (CUR) and its derivatives, i.e. diacetylcurcumin (DAC) and bis-dehydroxycurcumin (bDHC). This study is focused onto the development of curcuminoid complexes with gallium-68 employed as potential new radio-labelled probes to detect neoplastic tissues through imaging techniques such as positron emission tomography. To this purpose, the uptake of three Ga-curcuminoid complexes, namely Ga(CUR) 2 + , Ga(DAC) 2 + , Ga(bDHC) 2 + , by various tumour cell lines was compared with the uptake of the same compounds by normal human lymphocytes by flow cytometry using the intrinsic fluorescence of the curcuminoids. Ga(CUR) 2 + , and particularly Ga(DAC) 2 + , showed a higher uptake by colorectal carcinoma (HT29) and lymphoma (K562) cell lines than lymphocytes, while the uptake of Ga(bDHC) 2 + was higher in lymphocytes than in all the other cell lines. Based on the fluorescence data, Gallium-68 labelled complexes were then tested in HT29 cell line. 68 Ga(DAC) 2 + showed the highest uptake by HT29 cells (higher internalization with a lower externalization) and the highest affinity. The obtained results are promising and the findings foster further investigation on the development of curcumin-metal-based radiopharmaceuticals. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Homozygous deletion of the α- and β1-interferon genes in human leukemia and derived cell lines

    International Nuclear Information System (INIS)

    Diaz, M.O.; Ziemin, S.; Le Beau, M.M.; Pitha, P.; Smith, S.D.; Chilcote, R.R.; Rowley, J.D.

    1988-01-01

    The loss of bands p21-22 from one chromosome 9 homologue as a consequence of a deletion of the short arm [del(9p)], unbalanced translocation, or monosomy 9 is frequently observed in the malignant cells of patients with lymphoid neoplasias, including acute lymphoblastic leukemia and non-Hodgkin lymphoma. The α- and β 1 -interferon genes have been assigned to this chromosome region (9p21-22). The authors now present evidence of the homozygous deletion of the interferon genes in neoplastic hematopoietic cell lines and primary leukemia cells in the presence or absence of chromosomal deletions that are detectable at the level of the light microscope. In these cell lines, the deletion of the interferon genes is accompanied by a deficiency of 5'-methylthioadenosine phosphorylase, an enzyme of purine metabolism. These homozygous deletions may be associated with the loss of a tumor-suppressor gene that is involved in the development of these neoplasias. The relevant genes may be either the interferon genes themselves or a gene that has a tumor-suppressor function and is closely linked to them

  15. Characterization of Camptothecin-induced Genomic Changes in the Camptothecin-resistant T-ALL-derived Cell Line CPT-K5.

    Science.gov (United States)

    Kjeldsen, Eigil; Nielsen, Christine J F; Roy, Amit; Tesauro, Cinzia; Jakobsen, Ann-Katrine; Stougaard, Magnus; Knudsen, Birgitta R

    2018-01-01

    Acquisition of resistance to topoisomerase I (TOP1)-targeting camptothecin (CPT) derivatives is a major clinical problem. Little is known about the underlying chromosomal and genomic mechanisms. We characterized the CPT-K5 cell line expressing mutant CPT-resistant TOP1 and its parental T-cell derived acute lymphoblastic leukemia CPT-sensitive RPMI-8402 cell line by karyotyping and molecular genetic methods, including subtractive oligo-based array comparative genomic hybridization (soaCGH) analysis. Karyotyping revealed that CPT-K5 cells had acquired additional structural aberrations and a reduced modal chromosomal number compared to RPMI-8402. soaCGH analysis identified vast copy number alterations and >200 unbalanced DNA breakpoints distributed unevenly across the chromosomal complement in CPT-K5. In addition, the short tandem repeat alleles were found to be highly different between CPT-K5 and its parental cell line. We identified copy number alterations affecting genes important for maintaining genome integrity and reducing CPT-induced DNA damage. We show for the first time that short tandem repeats are targets for TOP1 cleavage, that can be differentially stimulated by CPT. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  16. Lulo cell line derived from Lutzomyia longipalpis (Diptera: Psychodidae): a novel model to assay Leishmania spp. and vector interaction.

    Science.gov (United States)

    Côrtes, Luzia Mc; Silva, Roger Mm; Pereira, Bernardo As; Guerra, Camila; Zapata, Angela C; Bello, Felio J; Finkelstein, Léa C; Madeira, Maria F; Brazil, Reginaldo P; Côrte-Real, Suzana; Alves, Carlos R

    2011-11-14

    Leishmania (Vianna) braziliensis, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) chagasi are important parasites in the scenario of leishmaniasis in Brazil. During the life cycle of these parasites, the promastigote forms adhere to the midgut epithelial microvillii of phlebotomine insects to avoid being secreted along with digestive products. Lulo cells are a potential model that will help to understand the features of this adhesion phenomenon. Here, we analyze the interaction between Leishmania spp. promastigotes and Lulo cells in vitro, specifically focusing on adhesion events occurring between three Leishmania species and this cell line. Confluent monolayers of Lulo cells were incubated with promastigotes and adhesion was assessed using both light microscopy and scanning electron microscopy. The results indicate that species from the subgenera Leishmania and Viannia have great potential to adhere to Lulo cells. The highest adherence rate was observed for L. (L.) chagasi after 24 h of incubation with Lulo cells (27.3 ± 1.8% of cells with adhered promastigotes), followed by L. (L.) amazonensis (16.0 ± 0.7%) and L. (V.) braziliensis (3.0 ± 0.7%), both after 48 h. In the ultrastructural analysis, promastigote adherence was also assessed by scanning electron microscopy, showing that, for parasites from both subgenera, adhesion occurs by both the body and the flagellum. The interaction of Lulo cells with Leishmania (L.) chagasi showed the participation of cytoplasmic projections from the former closely associating the parasites with the cells. We present evidence that Lulo cells can be useful in studies of insect-parasite interactions for Leishmania species.

  17. Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII

    Directory of Open Access Journals (Sweden)

    Marcela Cristina Correa de Freitas

    2014-06-01

    Full Text Available OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO and baby hamster kidney cells (BHK. Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K.METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones.RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293.CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.

  18. Glial cell line-derived neurotrophic factor (GDNF) induces neuritogenesis in the cochlear spiral ganglion via neural cell adhesion molecule (NCAM)

    Science.gov (United States)

    Euteneuer, Sara; Yang, Kuo H.; Chavez, Eduardo; Leichtle, Anke; Loers, Gabriele; Olshansky, Adel; Pak, Kwang; Schachner, Melitta; Ryan, Allen F.

    2013-01-01

    Glial cell line-derived neurotrophic factor (GDNF) increases survival and neurite extension of spiral ganglion neurons (SGNs), the primary neurons of the auditory system, via yet unknown signaling mechanisms. In other cell types, signaling is achieved by the GPI-linked GDNF family receptor α1 (GFRα1) via recruitment of transmembrane receptors: Ret (re-arranged during transformation) and/or NCAM (neural cell adhesion molecule). Here we show that GDNF enhances neuritogenesis in organotypic cultures of spiral ganglia from 5-day-old rats and mice. Addition of GFRα1-Fc increases this effect. GDNF/GFRα1-Fc stimulation activates intracellular PI3K/Akt and MEK/Erk signaling cascades as detected by Western blot analysis of cultures prepared from rats at postnatal days 5 (P5, before the onset of hearing) and 20 (P20, after the onset of hearing). Both cascades mediate GDNF stimulation of neuritogenesis, since application of the Akt inhibitor Wortmannin or the Erk inhibitor U0126 abolished GDNF/GFRα1-Fc stimulated neuritogenesis in P5 rats. Since cultures of P5 NCAM-deficient mice failed to respond by neuritogenesis to GDNF/GFRα1-Fc, we conclude that NCAM serves as a receptor for GDNF signaling responsible for neuritogenesis in early postnatal spiral ganglion. PMID:23262364

  19. Combined 5-FU and ChoKα inhibitors as a new alternative therapy of colorectal cancer: evidence in human tumor-derived cell lines and mouse xenografts.

    Directory of Open Access Journals (Sweden)

    Ana de la Cueva

    Full Text Available Colorectal cancer (CRC is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα, an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy.ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS and thymidine kinase (TK1 levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action.Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

  20. Cytometric comparisons between circulating tumor cells from prostate cancer patients and the prostate-tumor-derived LNCaP cell line

    Science.gov (United States)

    Lazar, Daniel C.; Cho, Edward H.; Luttgen, Madelyn S.; Metzner, Thomas J.; Loressa Uson, Maria; Torrey, Melissa; Gross, Mitchell E.; Kuhn, Peter

    2012-02-01

    Many important experiments in cancer research are initiated with cell line data analysis due to the ease of accessibility and utilization. Recently, the ability to capture and characterize circulating tumor cells (CTCs) has become more prevalent in the research setting. This ability to detect, isolate and analyze CTCs allows us to directly compare specific protein expression levels found in patient CTCs to cell lines. In this study, we use immunocytochemistry to compare the protein expression levels of total cytokeratin (CK) and androgen receptor (AR) in CTCs and cell lines from patients with prostate cancer to determine what translational insights might be gained through the use of cell line data. A non-enrichment CTC detection assay enables us to compare cytometric features and relative expression levels of CK and AR by indirect immunofluorescence from prostate cancer patients against the prostate cancer cell line LNCaP. We measured physical characteristics of these two groups and observed significant differences in cell size, fluorescence intensity and nuclear to cytoplasmic ratio. We hope that these experiments will initiate a foundation to allow cell line data to be compared against characteristics of primary cells from patients.

  1. Tricyclic Antidepressant Amitriptyline-induced Glial Cell Line-derived Neurotrophic Factor Production Involves Pertussis Toxin-sensitive Gαi/o Activation in Astroglial Cells.

    Science.gov (United States)

    Hisaoka-Nakashima, Kazue; Miyano, Kanako; Matsumoto, Chie; Kajitani, Naoto; Abe, Hiromi; Okada-Tsuchioka, Mami; Yokoyama, Akinobu; Uezono, Yasuhito; Morioka, Norimitsu; Nakata, Yoshihiro; Takebayashi, Minoru

    2015-05-29

    Further elaborating the mechanism of antidepressants, beyond modulation of monoaminergic neurotransmission, this study sought to elucidate the mechanism of amitriptyline-induced production of glial cell line-derived neurotrophic factor (GDNF) in astroglial cells. Previous studies demonstrated that an amitriptyline-evoked matrix metalloproteinase (MMP)/FGF receptor (FGFR)/FGFR substrate 2α (FRS2α)/ERK cascade is crucial for GDNF production, but how amitriptyline triggers this cascade remains unknown. MMP is activated by intracellular mediators such as G proteins, and this study sought to clarify the involvement of G protein signaling in amitriptyline-evoked GDNF production in rat C6 astroglial cells (C6 cells), primary cultured rat astrocytes, and normal human astrocytes. Amitriptyline-evoked GDNF mRNA expression and release were inhibited by pertussis toxin (PTX), a Gα(i/o) inhibitor, but not by NF449, a Gα(s) inhibitor, or YM-254890, a Gαq inhibitor. The activation of the GDNF production cascade (FGFR/FRS2α/ERK) was also inhibited by PTX. Deletion of Gα(ο1) and Gα(i3) by RNAi demonstrated that these G proteins play important roles in amitriptyline signaling. G protein activation was directly analyzed by electrical impedance-based biosensors (CellKey(TM) assay), using a label-free (without use of fluorescent proteins/probes or radioisotopes) and real time approach. Amitriptyline increased impedance, indicating Gα(i/o) activation that was suppressed by PTX treatment. The impedance evoked by amitriptyline was not affected by inhibitors of the GDNF production cascade. Furthermore, FGF2 treatment did not elicit any effect on impedance, indicating that amitriptyline targets PTX-sensitive Gα(i/o) upstream of the MMP/FGFR/FRS2α/ERK cascade. These results suggest novel targeting for the development of antidepressants. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Whole-exome sequencing of fibroblast and its iPS cell lines derived from a patient diagnosed with xeroderma pigmentosum

    Directory of Open Access Journals (Sweden)

    Kohji Okamura

    2015-12-01

    Full Text Available Cells from a patient with a DNA repair-deficiency disorder are anticipated to bear a large number of somatic mutations. Because such mutations occur independently in each cell, there is a high degree of mosaicism in patients' tissues. While major mutations that have been expanded in many cognate cells are readily detected by sequencing, minor ones are overlaid with a large depth of non-mutated alleles and are not detected. However, cell cloning enables us to observe such cryptic mutations as well as major mutations. In the present study, we focused on a fibroblastic cell line that is derived from a patient diagnosed with xeroderma pigmentosum (XP, which is an autosomal recessive disorder caused by a deficiency in nucleotide excision repair. By making a list of somatic mutations, we can expect to see a characteristic pattern of mutations caused by the hereditary disorder. We cloned a cell by generating an iPS cell line and performed a whole-exome sequencing analysis of the progenitor and its iPS cell lines. Unexpectedly, we failed to find causal mutations in the XP-related genes, but we identified many other mutations including homozygous deletion of GSTM1 and GSTT1. In addition, we found that the long arm of chromosome 9 formed uniparental disomy in the iPS cell line, which was also confirmed by a structural mutation analysis using a SNP array. Type and number of somatic mutations were different from those observed in XP patients. Taken together, we conclude that the patient might be affected by a different type of the disorder and that some of the mutations that we identified here may be responsible for exhibiting the phenotype. Sequencing and SNP-array data have been submitted to SRA and GEO under accession numbers SRP059858 and GSE55520, respectively.

  3. Sensitivity to cisplatin in primary cell lines derived from human glioma correlates with levels of EGR-1 expression

    Directory of Open Access Journals (Sweden)

    Ponti Donatella

    2011-03-01

    Full Text Available Abstract Background Less than 30% of malignant gliomas respond to adjuvant chemotherapy. Here, we have asked whether variations in the constitutive expression of early-growth response factor 1 (EGR-1 predicted acute cytotoxicity and clonogenic cell death in vitro, induced by six different chemotherapics. Materials and methods Cytotoxicity assays were performed on cells derived from fresh tumor explants of 18 human cases of malignant glioma. In addition to EGR-1, tumor cultures were investigated for genetic alterations and the expression of cancer regulating factors, related to the p53 pathway. Results We found that sensitivity to cisplatin correlates significantly with levels of EGR-1 expression in tumors with wild-type p53/INK4a/p16 status. Conclusion Increased knowledge of the mechanisms regulating EGR-1 expression in wild-type p53/INK4a/p16 cases of glioma may help in the design of new chemotherapeutic strategies for these tumors.

  4. Efficient propagation of progressive multifocal leukoencephalopathy-type JC virus in COS-7-derived cell lines stably expressing Tat protein of human immunodeficiency virus type 1.

    Science.gov (United States)

    Nukuzuma, Souichi; Nakamichi, Kazuo; Kameoka, Masanori; Sugiura, Shigeki; Nukuzuma, Chiyoko; Miyoshi, Isao; Takegami, Tsutomu

    2010-12-01

    The high incidence of progressive multifocal leukoencephalopathy (PML) in AIDS patients compared with many other immunosuppressive diseases suggests that HIV-1 infection is strictly related to the activation of JC virus (JCV) propagation. In this report, propagation of PML-type JCV in COS-7-derived cell lines stably expressing HIV-1 Tat (COS-tat cells) has been examined. In COS-tat cells, production of viral particles and replication of genomic DNA were markedly increased compared to COS-7 cells, as judged by HA and real-time PCR analyses. These results demonstrate that COS-tat cells provide a useful model system for studying HIV-1 Tat-mediated propagation of PML-type JCV. © 2010 The Societies and Blackwell Publishing Asia Pty Ltd.

  5. Gliotoxin Targets Nuclear NOTCH2 in Human Solid Tumor Derived Cell Lines In Vitro and Inhibits Melanoma Growth in Xenograft Mouse Model

    Directory of Open Access Journals (Sweden)

    Rainer Hubmann

    2017-07-01

    Full Text Available Deregulation of NOTCH2 signaling is implicated in a wide variety of human neoplasias. The current concept of targeting NOTCH is based on using gamma secretase inhibitors (GSI to regulate the release of the active NOTCH intracellular domain. However, the clinical outcome of GSI remains unsatisfactory. Therefore we analyzed human solid tumor derived cell lines for their nuclear NOTCH activity and evaluated the therapeutic potential of the NOTCH2 transactivation inhibitor gliotoxin in comparison to the representative GSI DAPT. Electrophoretic mobility shift assays (EMSA were used as a surrogate method for the detection of NOTCH/CSL transcription factor complexes. The effect of gliotoxin on cell viability and its clinical relevance was evaluated in vitro and in a melanoma xenograft mouse model. Cell lines derived from melanoma (518A2, hepatocellular carcinoma (SNU398, HCC-3, Hep3B, and pancreas carcinoma (PANC1 express high amounts of nuclear NOTCH2. Gliotoxin efficiently induced apoptosis in these cell lines whereas the GSI DAPT was ineffective. The specificity of gliotoxin was demonstrated in the well differentiated nuclear NOTCH negative cell line Huh7, which was resistant to gliotoxin treatment in vitro. In xenotransplanted 518A2 melanomas, a single day dosing schedule of gliotoxin was well tolerated without any study limiting side effects. Gliotoxin significantly reduced the tumor volume in early (83 mm3 vs. 115 mm3, p = 0.008 as well as in late stage (218 mm3 vs. 576 mm3, p = 0.005 tumor models. In conclusion, NOTCH2 appears to be a key target of gliotoxin in human neoplasias and gliotoxin deserves further evaluation as a potential therapeutic agent in cancer management.

  6. Epigenetic status of H19/IGF2 and SNRPN imprinted genes in aborted and successfully derived embryonic stem cell lines in non-human primates

    Directory of Open Access Journals (Sweden)

    Florence Wianny

    2016-05-01

    Full Text Available The imprinted genes of primate embryonic stem cells (ESCs often show altered DNA methylation. It is unknown whether these alterations emerge while deriving the ESCs. Here we studied the methylation patterns of two differentially methylated regions (DMRs, SNRPN and H19/IGF2 DMRs, during the derivation of monkey ESCs. We show that the SNRPN DMR is characteristically methylated at maternal alleles, whereas the H19/IGF2 DMR is globally highly methylated, with unusual methylation on the maternal alleles. These methylation patterns remain stable from the early stages of ESC derivation to late passages of monkey ESCs and following differentiation. Importantly, the methylation status of H19/IGF2 DMR and the expression levels of IGF2, H19, and DNMT3B mRNAs in early embryo-derived cells were correlated with their capacity to generate genuine ESC lines. Thus, we propose that these markers could be useful to predict the outcomes of establishing an ESC line in primates.

  7. Synthesis of an anthraquinone derivative (DHAQC and its effect on induction of G2/M arrest and apoptosis in breast cancer MCF-7 cell line

    Directory of Open Access Journals (Sweden)

    Yeap SK

    2015-02-01

    Full Text Available SweeKeong Yeap,1 Muhammad Nadeem Akhtar,2 Kian Lam Lim,3 Nadiah Abu,4,5 Wan Yong Ho,6 Seema Zareen,2 Kiarash Roohani,1 Huynh Ky,4 Sheau Wei Tan,1 Nordin Lajis,7 Noorjahan Banu Alitheen1,4 1Institute of Bioscience, Universiti Putra Malaysia, Selangor Darul Ehsan, Malaysia; 2Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Kuantan, Pahang, Malaysia; 3Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Selangor Darul Ehsan, Malaysia; 4Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Selangor Darul Ehsan, Malaysia; 5Bright Sparks Unit, University of Malaya, Kuala Lumpur, Malaysia; 6School of Biomedical Sciences, University of Nottingham Malaysia Campus, Selangor Darul Ehsan, Malaysia; 7Scientific Chairs Unit, Taibah University, Medina, Saudi Arabia Abstract: Anthraquinones are an important class of naturally occurring biologically active compounds. In this study, anthraquinone derivative 1,3-dihydroxy-9,10-anthraquinone-2-carboxylic acid (DHAQC (2 was synthesized with 32% yield through the Friedel–Crafts condensation reaction. The mechanisms of cytotoxicity of DHAQC (2 in human breast cancer MCF-7 cells were further investigated. Results from the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay showed that DHAQC (2 exhibited potential cytotoxicity and selectivity in the MCF-7 cell line, comparable with the naturally occurring anthraquinone damnacanthal. DHAQC (2 showed a slightly higher IC50 (inhibitory concentration with 50% cell viability value in the MCF-7 cell line compared to damnacanthal, but it is more selective in terms of the ratio of IC50 on MCF-7 cells and normal MCF-10A cells. (selective index for DHAQC (2 was 2.3 and 1.7 for damnacanthal. The flow cytometry cell cycle analysis on the MCF-7 cell line treated with the IC50 dose of DHAQC (2 for 48 hours showed that DHAQC (2 arrested MCF-7 cell line at the G2/M phase in association with an

  8. Signaling via class IA Phosphoinositide 3-kinases (PI3K in human, breast-derived cell lines.

    Directory of Open Access Journals (Sweden)

    Veronique Juvin

    Full Text Available We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K in human breast-derived MCF10a (and iso-genetic derivatives and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, β, δ and γ and p50-101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5-trisphosphate (PtdIns(3,4,5P3 that can activate effectors, eg protein kinase B (PKB, and responses, eg migration. The PtdIns(3,4,5P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110β>>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not β- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not β- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K, basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN(-/- cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110β, but not α- or δ- activity; in PTEN(-/- MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, via acutely

  9. Evaluation of the sensitizing potential of antibiotics in vitro using the human cell lines THP-1 and MUTZ-LC and primary monocyte‐derived dendritic cells

    Energy Technology Data Exchange (ETDEWEB)

    Sebastian, Katrin, E-mail: ksebastian@ukaachen.de [Department of Dermatology and Allergology, RWTH Aachen University Hospital, D-52074 Aachen (Germany); Ott, Hagen [Department of Dermatology and Allergology, RWTH Aachen University Hospital, D-52074 Aachen (Germany); Zwadlo-Klarwasser, Gabriele [IZKF (BIOMAT), RWTH Aachen University Hospital, D-52074 Aachen (Germany); Skazik-Voogt, Claudia; Marquardt, Yvonne; Czaja, Katharina; Merk, Hans F.; Baron, Jens Malte [Department of Dermatology and Allergology, RWTH Aachen University Hospital, D-52074 Aachen (Germany)

    2012-08-01

    Since the 7th amendment to the EU cosmetics directive foresees a complete ban on animal testing, alternative in vitro methods have been established to evaluate the sensitizing potential of small molecular weight compounds. To find out whether these novel in vitro assays are also capable to predict the sensitizing potential of small molecular weight drugs, model compounds such as beta-lactams and sulfonamides – which are the most frequent cause of adverse drug reactions – were co-incubated with THP-1, MUTZ-LC, or primary monocyte‐derived dendritic cells for 48 h and subsequent expression of selected marker genes (IL-8, IL-1β, CES1, NQO1, GCLM, PIR and TRIM16) was studied by real time PCR. Benzylpenicillin and phenoxymethylpenicillin were recognized as sensitizing compounds because they are capable to induce the mRNA expression of these genes in moDCs and, except for IL-8, in THP-1 cells but not in MUTZ-LC. Ampicillin stimulated the expression of some marker genes in moDCs and THP-1 cells. SMX did not affect the expression of these genes in THP-1, however, in moDCs, at least PIR was enhanced and there was an increase of the release of IL-8. These data reveal that novel in vitro DC based assays might play a role in the evaluation of the allergenic potential of novel drug compounds, but these systems seem to lack the ability to detect the sensitizing potential of prohaptens that require metabolic activation prior to sensitization and moDCs seem to be superior with regard to the sensitivity compared with THP-1 and MUTZ-3 cell lines. -- Highlights: ► We tested the sensitizing potential of small molecular weight drugs in vitro. ► In vitro assays were performed with moDCs and THP-1 cells. ► Beta-lactam antibiotics can be recognized as sensitizing compounds. ► They affect the expression of metabolic enzymes, cytokines and transcription factors. ► Sulfamethoxazole has no measurable effect on THP-1 cells and moDCs.

  10. Evaluation of the sensitizing potential of antibiotics in vitro using the human cell lines THP-1 and MUTZ-LC and primary monocyte‐derived dendritic cells

    International Nuclear Information System (INIS)

    Sebastian, Katrin; Ott, Hagen; Zwadlo-Klarwasser, Gabriele; Skazik-Voogt, Claudia; Marquardt, Yvonne; Czaja, Katharina; Merk, Hans F.; Baron, Jens Malte

    2012-01-01

    Since the 7th amendment to the EU cosmetics directive foresees a complete ban on animal testing, alternative in vitro methods have been established to evaluate the sensitizing potential of small molecular weight compounds. To find out whether these novel in vitro assays are also capable to predict the sensitizing potential of small molecular weight drugs, model compounds such as beta-lactams and sulfonamides – which are the most frequent cause of adverse drug reactions – were co-incubated with THP-1, MUTZ-LC, or primary monocyte‐derived dendritic cells for 48 h and subsequent expression of selected marker genes (IL-8, IL-1β, CES1, NQO1, GCLM, PIR and TRIM16) was studied by real time PCR. Benzylpenicillin and phenoxymethylpenicillin were recognized as sensitizing compounds because they are capable to induce the mRNA expression of these genes in moDCs and, except for IL-8, in THP-1 cells but not in MUTZ-LC. Ampicillin stimulated the expression of some marker genes in moDCs and THP-1 cells. SMX did not affect the expression of these genes in THP-1, however, in moDCs, at least PIR was enhanced and there was an increase of the release of IL-8. These data reveal that novel in vitro DC based assays might play a role in the evaluation of the allergenic potential of novel drug compounds, but these systems seem to lack the ability to detect the sensitizing potential of prohaptens that require metabolic activation prior to sensitization and moDCs seem to be superior with regard to the sensitivity compared with THP-1 and MUTZ-3 cell lines. -- Highlights: ► We tested the sensitizing potential of small molecular weight drugs in vitro. ► In vitro assays were performed with moDCs and THP-1 cells. ► Beta-lactam antibiotics can be recognized as sensitizing compounds. ► They affect the expression of metabolic enzymes, cytokines and transcription factors. ► Sulfamethoxazole has no measurable effect on THP-1 cells and moDCs.

  11. A recurrent human papillomavirus integration site at chromosome region 12q14-q15 in SW756 and SK-v cell lines derived from genital tumors

    International Nuclear Information System (INIS)

    Sastre-Garau, X.; Couturier, J.; Favre, M.; Orth, G.

    1995-01-01

    The SW756 cell line, derived from an invasive cancer of the uterine cervix, harbours integrated human papillomavirus (HPV) 18 DNA sequences which have been located in chromosome band 12q13. By in situ hybridization experiments with tritiated and digoxigenin-labelled HPV18 probes on R-banded chromosomes, we now localize the integrated viral sequences in 12q14-q15. Interestingly, we have previously localized integrated HPV16 sequences in the same chromosomal region in SK-v cells, derived from a pre-invasive vulvar neoplasia. The chromosomal region 12q14-q15 could thus correspond to a preferential site for the integration of HPV DNA in genital tumors. (authors). 29 refs., 2 figs

  12. Synergistic Cytotoxicity Effect by Combination Treatment of Polyketide Derivatives from Annona muricata Linn Leaves and Doxorubicin as Potential Anticancer Material on Raji Cell Line

    Science.gov (United States)

    Artanti, A. N.; Astirin, O. P.; Prayito, A.; Fisma, R.; Prihapsara, F.

    2018-03-01

    Nasopharynx cancer is one of the most deadly cancer. The main priority of nasopharynx cancer treatment is the use of chemotherapeutic agents, especially doxorubicin. However, doxorubicin might also lead to diverse side effect. An approach recently develop to overcome side effect of doxorubicin is to used of combined chemotherapeutic agent. One of the compounds found effication as an anticancer agent on nasopharynx cancer is acetogenin, a polyketide compound that is abundant in Annona muricata L. leaves. This study has been done to examine polyketide derivatives was isolated from Annona muricata L. which has potency to induce apoptosis by p53 expression on raji cell line. The determination of cytotoxic combination activity from polyketide derivative and doxorubicin was evaluated using MTT assay to obtain the value of CI (combination index). Data analysis showed that combination of polyketide derivative from Annona muricata L. (14,4 µg/ml) and doxorubicin with all of concentration performed synergistic effect on raji cell line with CI value from 0.13 – 0.65.

  13. Establishment and cryopreservation of a skin fibroblast cell line derived from Yunnan semi-fine wool sheep in the presence of synthetic ice blocker.

    Science.gov (United States)

    Wu, Shuai Shuai; Li, Dong Jiang; Lv, Chun Rong; Yang, Hong Yuan; Zhu, Lan; Li, Wei Juan; Quan, Guo Bo; Hong, Qiong Hua

    2013-01-01

    In this study, the fibroblasts cell line derived from ear marginal tissue of Yunnan semi-fine wool sheep was successfully established using the primary explants technique and cryopreservation technology. Additionally, the protective effect of synthetic ice blocker (SIB) including 1, 3-cyclohexanediol (1, 3-CHD) and 1, 4-cyclohexanediol (1, 4-CHD) on frozen fibroblast cells was also assessed and compared. Propidium iodide (PI) was used to stain the dead cells following cryopreservation and thawing. The results showed that compared with Medium 199 (M199) and Dulbecco's modified Eagle's medium : Nutrient Mixture F-12 (1 : 1) Mixture (DMEM/F12), Dulbecco's modified Eagle's medium (DMEM) may be more suitable for the primary culture of fibroblast cells of Yunnan semi-fine wool sheep. The growth curve of cells is a typical "S" type. After subculture for four days, the cells entered the plateau phase and began to degenerate. Biological analysis showed that the population doubling time (PDT) for subculturing fibroblast cells was approximately 26h. The Karyotyping data indicated that the percentage of fibroblast cells with normal chromosome number 2n = 54 was over 90% following subculture for 10 passages. Moreover, the tests for bacteria, fungi, viruses and mycoplasma were negative. After serial subculture for 5 generations, the fibroblast cells were cryopreserved in the presence or absence of 1, 3-CHD or 1, 4-CHD. The data indicated that with increase of the synthetic ice blocker concentrations, the viability of frozen-thawed fibroblast cells was firstly increased and then decreased. When the concentration of 1, 3-CHD or 1, 4-CHD was 50 mM, the viable percentage of frozen-thawed fibroblast cells was 91.93% +/- 2.24% and 94.13% +/- 0.55% respectively and significantly higher than that of the cells frozen in the absence of synthetic ice blockers (88.10% +/- 1.49%, P skin fibroblast cell line of Yunnan semi-fine wool sheep was firstly established in this study. Additionally

  14. NTRK1 rearrangement in colorectal cancer patients: evidence for actionable target using patient-derived tumor cell line.

    Science.gov (United States)

    Lee, Su Jin; Li, Gang Gary; Kim, Seung Tae; Hong, Min Eui; Jang, Jiryeon; Yoon, Nara; Ahn, Soo Min; Murphy, Danielle; Christiansen, Jason; Wei, Ge; Hornby, Zachary; Lee, Dong Woo; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Hong, Sung No; Kim, Seok-Hyeong; Kang, Won Ki; Park, Keunchil; Park, Woong Yang; Kim, Kyoung-Mee; Lee, Jeeyun

    2015-11-17

    We have investigated the incidence of NTRK1 rearrangements in metastatic gastrointestinal cancer patients and demonstrated the potential for clinical response of these patients to targeted therapy. We prospectively collected tumor tissue specimens for one year and simultaneously generated patient-derived tumor cells (PDCs). Specimens were initially screened for TrkA protein expression using TrkA immunohistochemistry (IHC). In the case of TrkA IHC positive results, samples were further examined by fluorescence in situ hybridization (FISH) and next generation sequencing (NGS) to confirm the presence of NTRK1 rearrangements. From January 2014 to December 2014, a total of 74 metastatic colorectal cancer (CRC) patients and 66 gastric cancer (GC) patients were initially screened by TrkA IHC. Two of the 74 CRC patients (2.7%) and one of the 66 GC patients (1.5%) were positive for TrkA expression by IHC. All three IHC positive cases had evidence of NTRK1 rearrangements by FISH. NGS was performed on the 3 IHC positive cases and confirmed TPM3-NTRK1 rearrangements in the two CRC cases. One GC patient with TrkA expression by IHC did not harbor an NTRK1 rearrangement. PDCs established from the NTRK1 positive CRC patients were positive for the NTRK1 rearrangement. Entrectinib, a pan-TRK inhibitor, profoundly inhibited cell proliferation of NTRK1-rearranged PDCs with such inhibition associated with inactivation of TrkA, and down-regulation of downstream signaling pathways. TrkA IHC is an effective, initial screening method for NTRK1 rearrangement detection in the clinic. Inhibition of the TrkA kinase is a promising targeted therapy for cancer patients whose tumors harbor a NTRK1 rearrangement.

  15. Patient-Derived Antibody Targets Tumor Cells

    Science.gov (United States)

    An NCI Cancer Currents blog on an antibody derived from patients that killed tumor cells in cell lines of several cancer types and slowed tumor growth in mouse models of brain and lung cancer without evidence of side effects.

  16. Cytotoxicity and Apoptosis Inducing Activities of 2-Amino-4H-chromene-3-carbonitrile Derivatives Loaded on Gold Nanoparticles Against Human Breast Cancer Cell Line T47D.

    Science.gov (United States)

    Saffari, Zahra; Zarabi, Maryam Farahnak; Aryapour, Hasan; Foroumadi, Alireza; Farhangi, Ali; Ghassemi, Soheil; Akbarzadeh, Azim

    2015-04-01

    Chemotherapy drugs, used for prevention of uncontrolled cell proliferation in certain tissues as well as inducing apoptosis in tumor cells, are important candidates for treatment of cancer. The synthesized 2-amino-4H-chromene-3-carbonitrile derivatives effective on cancerous cells resistant to other drugs such as Paclitaxel were used due to their ability in induction of apoptosis. The growth inhibitory and inducing apoptosis activities were determined. In order to make it target-oriented, the best compound was conjugated with gold nanoparticles (NPs) by aspartic acid with chemical reduction method. Cytotoxicity effect of 2-amino-4H-chromene-3-carbonitrile derivatives against the T47D breast cancer cell line was determined by MTT assay. The synthesis of gold NPs was confirmed by transmission electron microscopy, UV-Vis and dynamic light scattering. To assess the effects of compounds on the process of apoptosis, staining methods with acridine orange-ethidium bromide and Hoechst staining by fluorescence microscopy and DNA fragmentation by the diphenylamine method were used. The synthesized compounds containing two NH2 groups on benzene rings, demonstrated more cytotoxicity effect. The effect of conjugation with gold NPs and the induction of apoptosis were studied with the best compound. The cytotoxicity effects of the synthesized 2-amino-4H-chromene-3-carbonitrile compounds were changed by replacement of NO2 group on thiol ring with different chemical groups on the benzene ring. Analyses of treated cell lines by conjugated and non-conjugated forms of compounds verified their ability in inducing apoptosis while conjugated form demonstrated higher apoptosis.

  17. Comparative cytotoxicity of fumonisin B1 in two cell lines derived from normal human bronchial epithelial cells using four distinct bioassay techniques.

    Science.gov (United States)

    Lewis, C; Smith, J; Anderson, J; Freshney, R

    1999-06-01

    This study focuses on the cytotoxic effects of fumonisin B1 (FB1) on both immortalised and immortalised and subsequently transfected normal human bronchial epithelial (NHBE) cells of human origin using four bioassays. While the MTT, Neutral Red and hexosaminidase colorimetric assays showed little difference between the toxic effects on the two related cell lines, the clonogenic assay, measuring cell survival and proliferation, indicated that FB1 had a more toxic effect on the nontransfected cells. This kind ofin vitro approach using cells which retain many characteristics of normal cell growth and differentiation can go some way to developing evaluation models for food safety in the case of mycotoxin contamination without resorting totally to whole animal testing. Nevertheless, one or two cytotoxicity tests may be inadequate for a complete appraisal of toxic potential: rather, as wide a range of methodologies as feasible should be employed initially before meaningful conclusions may be drawn.

  18. Proteomic investigation of embryonic rat heart-derived H9c2 cell line sheds new light on the molecular phenotype of the popular cell model.

    Science.gov (United States)

    Lenčo, Juraj; Lenčová-Popelová, Olga; Link, Marek; Jirkovská, Anna; Tambor, Vojtěch; Potůčková, Eliška; Stulík, Jiří; Šimůnek, Tomáš; Štěrba, Martin

    2015-12-10

    Due to their cardiac origin, H9c2 cells rank among the most popular cell lines in current cardiovascular research, yet molecular phenotype remains elusive. Hence, in this study we used proteomic approach to describe molecular phenotype of H9c2 cells in their undifferentiated (i.e., most frequently used) state, and its functional response to cardiotoxic drug doxorubicin. Of 1671 proteins identified by iTRAQ IEF/LC-MSMS analysis, only 12 proteins were characteristic for striated muscle cells and none was cardiac phenotype-specific. Targeted LC-SRM and western blot analyses confirmed that undifferentiated H9c2 cells are phenotypically considerably different to both primary neonatal cardiomyocytes and adult myocardium. These cells lack proteins essential for formation of striated muscle myofibrils or they express only minor amounts thereof. They also fail to express many proteins important for metabolism of muscle cells. The challenge with clinically relevant concentrations of doxorubicin did not induce a proteomic signature that has been previously noted in primary cardiomyocytes or adult hearts. Instead, several alterations previously described in other cells of mesodermal origin, such as fibroblasts, were observed (e.g., severe down-regulation of collagen synthesis pathway). In conclusion, the molecular phenotype of H9c2 cells resembles very immature myogenic cells with skeletal muscle commitment upon differentiation and thus, translatability of findings obtained in these cells deserves caution. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. The high-risk HPV E6 target scribble (hScrib is required for HPV E6 expression in cervical tumour-derived cell lines

    Directory of Open Access Journals (Sweden)

    Christian Kranjec

    2016-12-01

    Full Text Available The ability of high-risk HPV E6 oncoproteins to target cellular proteins which harbor PDZ domains is believed to play an important role in the virus life cycle and to influence the ability of these viruses to bring about malignant transformation. Whilst many of these PDZ proteins are potential tumour suppressors, involved in the control of cell polarity and cell-contact, recent studies suggest that mislocalisation or overexpression might result in the emergence of oncogenic functions. This has been shown most clearly for two E6 targets, hDlg and hScrib. In this study we show that hScrib plays such a role in HeLa cells, where its expression is required for maintaining high levels of HPV-18 E6 protein. Loss of hScrib has no effect on E6 stability but results in lower levels of E6 transcription and a reduced rate of E6 translation. We further show that, in the context of cervical tumour-derived cell lines, both hScrib and E6 cooperate in the activation of the S6 kinase signaling pathway, and thereby contribute towards maintaining high rates of protein translation. These results indicate that the residual hScrib that is present within HPV transformed cells is pro-oncogenic, and highlights the dual functions of E6 cell polarity targets. Keywords: HPV E6, hScrib, S6 kinase, Protein translation

  20. Differential growth of U and M type infectious haematopoietic necrosis virus in a rainbow trout–derived cell line, RTG-2

    Science.gov (United States)

    Kurath, Gael; Purcell, Maureen K.; Wargo, Andrew; Park, Jeong Woo; Moon, Chang Hoon

    2010-01-01

    Infectious haematopoietic necrosis virus (IHNV) is one of the most important viral pathogens of salmonids. In rainbow trout, IHNV isolates in the M genogroup are highly pathogenic, while U genogroup isolates are significantly less pathogenic. We show here that, at a multiplicity of infection (MOI) of 1, a representative U type strain yielded 42-fold less infectious virus than an M type strain in the rainbow trout–derived RTG-2 cell line at 24 h post-infection (p.i.). However, at an MOI of 10, there was only fivefold difference in the yield of infectious virus between the U and M strains. Quantification of extracellular viral genomic RNA suggested that the number of virus particles released from cells infected with the U strain at a MOI of 1 was 47-fold lower than from M-infected cells, but U and M virions were equally infectious by particle to infectivity ratios. At an MOI of 1, U strain intracellular viral genome accumulation and transcription were 37- and 12-fold lower, respectively, than those of the M strain at 24 h p.i. Viral nucleocapsid (N) protein accumulation in U strain infections was fivefold lower than in M strain infections. These results suggest that the block in U type strain growth in RTG-2 cells was because of the effects of reduced genome replication and transcription. The reduced growth of the U strain does not seem to be caused by defective genes, because the U and M strains grew equally well in the permissive epithelioma papulosum cyprini cell line at an MOI of 1. This suggests that host-specific factors in RTG-2 cells control the growth of the IHNV U and M strains differently, leading to growth restriction of the U type virus during the RNA synthesis step.

  1. BHD Tumor Cell Line and Renal Cell Carcinoma Line | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    Scientists at the National Cancer Institute  have developed a novel renal cell carcinoma (RCC) cell line designated UOK257, which was derived from the surgical kidney tissue of a patient with hereditary Birt-Hogg-Dube''''(BHD) syndrome and companion cell line UOK257-2 in which FLCN expression has been restored by lentivirus infection. The NCI Urologic Oncology Branch seeks parties interested in licensing or collaborative research to co-develop, evaluate, or commercialize kidney cancer tumor cell lines.

  2. Radiosensitivity of mesothelioma cell lines

    International Nuclear Information System (INIS)

    Haekkinen, A.M.; Laasonen, A.; Linnainmaa, K.; Mattson, K.; Pyrhoenen, S.

    1996-01-01

    The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters α and β of the linear quadratic model (LQ-model) and mean inactivation dose (D MID ) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean α value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The α/β ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.)

  3. Generation of spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cell line SCA3.A11

    DEFF Research Database (Denmark)

    Hansen, Susanne Kofoed; Borland, Helena; Hasholt, Lis Frydenreich

    2016-01-01

    , SOX2, KLF4, OCT4 and short hairpin RNA targeting P53. The resulting iPSCs had normal karyotype, were free of genomically integrated episomal plasmids, expressed pluripotency markers, could differentiate into the three germ layers in vitro and retained the disease-causing ATXN3 mutation. This iPSC line...

  4. CLO : The cell line ontology

    NARCIS (Netherlands)

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

    2014-01-01

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

  5. Characterization of a new small cell lung cancer (SCLC cell line STP54 derived from a metastatic bioptate of a combined type of SCLC with Non-SCLC component.

    Directory of Open Access Journals (Sweden)

    Joanna Domagała-Kulawik

    2009-05-01

    Full Text Available Small cell lung cancer constitutes 15-20% cases of lung cancers, currently the leading cause of death from malignant diseases. It also causes the demise of >90% of affected individuals in 5 years. We have established a new SCLC cell line STP54 derived from fine needle aspirate of metastatic supraclavicular lymph node of 54 -year-old women for model experiments. The primary tumor was diagnosed by histopathological examination as combined type of small cell lung cancer with a non-small cell component. We cultured the cancer cells in the RPMI 1640 medium. In the long-term culture only the small cell component survived. The cell line was established after 30 passages and then characterized by performing cell morphology, cell growth analysis, tumorigenicity in vitro and flow cytometry analysis of selected markers (like NCAM, cytokeratines, HLA-ABC, Fas, Bcl-2, p53, CXCR4, CD210. The cells were growing in floating aggregates and show features suggesting its invasiveness. We suggest that this new cell line may serve as a valuable tool for further studies on lung tumor biology, molecular pathogenesis and metastatic mechanism.

  6. Generation of spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cell line SCA3.B11

    DEFF Research Database (Denmark)

    Hansen, Susanne Kofoed; Borland, Helena; Hasholt, Lis Frydenreich

    2016-01-01

    -MYC, LIN28, SOX2, KLF4, OCT4 and short hairpin RNA targeting P53. The resulting iPSCs had normal karyotype, were free of integrated episomal plasmids, expressed pluripotency markers, could differentiate into the three germ layers in vitro and retained the disease-causing ATXN3 mutation. Potentially, this i......PSC line could be a useful tool for the investigation of SCA3 disease mechanisms....

  7. The Effects of Low-Dose Bisphenol A and Bisphenol F on Neural Differentiation of a Fetal Brain-Derived Neural Progenitor Cell Line.

    Science.gov (United States)

    Fujiwara, Yuki; Miyazaki, Wataru; Koibuchi, Noriyuki; Katoh, Takahiko

    2018-01-01

    Environmental chemicals are known to disrupt the endocrine system in humans and to have adverse effects on several organs including the developing brain. Recent studies indicate that exposure to environmental chemicals during gestation can interfere with neuronal differentiation, subsequently affecting normal brain development in newborns. Xenoestrogen, bisphenol A (BPA), which is widely used in plastic products, is one such chemical. Adverse effects of exposure to BPA during pre- and postnatal periods include the disruption of brain function. However, the effect of BPA on neural differentiation remains unclear. In this study, we explored the effects of BPA or bisphenol F (BPF), an alternative compound for BPA, on neural differentiation using ReNcell, a human fetus-derived neural progenitor cell line. Maintenance in growth factor-free medium initiated the differentiation of ReNcell to neuronal cells including neurons, astrocytes, and oligodendrocytes. We exposed the cells to BPA or BPF for 3 days from the period of initiation and performed real-time PCR for neural markers such as β III-tubulin and glial fibrillary acidic protein (GFAP), and Olig2. The β III-tubulin mRNA level decreased in response to BPA, but not BPF, exposure. We also observed that the number of β III-tubulin-positive cells in the BPA-exposed group was less than that of the control group. On the other hand, there were no changes in the MAP2 mRNA level. These results indicate that BPA disrupts neural differentiation in human-derived neural progenitor cells, potentially disrupting brain development.

  8. The Effects of Low-Dose Bisphenol A and Bisphenol F on Neural Differentiation of a Fetal Brain-Derived Neural Progenitor Cell Line

    Directory of Open Access Journals (Sweden)

    Yuki Fujiwara

    2018-02-01

    Full Text Available Environmental chemicals are known to disrupt the endocrine system in humans and to have adverse effects on several organs including the developing brain. Recent studies indicate that exposure to environmental chemicals during gestation can interfere with neuronal differentiation, subsequently affecting normal brain development in newborns. Xenoestrogen, bisphenol A (BPA, which is widely used in plastic products, is one such chemical. Adverse effects of exposure to BPA during pre- and postnatal periods include the disruption of brain function. However, the effect of BPA on neural differentiation remains unclear. In this study, we explored the effects of BPA or bisphenol F (BPF, an alternative compound for BPA, on neural differentiation using ReNcell, a human fetus-derived neural progenitor cell line. Maintenance in growth factor-free medium initiated the differentiation of ReNcell to neuronal cells including neurons, astrocytes, and oligodendrocytes. We exposed the cells to BPA or BPF for 3 days from the period of initiation and performed real-time PCR for neural markers such as β III-tubulin and glial fibrillary acidic protein (GFAP, and Olig2. The β III-tubulin mRNA level decreased in response to BPA, but not BPF, exposure. We also observed that the number of β III-tubulin-positive cells in the BPA-exposed group was less than that of the control group. On the other hand, there were no changes in the MAP2 mRNA level. These results indicate that BPA disrupts neural differentiation in human-derived neural progenitor cells, potentially disrupting brain development.

  9. Graphene oxide and reduced graphene oxide induced neural pheochromocytoma-derived PC12 cell lines apoptosis and cell cycle alterations via the ERK signaling pathways.

    Science.gov (United States)

    Kang, Yiyuan; Liu, Jia; Wu, Junrong; Yin, Qian; Liang, Huimin; Chen, Aijie; Shao, Longquan

    2017-01-01

    Given the novel applications of graphene materials in biomedical and electronics industry, the health hazards of these particles have attracted extensive worldwide attention. Although many studies have been performed on graphene material-induced toxic effects, toxicological data for the effect of graphene materials on the nervous system are lacking. In this study, we focused on the biological effects of graphene oxide (GO) and reduced graphene oxide (rGO) materials on PC12 cells, a type of traditional neural cell line. We found that GO and rGO exerted significant toxic effects on PC12 cells in a dose- and time-dependent manner. Moreover, apoptosis appeared to be a response to toxicity. A potent increase in the number of PC12 cells at G0/G1 phase after GO and rGO exposure was detected by cell cycle analysis. We found that phosphorylation levels of ERK signaling molecules, which are related to cell cycle regulation and apoptosis, were significantly altered after GO and rGO exposure. In conclusion, our results show that GO has more potent toxic effects than rGO and that apoptosis and cell cycle arrest are the main toxicity responses to GO and rGO treatments, which are likely due to ERK pathway regulation.

  10. Tissue Banks and Cell Lines Derived from Patients with a High Risk of Breast Cancer and in situ Disease

    National Research Council Canada - National Science Library

    Gusterson, Barry

    1998-01-01

    .... These cells are from patients who are mutation carriers of BRCA1, BRCA2, and p53. Samples have been received from 46 women, five of which have a Li-Fraumeni - phenotype, with confirmed mutations...

  11. Exposure to nickel, chromium, or cadmium causes distinct changes in the gene expression patterns of a rat liver derived cell line.

    Directory of Open Access Journals (Sweden)

    Matthew G Permenter

    Full Text Available Many heavy metals, including nickel (Ni, cadmium (Cd, and chromium (Cr are toxic industrial chemicals with an exposure risk in both occupational and environmental settings that may cause harmful outcomes. While these substances are known to produce adverse health effects leading to disease or health problems, the detailed mechanisms remain unclear. To elucidate the processes involved in the toxicity of nickel, cadmium, and chromium at the molecular level and to perform a comparative analysis, H4-II-E-C3 rat liver-derived cell lines were treated with soluble salts of each metal using concentrations derived from viability assays, and gene expression patterns were determined with DNA microarrays. We identified both common and unique biological responses to exposure to the three metals. Nickel, cadmium, chromium all induced oxidative stress with both similar and unique genes and pathways responding to this stress. Although all three metals are known to be genotoxic, evidence for DNA damage in our study only exists in response to chromium. Nickel induced a hypoxic response as well as inducing genes involved in chromatin structure, perhaps by replacing iron in key proteins. Cadmium distinctly perturbed genes related to endoplasmic reticulum stress and invoked the unfolded protein response leading to apoptosis. With these studies, we have completed the first gene expression comparative analysis of nickel, cadmium, and chromium in H4-II-E-C3 cells.

  12. Human omental adipose-derived mesenchymal stem cell-conditioned medium alters the proteomic profile of epithelial ovarian cancer cell lines in vitro

    Directory of Open Access Journals (Sweden)

    Zhang YL

    2017-03-01

    Full Text Available Yanling Zhang,1,* Weihong Dong,1,* Junjie Wang,2 Jing Cai,1 Zehua Wang1 1Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 2Department of Obstetrics and Gynecology, Renhe Hospital, China Three Gorges University, Yichang, People’s Republic of China *These authors contributed equally to this work Abstract: Mesenchymal stem cells (MSCs have been reported to participate in the formation of supportive tumor stroma. The abilities of proliferation and invasion of human epithelial ovarian cancer (EOC cells were significantly enhanced when indirectly cocultured with human omental adipose-derived MSCs (O-ADSCs in vitro. However, the underlying mechanisms remain poorly understood. In this study, EOC cells were cultured with conditioned medium (CM from O-ADSCs (O-ADSC, and the effect of O-ADSC CM on the proteomic profile of EOC cells was assessed by two-dimensional gel electrophoresis (2-DE, followed by liquid chromatography and tandem mass spectrometry. The 2-DE assays revealed a global increase in protein expression in the EOC cells treated with CM. Nine proteins were identified from 11 selected protein spots with differential expression after treatment with CM from O-ADSCs. All the nine proteins have been linked to carcinoma and apoptosis, and the migration ability of tumor cells can be regulated by these proteins. Moreover, the upregulation of prohibitin and serine/arginine-rich splicing factor 1 in EOC cells treated with CM was further confirmed by quantitative real-time polymerase chain reaction. These results suggest that O-ADSCs affect the proteomic profile of EOC cells via paracrine mechanism in favor of EOC progression. Keywords: ovarian cancer, mesenchymal stromal cells, mesenchymal stem cells, omentum, proteomic

  13. Efficient Transduction of Feline Neural Progenitor Cells for Delivery of Glial Cell Line-Derived Neurotrophic Factor Using a Feline Immunodeficiency Virus-Based Lentiviral Construct

    Directory of Open Access Journals (Sweden)

    X. Joann You

    2011-01-01

    Full Text Available Work has shown that stem cell transplantation can rescue or replace neurons in models of retinal degenerative disease. Neural progenitor cells (NPCs modified to overexpress neurotrophic factors are one means of providing sustained delivery of therapeutic gene products in vivo. To develop a nonrodent animal model of this therapeutic strategy, we previously derived NPCs from the fetal cat brain (cNPCs. Here we use bicistronic feline lentiviral vectors to transduce cNPCs with glial cell-derived neurotrophic factor (GDNF together with a GFP reporter gene. Transduction efficacy is assessed, together with transgene expression level and stability during induction of cellular differentiation, together with the influence of GDNF transduction on growth and gene expression profile. We show that GDNF overexpressing cNPCs expand in vitro, coexpress GFP, and secrete high levels of GDNF protein—before and after differentiation—all qualities advantageous for use as a cell-based approach in feline models of neural degenerative disease.

  14. Establishment and characterization of two human breast carcinoma cell lines by spontaneous immortalization: Discordance between Estrogen, Progesterone and HER2/neu receptors of breast carcinoma tissues with derived cell lines

    Directory of Open Access Journals (Sweden)

    Kamalidehghan Behnam

    2012-10-01

    Full Text Available Abstract Background Breast cancer is one of the most common cancers among women throughout the world. Therefore, established cell lines are widely used as in vitro experimental models in cancer research. Methods Two continuous human breast cell lines, designated MBC1 and MBC2, were successfully established and characterized from invasive ductal breast carcinoma tissues of Malaysian patients. MBC1 and MBC2 have been characterized in terms of morphology analysis, population doubling time, clonogenic formation, wound healing assay, invasion assay, cell cycle, DNA profiling, fluorescence immunocytochemistry, Western blotting and karyotyping. Results MBC1 and MBC2 exhibited adherent monolayer epithelial morphology at a passage number of 150. Receptor status of MBC1 and MBC2 show (ER+, PR+, HER2+ and (ER+, PR-, HER2+, respectively. These results are in discordance with histopathological studies of the tumoral tissues, which were triple negative and (ER-, PR-, HER2+ for MBC1 and MBC2, respectively. Both cell lines were capable of growing in soft agar culture, which suggests their metastatic potential. The MBC1 and MBC2 metaphase spreads showed an abnormal karyotype, including hyperdiploidy and complex rearrangements with modes of 52–58 chromosomes per cell. Conclusions Loss or gain in secondary properties, deregulation and specific genetic changes possibly conferred receptor changes during the culturing of tumoral cells. Thus, we hypothesize that, among heterogenous tumoral cells, only a small minority of ER+/PR+/HER2+ and ER+/PR-/HER2+ cells with lower energy metabolism might survive and adjust easily to in vitro conditions. These cell lines will pave the way for new perspectives in genetic and biological investigations, drug resistance and chemotherapy studies, and would serve as prototype models in Malaysian breast carcinogenesis investigations.

  15. Newly-derived neuroblastoma cell lines propagated in serum-free media recapitulate the genotype and phenotype of primary neuroblastoma tumours

    NARCIS (Netherlands)

    Bate-Eya, Laurel T.; Ebus, Marli E.; Koster, Jan; den Hartog, Ilona J. M.; Zwijnenburg, Danny A.; Schild, Linda; van der Ploeg, Ida; Dolman, M. Emmy M.; Caron, Huib N.; Versteeg, Rogier; Molenaar, Jan J.

    2014-01-01

    Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of

  16. Pigment epithelium-derived factor (PEDF) regulates metabolism and insulin secretion from a clonal rat pancreatic beta cell line BRIN-BD11 and mouse islets.

    Science.gov (United States)

    Chen, Younan; Carlessi, Rodrigo; Walz, Nikita; Cruzat, Vinicius Fernandes; Keane, Kevin; John, Abraham N; Jiang, Fang-Xu; Carnagarin, Revathy; Dass, Crispin R; Newsholme, Philip

    2016-05-05

    Pigment epithelium-derived factor (PEDF) is a multifunctional glycoprotein, associated with lipid catabolism and insulin resistance. In the present study, PEDF increased chronic and acute insulin secretion in a clonal rat β-cell line BRIN-BD11, without alteration of glucose consumption. PEDF also stimulated insulin secretion from primary mouse islets. Seahorse flux analysis demonstrated that PEDF did not change mitochondrial respiration and glycolytic function. The cytosolic presence of the putative PEDF receptor - adipose triglyceride lipase (ATGL) - was identified, and ATGL associated stimulation of glycerol release was robustly enhanced by PEDF, while intracellular ATP levels increased. Addition of palmitate or ex vivo stimulation with inflammatory mediators induced β-cell dysfunction, effects not altered by the addition of PEDF. In conclusion, PEDF increased insulin secretion in BRIN-BD11 and islet cells, but had no impact on glucose metabolism. Thus elevated lipolysis and enhanced fatty acid availability may impact insulin secretion following PEDF receptor (ATGL) stimulation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  17. Cloning of fusion protein gene of Newcastle disease virus into a baculovirus derived bacmid shuttle vector, in order to express it in insect cell line

    Directory of Open Access Journals (Sweden)

    Hashemzadeh MS

    2015-05-01

    Full Text Available Abstract Background: Newcastle disease virus (NDV is one of the major pathogens in poultry and vaccination is intended to control the disease, as an effective solution, yet. Fusion protein (F on surface of NDV, has a fundamental role in virus pathogenicity and can induce protective immunity, alone. With this background, here our aim was to construct a baculovirus derived recombinant bacmid shuttle vector (encoding F-protein in order to express it in insect cell line. Materials and Methods: In this experimental study, at first complete F gene from avirulent strain La Sota of NDV was amplified by RT-PCR to produce F cDNA. The amplicon was cloned into T/A cloning vector and afterwards into pFastBac Dual donor plasmid. After the verification of cloning process by two methods, PCR and enzymatic digestion analysis, the accuracy of F gene sequence was confirmed by sequencing. Finally, F-containing recombinant bacmid was subsequently generated in DH10Bac cell and the construct production was confirmed by a special PCR panel, using F specific primers and M13 universal primers. Results: Analysis of confirmatory tests showed that the recombinant bacmid, expressing of F-protein gene in correct sequence and framework, has been constructed successfully. Conclusion: The product of this F-containing recombinant bacmid, in addition to its independent application in the induction of protective immunity, can be used with the other individual recombinant baculoviruses, expressing HN and NP genes to produce NDV-VLPs in insect cell line.

  18. Measuring and modeling of binary mixture effects of pharmaceuticals and nickel on cell viability/cytotoxicity in the human hepatoma derived cell line HepG2

    International Nuclear Information System (INIS)

    Rudzok, S.; Schlink, U.; Herbarth, O.; Bauer, M.

    2010-01-01

    The interaction of drugs and non-therapeutic xenobiotics constitutes a central role in human health risk assessment. Still, available data are rare. Two different models have been established to predict mixture toxicity from single dose data, namely, the concentration addition (CA) and independent action (IA) model. However, chemicals can also act synergistic or antagonistic or in dose level deviation, or in a dose ratio dependent deviation. In the present study we used the MIXTOX model (EU project ENV4-CT97-0507), which incorporates these algorithms, to assess effects of the binary mixtures in the human hepatoma cell line HepG2. These cells possess a liver-like enzyme pattern and a variety of xenobiotic-metabolizing enzymes (phases I and II). We tested binary mixtures of the metal nickel, the anti-inflammatory drug diclofenac, and the antibiotic agent irgasan and compared the experimental data to the mathematical models. Cell viability was determined by three different methods the MTT-, AlamarBlue (registered) and NRU assay. The compounds were tested separately and in combinations. We could show that the metal nickel is the dominant component in the mixture, affecting an antagonism at low-dose levels and a synergism at high-dose levels in combination with diclofenac or irgasan, when using the NRU and the AlamarBlue assay. The dose-response surface of irgasan and diclofenac indicated a concentration addition. The experimental data could be described by the algorithms with a regression of up to 90%, revealing the HepG2 cell line and the MIXTOX model as valuable tool for risk assessment of binary mixtures for cytotoxic endpoints. However the model failed to predict a specific mode of action, the CYP1A1 enzyme activity.

  19. Tetraethylammonium block of water flux in Aquaporin-1 channels expressed in kidney thin limbs of Henle's loop and a kidney-derived cell line.

    Directory of Open Access Journals (Sweden)

    Pannabecker Thomas L

    2002-03-01

    Full Text Available Abstract Background Aquaporin-1 (AQP1 channels are constitutively active water channels that allow rapid transmembrane osmotic water flux, and also serve as cyclic-GMP-gated ion channels. Tetraethylammonium chloride (TEA; 0.05 to 10 mM was shown previously to inhibit the osmotic water permeability of human AQP1 channels expressed in Xenopus oocytes. The purpose of the present study was to determine if TEA blocks osmotic water flux of native AQP1 channels in kidney, and recombinant AQP1 channels expressed in a kidney derived MDCK cell line. We also demonstrate that TEA does not inhibit the cGMP-dependent ionic conductance of AQP1 expressed in oocytes, supporting the idea that water and ion fluxes involve pharmacologically distinct pathways in the AQP1 tetrameric complex. Results TEA blocked water permeability of AQP1 channels in kidney and kidney-derived cells, demonstrating this effect is not limited to the oocyte expression system. Equivalent inhibition is seen in MDCK cells with viral-mediated AQP1 expression, and in rat renal descending thin limbs of Henle's loops which abundantly express native AQP1, but not in ascending thin limbs which do not express AQP1. External TEA (10 mM does not block the cGMP-dependent AQP1 ionic conductance, measured by two-electrode voltage clamp after pre-incubation of oocytes in 8Br-cGMP (10–50 mM or during application of the nitric oxide donor, sodium nitroprusside (2–4 mM. Conclusions TEA selectively inhibits osmotic water permeability through native and heterologously expressed AQP1 channels. The pathways for water and ions in AQP1 differ in pharmacological sensitivity to TEA, and are consistent with the idea of independent solute pathways within the channel structure. The results confirm the usefulness of TEA as a pharmacological tool for the analysis of AQP1 function.

  20. Factors affecting the sensitivity of human-derived esophageal carcinoma cell lines to 5-fluorouracil and cisplatin

    Science.gov (United States)

    MINEGAKI, TETSUYA; TAKARA, KOHJI; HAMAGUCHI, RYOHEI; TSUJIMOTO, MASAYUKI; NISHIGUCHI, KOHSHI

    2013-01-01

    Effective chemotherapy against esophageal carcinoma is considered achievable with a combination of 5-fluorouracil (5-FU) and cisplatin (CDDP). However, chemo-therapy remains ineffective in certain patients. The aim of this study was to clarify the factors which affect sensitivity to 5-FU and CDDP. The effects of factors known to influence sensitivity to 5-FU and CDDP, namely transporters, DNA repair enzymes and metabolic enzymes, were examined. mRNA levels of four transporters, SLC22A2, SLC23A2, ABCB1 and ABCC2, two DNA repair-related enzymes, Rad51 and MSH2, and one metabolic enzyme, dihydropyrimidine dehydrogenase (DPYD), showed a strong correlation (|r|>0.7) with IC50 values for 5-FU. In addition, the mRNA levels of ABCC2, MSH2 and DPYD showed a strong correlation (|r|>0.7) with the IC50 values for CDDP. Gimeracil, a DPYD inhibitor, enhanced the sensitivity of some cells to 5-FU but decreased the sensitivity of all the cells to CDDP. The inhibitory effects of ABCC2 with MK571 did not correspond to those observed in the correlation analysis. In conclusion, mRNA levels of SLC22A2, SLC23A2, ABCB1, ABCC2, Rad51, MSH2 and DPYD were confirmed to be strongly correlated with IC50 values for 5-FU, and mRNA levels of ABCC2, MSH2 and DPYD were confirmed to be strongly correlated with IC50 values for CDDP. In addition, the inhibition of DPYD appeared to affect the cytotoxicity of CDDP. PMID:23420099

  1. Inhibition of Mevalonate Pathway and Synthesis of the Storage Lipids in Human Liver-Derived and Non-liver Cell Lines by Lippia alba Essential Oils.

    Science.gov (United States)

    Montero-Villegas, Sandra; Polo, Mónica; Galle, Marianela; Rodenak-Kladniew, Boris; Castro, María; Ves-Losada, Ana; Crespo, Rosana; García de Bravo, Margarita

    2017-01-01

    The essential oils (EOs) of Lippia alba, an herb extensively used as a folk medicine in Latin America, are today promoted as an effective means of eliminating problems caused by hyperlipemia. We hypothesized that L.alba EOs inhibited cholesterol and triacylglycerols synthesis and decreased the intracellular depots of those lipids (lipid droplets), mechanisms involving the induction of a hypolipidemic response. Our aim was, therefore, to evaluate the hypolipogenic capability of the EOs of four L. alba chemotypes on liver-derived (HepG2) and non-liver (A549) human cell lines and to identify the potential biochemical targets of those chemotypes, particularly within the mevalonate pathway (MP). [ 14 C]Acetate was used as radioactive precursor for assays. Lipid analyses were performed by thin-layer and capillary gas chromatography, lipid droplets analyzed by fluorescence microscopy, and HMGCR levels determined by Western blot. In both cell lines, all four chemotypes exerted hypocholesterogenic effects within a concentration range of 3.2-32 µg/mL. Nonsaponifiable lipids manifested a decrease in incorporation of [ 14 C]acetate into squalene, lanosterol, lathosterol, and cholesterol, but not into ubiquinone, thus suggesting an inhibition of enzymes in the MP downstream from farnesyl pyrophosphate. The tagetenone chemotype, the most efficacious hypocholesterogenic L. alba EO, lowered HMGCR protein levels; inhibited triacylglycerols, cholesteryl esters, and phospholipids synthesis; and diminished lipid droplets in size and volume. These results revealed that L. alba EOs inhibited different lipogenic pathways and such lipid-lowering effects could prove essential to prevent cardiovascular diseases.

  2. Radiosensitivity of mesothelioma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Haekkinen, A.M. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland); Laasonen, A. [Dept. of Pathology, Central Hospital of Etelae-Pohjanmaa, Seinaejoki (Finland); Linnainmaa, K. [Dept. of Industrial Hygiene and Toxicology, Inst. of Occupational Health, Helsinki (Finland); Mattson, K. [Dept. Pulmonary Medicine, Univ. Central Hospital, Helsinki (Finland); Pyrhoenen, S. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland)

    1996-10-01

    The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters {alpha} and {beta} of the linear quadratic model (LQ-model) and mean inactivation dose (D{sub MID}) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean {alpha} value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The {alpha}/{beta} ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.).

  3. Effects of gamma-rays and glucose analogs on the energy metabolism of a cell line derived from human cerebral glioma

    International Nuclear Information System (INIS)

    Dwarakanath, B.S.

    1991-01-01

    Effects of gamma-rays and glucose analogs, 2-deoxy-D-glucose (2-DG), 5-thio-D-glucose (5-TG) and 3-O-methyl glucose (3-O-MG) on cellular energy metabolism have been studied in a cell line, derived from a human cerebral glioma, by analysing intermediates of glycolysis and some important nucleotides (ATP, NAD etc.) using the technique of isotachophoresis. Gamma-irradiation induced a transient decrease in the nucleotide levels accompanied by an accumulation of sugar phosphates, the nucleotide levels recovering in a few hours post-irradiation. 2-DG inhibited glycolysis and reduced the nucleotide levels of irradiated as well as unirradiated cells in a concentration-dependent manner both in presence and absence of respiration, whereas 5-TG and 3-O M G did not show significant effects in the presence of respiration. Reduced energy status observed with 2-DG under respiratory proficient conditions was completely reversed in 2 hr following its removal, whereas such a recovery was not observed in the absence of respiration. These results have important implications in the energy-linked modifications of tumor radiation response using glucose analogs. (author). 36 refs., 6 figs., 4 tabs

  4. Role of glial cell line-derived neurotrophic factor (GDNF)-neural cell adhesion molecule (NCAM) interactions in induction of neurite outgrowth and identification of a binding site for NCAM in the heel region of GDNF

    DEFF Research Database (Denmark)

    Nielsen, Janne; Gotfryd, Kamil; Li, Shizhong

    2009-01-01

    The formation of appropriate neuronal circuits is an essential part of nervous system development and relies heavily on the outgrowth of axons and dendrites and their guidance to their respective targets. This process is governed by a large array of molecules, including glial cell line......-derived neurotrophic factor (GDNF) and the neural cell adhesion molecule (NCAM), the interaction of which induce neurite outgrowth. In the present study the requirements for NCAM-mediated GDNF-induced neurite outgrowth were investigated in cultures of hippocampal neurons, which do not express Ret. We demonstrate...

  5. Biophysical Profiling of Tumor Cell Lines

    Directory of Open Access Journals (Sweden)

    Frederick Coffman

    2011-01-01

    Full Text Available Despite significant differences in genetic profiles, cancer cells share common phenotypic properties, including membrane-associated changes that facilitate invasion and metastasis. The Corning Epic® optical biosensor was used to monitor dynamic mass rearrangements within and proximal to the cell membrane in tumor cell lines derived from cancers of the colon, bone, cervix, lung and breast. Data was collected in real time and required no exogenously added signaling moiety (signal-free technology. Cell lines displayed unique profiles over the time-courses: the time-courses all displayed initial signal increases to maximal values, but the rate of increase to those maxima and the value of those maxima were distinct for each cell line. The rate of decline following the maxima also differed among cell lines. There were correlations between the signal maxima and the observed metastatic behavior of the cells in xenograft experiments; for most cell types the cells that were more highly metastatic in mice had lower time-course maxima values, however the reverse was seen in breast cancer cells. The unique profiles of these cell lines and the correlation of at least one profile characteristic with metastatic behavior demonstrate the potential utility of biophysical tumor cell profiling in the study of cancer biology.

  6. Efficient cDNA cloning by direct phenotypic correction of a mutant human cell line (HPRT-) using an Epstein-Barr virus derived cDNA expression vector.

    NARCIS (Netherlands)

    P.B.G.M. Belt; W. Jongmans; J. de Wit (Jan); J.H.J. Hoeijmakers (Jan); C.M.P. Backendorf (Claude); P. van de Putte (Pieter)

    1991-01-01

    textabstractHuman cells are, in general, poor recipients of foreign DNA, which has severely hampered the cloning of genes by direct phenotypic correction of deficient human cell lines after DNA mediated gene transfer. In this communication a methodology is presented which largely circumvents this

  7. Effects of Environmental Enrichment on Nicotine Sensitization in Rats Neonatally Treated with Quinpirole: Analyses of Glial Cell Line-Derived Neurotrophic Factor and Implications towards Schizophrenia.

    Science.gov (United States)

    Brown, Russell W; Schlitt, Marjorie A; Owens, Alex S; DePreter, Caitlynn C; Cummins, Elizabeth D; Kirby, Seth L; Gill, W Drew; Burgess, Katherine C

    2018-01-01

    The current study analyzed the effects of environmental enrichment versus isolation housing on the behavioral sensitization to nicotine in the neonatal quinpirole (NQ; dopamine D2-like agonist) model of dopamine D2 receptor supersensitivity, a rodent model of schizophrenia. NQ treatment in rats increases dopamine D2 receptor sensitivity throughout the animal's lifetime, consistent with schizophrenia. Animals were administered NQ (1 mg/kg) or saline (NS) from postnatal day (P)1 to P21, weaned, and immediately placed into enriched housing or isolated in wire cages throughout the experiment. Rats were behaviorally sensitized to nicotine (0.5 mg/kg base) or saline every consecutive day from P38 to P45, and brain tissue was harvested at P46. Results revealed that neither housing condition reduced nicotine sensitization in NQ rats, whereas enrichment reduced sensitization to nicotine in NS-treated animals. The nucleus accumbens (NAcc) was analyzed for glial cell line-derived neurotrophic factor (GDNF), a neurotrophin important in dopamine plasticity. Results were complex, and revealed that NAcc GDNF was increased in animals given nicotine, regardless of housing condition. Further, enrichment increased GDNF in NQ rats regardless of adolescent drug treatment and in NS-treated rats given nicotine, but did not increase GDNF in NS-treated controls compared to the isolated housing condition. This study demonstrates that environmental experience has a prominent impact on the behavioral and the neural plasticity NAcc response to nicotine in adolescence. © 2018 S. Karger AG, Basel.

  8. Bisphenol A and Its Derivatives Induce Degradation of HIF-1alpha via the Lysosomal Pathway in Human Hepatocarcinoma Cell Line, Hep3B.

    Science.gov (United States)

    Kobayashi, Yukino; Oguro, Ami; Imaoka, Susumu

    2018-01-01

    Bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)propane), one of the phenolic compounds widely used in the manufacture of plastic and epoxy resins, is known as an endocrine disruptor. In a previous study, we found that BPA induced hypoxia inducible factor-1alpha (HIF-1alpha) degradation by dissociation from heat shock protein 90 (Hsp90). In this study, to investigate the structural requirements for degradation of HIF-1alpha, we estimated the effect of BPA derivatives (BPE, BPF, BPB, Dimethyl butylidene diphenol (DMBDP), Ethyl hexylidene diphenol (EHDP), Bishydroxyphenyl cyclohexane (BHCH), and Methyl benzylidene bisphenol (MBBP)) on HIF-1alpha protein degradation, using human hepatocarcinoma cell line, Hep3B. BPB, DMBDP, BHCH, and MBBP decreased HIF-1alpha protein levels more efficiently than BPA, but BPE, BPF, and EHDP did not affect HIF-1alpha protein levels. BPA degraded HIF-1alpha even in the presence of MG132, a proteasome inhibitor. In this study, we found that ammonium chloride (NH 4 Cl), a lysosomal enzyme inhibitor, efficiently restored the decrease in HIF-1alpha protein levels by BPA. Recent studies indicated that HIF-1alpha is degraded by the lysosomal pathway as well as the proteasomal pathway. Therefore, we investigated the levels of heat shock cognate 70 kDa protein (HSC70) protein after treatment with BPA. We found that BPA induced HSC70 protein and overexpression of HSC70 enhanced HIF-1alpha degradation in Hep3B cells. These results suggested that BPA causes the degradation of HIF-1alpha by induction of HSC70, leading lysosomal degradation of HIF-1alpha.

  9. Extracellular calcium (Ca2+(o))-sensing receptor in a murine bone marrow-derived stromal cell line (ST2): potential mediator of the actions of Ca2+(o) on the function of ST2 cells

    Science.gov (United States)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+(o)) homeostasis by mediating the actions of Ca2+(o) on parathyroid gland and kidney. Bone marrow stromal cells support the formation of osteoclasts from their progenitors as well as the growth of hematopoietic stem cells by secreting humoral factors and through cell to cell contact. Stromal cells also have the capacity to differentiate into bone-forming osteoblasts. Bone resorption by osteoclasts probably produces substantial local increases in Ca2+(o) that could provide a signal for stromal cells in the immediate vicinity, leading us to determine whether such stromal cells express the CaR. In this study, we used the murine bone marrow-derived, stromal cell line, ST2. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in ST2 cells. We also identified CaR transcripts in ST2 cells by Northern analysis using a CaR-specific probe and by RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of ST2 cells to high Ca2+(o) (4.8 mM) or to the polycationic CaR agonists, neomycin (300 microM) or gadolinium (100 microM), stimulated both chemotaxis and DNA synthesis in ST2 cells. Therefore, taken together, our data strongly suggest that the bone marrow-derived stromal cell line, ST2, possesses both CaR protein and messenger RNA that are very similar if not identical to those in parathyroid and kidney. Furthermore, as ST2 cells have the potential to differentiate into osteoblasts, the CaR in stromal cells could participate in bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local, osteoclast-mediated release of Ca2+(o) and, thereafter, initiating bone formation after their differentiation into osteoblasts.

  10. SRS06, a new semisynthetic andrographolide derivative with improved anticancer potency and selectivity, inhibits nuclear factor-κB nuclear binding in the A549 non-small cell lung cancer cell line.

    Science.gov (United States)

    Lim, Jonathan Chee Woei; Jeyaraj, Ethel Jeyaseela; Sagineedu, Sreenivasa Rao; Wong, Wai Shiu Fred; Stanslas, Johnson

    2015-01-01

    Andrographolide has been reported with anticancer and anti-inflammatory properties through the inhibition of the activity of signaling molecules such as v-Src, nuclear factor-κB (NF-κB), STAT3, and PI3K. NF-κB has been proven to promote cancer cell survival, and targeting this pathway will halt the growth of cancer cells. Efforts have been made to produce semisynthetic derivatives of andrographolide with improved anticancer potency and selectivity. Subsequently, the effect of a selected derivative, 3,14,19-tripropionylandrographolide (SRS06), was tested for its action against NF-κB. Screening against 60 US National Cancer Institute (NCI) human cancer cell lines representing leukemia and non-small cell lung (NSCL), colon, CNS, melanoma, ovarian, renal, prostate, and breast cancers was performed to determine the tumor type selectivity and potency of SRS06. Microculture tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and sulforhodamine B assays were used to determine the in vitro anticancer activity, while Western blot studies were performed to ascertain the inhibitory effect of SRS06 on the NF-κB signaling cascade. The TransAM™ p65 assay kit was used to determine NF-κB p65 DNA binding activity in the NSCL cancer cell line A549. From the NCI screening, SRS06 was found to exhibit potent growth-inhibitory effects on multiple cancer cell lines with 10-fold lower 50% growth inhibition (GI50) compared with andrographolide. It was also discerned that the compound preferentially targeted melanoma, CNS, renal, colon, ovarian, prostate, and NSCL cancer cell lines. The DNA fragmentation assay indicated that the main mode of cell death of SRS06-treated A549 cells was via apoptosis. At 5 µmol/l the compound decreased NF-κB protein expression and caused a significant reduction in the nuclear p65 DNA binding activity. SRS06 displayed improved anticancer selectivity and potency when compared with andrographolide. We alluded its anticancer

  11. Inhibition of nuclear factor-kappa B enhances the tumor growth of ovarian cancer cell line derived from a low-grade papillary serous carcinoma in p53-independent pathway

    International Nuclear Information System (INIS)

    Xiao, Xue; Yang, Gong; Bai, Peng; Gui, Shunping; Nyuyen, Tri M. Bui; Mercado-Uribe, Imelda; Yang, Mei; Zou, Juan; Li, Qintong; Xiao, Jianguo; Chang, Bin; Liu, Guangzhi; Wang, He; Liu, Jinsong

    2016-01-01

    NF-kB can function as an oncogene or tumor suppressor depending on cancer types. The role of NF-kB in low-grade serous ovarian cancer, however, has never been tested. We sought to elucidate the function of NF-kB in the low-grade serous ovarian cancer. The ovarian cancer cell line, HOC-7, derived from a low-grade papillary serous carcinoma. Introduction of a dominant negative mutant, IkBαM, which resulted in decrease of NF-kB function in ovarian cancer cell lines. The transcription ability, tumorigenesis, cell proliferation and apoptosis were observed in derivative cell lines in comparison with parental cells. Western blot analysis indicated increased expression of the anti-apoptotic proteins Bcl-xL and reduced expression of the pro-apoptotic proteins Bax, Bad, and Bid in HOC-7/IĸBαM cell. Further investigations validate this conclusion in KRAS wildtype cell line SKOV3. Interesting, NF-kB can exert its pro-apoptotic effect by activating mitogen-activated protein kinase (MAPK) phosphorylation in SKOV3 ovarian cancer cell, whereas opposite changes detected in p-MEK in HOC-7 ovarian cancer cell, the same as some chemoresistant ovarian cancer cell lines. In vivo animal assay performed on BALB/athymic mice showed that injection of HOC-7 induced subcutaneous tumor growth, which was completely regressed within 7 weeks. In comparison, HOC-7/IĸBαM cells caused sustained tumor growth and abrogated tumor regression, suggesting that knock-down of NF-kB by IĸBαM promoted sustained tumor growth and delayed tumor regression in HOC-7 cells. Our results demonstrated that NF-kB may function as a tumor suppressor by facilitating regression of low grade ovarian serous carcinoma through activating pro-apoptotic pathways

  12. Inhibition of nuclear factor-kappa B enhances the tumor growth of ovarian cancer cell line derived from a low-grade papillary serous carcinoma in p53-independent pathway.

    Science.gov (United States)

    Xiao, Xue; Yang, Gong; Bai, Peng; Gui, Shunping; Nyuyen, Tri M Bui; Mercado-Uribe, Imelda; Yang, Mei; Zou, Juan; Li, Qintong; Xiao, Jianguo; Chang, Bin; Liu, Guangzhi; Wang, He; Liu, Jinsong

    2016-08-02

    NF-kB can function as an oncogene or tumor suppressor depending on cancer types. The role of NF-kB in low-grade serous ovarian cancer, however, has never been tested. We sought to elucidate the function of NF-kB in the low-grade serous ovarian cancer. The ovarian cancer cell line, HOC-7, derived from a low-grade papillary serous carcinoma. Introduction of a dominant negative mutant, IkBαM, which resulted in decrease of NF-kB function in ovarian cancer cell lines. The transcription ability, tumorigenesis, cell proliferation and apoptosis were observed in derivative cell lines in comparison with parental cells. Western blot analysis indicated increased expression of the anti-apoptotic proteins Bcl-xL and reduced expression of the pro-apoptotic proteins Bax, Bad, and Bid in HOC-7/IĸBαM cell. Further investigations validate this conclusion in KRAS wildtype cell line SKOV3. Interesting, NF-kB can exert its pro-apoptotic effect by activating mitogen-activated protein kinase (MAPK) phosphorylation in SKOV3 ovarian cancer cell, whereas opposite changes detected in p-MEK in HOC-7 ovarian cancer cell, the same as some chemoresistant ovarian cancer cell lines. In vivo animal assay performed on BALB/athymic mice showed that injection of HOC-7 induced subcutaneous tumor growth, which was completely regressed within 7 weeks. In comparison, HOC-7/IĸBαM cells caused sustained tumor growth and abrogated tumor regression, suggesting that knock-down of NF-kB by IĸBαM promoted sustained tumor growth and delayed tumor regression in HOC-7 cells. Our results demonstrated that NF-kB may function as a tumor suppressor by facilitating regression of low grade ovarian serous carcinoma through activating pro-apoptotic pathways.

  13. Tumor-derived hepatocyte growth factor is associated with poor prognosis of patients with glioma and influences the chemosensitivity of glioma cell line to cisplatin in vitro

    Directory of Open Access Journals (Sweden)

    Guo You-feng

    2012-06-01

    Full Text Available Abstract Background We examined the association of tumor-derived hepatocyte growth factor (HGF with the clinicopathological features of gliomas and investigated the effect of HGF inhibition on the biological behavior of tumor cells in vitro in order to determine whether HGF is a valuable prognostic predictor for glioma patients. Methods Seventy-six cases of glioma were collected. The tumor-derived HGF expression, cell proliferation index (PI and intratumoral microvessels were evaluated by immunohistochemistry. Correlation between immunostaining and clinicopathological parameters, as well as the follow-up data of patients, was analyzed statistically. U87MG glioma cells were transfected with short interference (si-RNA for HGF, and the cell viability, migratory ability and chemosensitivity to cisplatin were evaluated in vitro. Results Both high HGF expression in tumor cells (59.2%, 45/76 and high PI were significantly associated with high-grade glioma and increased microvessels in tumors (P P = 0.004 and high-expression of HGF (P = 0.008 emerged as independent prognostic factors for the overall survival of glioma patients. The tumor-derived HGF mRNA and protein expressions were significantly decreased in vitro after transfection of HGF siRNA. HGF siRNA inhibited the cell growth and reduced cell migratory ability. Moreover, HGF siRNA transfection enhanced the chemosensitivity of U87MG glioma cells to cisplatin. Conclusion This study indicated that there was significant correlation among tumor cell-derived HGF, cell proliferation and microvessel proliferation in gliomas. HGF might influence tumor progression by modulating the cell growth, migration and chemoresistance to drugs. Increased expression of HGF may be a valuable predictor for prognostic evaluation of glioma patients.

  14. Validation of an in vitro cytotoxicity test for four heavy metals using cell lines derived from a green sea turtle (Chelonia mydas).

    Science.gov (United States)

    Tan, Fengxia; Wang, Min; Wang, Weimin; Alonso Aguirre, A; Lu, Yuanan

    2010-06-01

    Ten cell lines established from juvenile green sea turtles were tested and evaluated for their cytotoxic responses to four heavy metals: cadmium (Cd), chromium (Cr), zinc (Zn), and copper (Cu). Following a 24-h exposure to these metal salts at selected concentrations, test cells were comparatively characterized by morphology, viability, and proliferation. Experimental results indicated that all these metal salts were cytotoxic to these turtle cell lines at varied concentrations. Calculated 10% and 50% inhibitory concentration (IC(10) and IC(50)) values revealed that the cytotoxicities of Cd and Cr were significantly more potent than the other two metal salts (p metal contamination and may be used for initial environmental monitoring, while the highly tolerant nature of GT-LV and GT-EYE cells to the tested heavy metals suggest their potential use as an emergency last-resort indicator of potential metal-related adverse effect on human health.

  15. Establishment, growth and in vivo differentiation of a new clonal human cell line, EM-G3, derived from breast cancer progenitors

    Czech Academy of Sciences Publication Activity Database

    Brožová, Markéta; Kleibl, Z.; Netíková, I.; Ševčík, J.; Scholzová, E.; Březinová, J.; Chaloupková, Alena; Veselý, Pavel; Dundr, P.; Zadinová, M.; Krásná, Luboslava; Matoušková, Eva

    2007-01-01

    Roč. 103, č. 2 (2007), s. 247-257 ISSN 0167-6806 R&D Projects: GA MZd NR8145; GA MZd(CZ) NR8345 Grant - others:Univerzita Karlova(CZ) 93/2005/C Institutional research plan: CEZ:AV0Z50520514 Keywords : breast cancer * cell line * progenitor cells * differentiation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.453, year: 2007

  16. Association of ABCC2 and CDDP-Resistance in Two Sublines Resistant to CDDP Derived from a Human Nasopharyngeal Carcinoma Cell Line

    Directory of Open Access Journals (Sweden)

    Si Ming Xie

    2010-01-01

    Full Text Available Cisplatin (CDDP is one of the most active drugs to treat nasopharyngeal carcinoma (NPC patients. To further understand the mechanisms of CDDP-resistance in NPC, two CDDP-resistant sublines (CNE2-CDDP and CNE2-CDDP-5Fu derived from parental NPC cell line CNE2 were established. It was found that at the IC50 level, the resistance of CNE2-CDDP and CNE2-CDDP-5Fu against CDDP was 2.63-fold and 5.35-fold stronger than that of parental CNE2, respectively. Of the four ABC transporters (ABCB1, ABCC1, ABCC2 and ABCG2 related to MDR, only ABCC2 was found to be elevated both in CDDP-resistant sublines, with ABCC2 located in nucleus of CNE2-CDDP-5Fu but not in CNE2-CDDP and parental CNE2. Further research showed that compared to untreated CNE2, the intracellular levels of CDDP were decreased by 2.03-fold in CNE2-CDDP and 2.78-fold in CNE2-CDDP-5Fu. After treatment with PSC833, a modulator of MDR associated transporters including ABCC2, the intracellular level of CDDP was increased in CDDP-resistant sublines, and the resistance to CDDP was partially reversed from 2.63-fold to 1.62-fold in CNE2-CDDP and from 5.35-fold to 4.62-fold in CNE2-CDDP-5Fu. These data indicate that ABCC2 may play an important role in NPC resistant to CDDP.

  17. Clearance and toxicity of recombinant methionyl human glial cell line-derived neurotrophic factor (r-metHu GDNF) following acute convection-enhanced delivery into the striatum.

    Science.gov (United States)

    Taylor, Hannah; Barua, Neil; Bienemann, Alison; Wyatt, Marcella; Castrique, Emma; Foster, Rebecca; Luz, Matthias; Fibiger, Christian; Mohr, Erich; Gill, Steven

    2013-01-01

    Despite promising early results, clinical trials involving the continuous delivery of recombinant methionyl human glial cell line-derived neurotrophic factor (r-metHuGDNF) into the putamen for the treatment of Parkinson's disease have shown evidence of poor distribution and toxicity due to point-source accumulation. Convection-enhanced delivery (CED) has the potential to facilitate more widespread and clinically effective drug distribution. We investigated acute CED of r-metHuGDNF into the striatum of normal rats in order to assess tissue clearance, toxicity (neuron loss, gliosis, microglial activation, and decreases in synaptophysin), synaptogenesis and neurite-outgrowth. We investigated a range of clinically relevant infused concentrations (0.1, 0.2, 0.6 and 1.0 µg/µL) and time points (2 and 4 weeks) in order to rationalise a dosing regimen suitable for clinical translation. Two weeks after single dose CED, r-metHuGDNF was below the limit of detection by ELISA but detectable by immunohistochemistry when infused at low concentrations (0.1 and 0.2 µg/µL). At these concentrations, there was no associated neuronal loss (neuronal nuclei, NeuN, immunohistochemistry) or synaptic toxicity (synaptophysin ELISA). CED at an infused concentration of 0.2 µg/µL was associated with a significant increase in synaptogenesis (partificial cerebral spinal fluid (aCSF) control was observed with any infused concentration. The results of this study suggest that acute CED of low concentrations of GDNF, with dosing intervals determined by tissue clearance, has most potential for effective clinical translation by optimising distribution and minimising the risk of toxic accumulation.

  18. Diet-induced obesity has neuroprotective effects in murine gastric enteric nervous system: involvement of leptin and glial cell line-derived neurotrophic factor.

    Science.gov (United States)

    Baudry, Charlotte; Reichardt, François; Marchix, Justine; Bado, André; Schemann, Michael; des Varannes, Stanislas Bruley; Neunlist, Michel; Moriez, Raphaël

    2012-02-01

    Nutritional factors can induce profound neuroplastic changes in the enteric nervous system (ENS), responsible for changes in gastrointestinal (GI) motility. However, long-term effects of a nutritional imbalance leading to obesity, such as Western diet (WD), upon ENS phenotype and control of GI motility remain unknown. Therefore, we investigated the effects of WD-induced obesity (DIO) on ENS phenotype and function as well as factors involved in functional plasticity. Mice were fed with normal diet (ND) or WD for 12 weeks. GI motility was assessed in vivo and ex vivo. Myenteric neurons and glia were analysed with immunohistochemical methods using antibodies against Hu, neuronal nitric oxide synthase (nNOS), Sox-10 and with calcium imaging techniques. Leptin and glial cell line-derived neurotrophic factor (GDNF) were studied using immunohistochemical, biochemical or PCR methods in mice and primary culture of ENS. DIO prevented the age-associated decrease in antral nitrergic neurons observed in ND mice. Nerve stimulation evoked a stronger neuronal Ca(2+) response in WD compared to ND mice. DIO induced an NO-dependent increase in gastric emptying and neuromuscular transmission in the antrum without any change in small intestinal transit. During WD but not ND, a time-dependent increase in leptin and GDNF occurred in the antrum. Finally, we showed that leptin increased GDNF production in the ENS and induced neuroprotective effects mediated in part by GDNF. These results demonstrate that DIO induces neuroplastic changes in the antrum leading to an NO-dependent acceleration of gastric emptying. In addition, DIO induced neuroplasticity in the ENS is likely to involve leptin and GDNF.

  19. The Hexane Fraction of Bursera microphylla A Gray Induces p21-Mediated Antiproliferative and Proapoptotic Effects in Human Cancer–Derived Cell Lines

    Science.gov (United States)

    Adorisio, Sabrina; Fierabracci, Alessandra; Gigliarelli, Giulia; Muscari, Isabella; Cannarile, Lorenza; Liberati, Anna Marina; Marcotullio, Maria Carla; Riccardi, Carlo; Curini, Massimo; Robles Zepeda, Ramon Enrique; Delfino, Domenico V.

    2017-01-01

    Bursera microphylla (BM), one of the common elephant trees, is widely distributed in the Sonoran desert in Mexico. The Seri ethnic group in the Sonoran desert uses BM as an anti-inflammatory and painkiller drug for the treatment of sore throat, herpes labialis, abscessed tooth, and wound healing. Dried stems and leaves of BM are used in a tea to relieve painful urination and to stimulate bronchial secretion. Furthermore, BM is used for fighting venereal diseases. To investigate the effects of the hexane fraction of resin methanol extract (BM-H) on cell growth, the acute myeloid cell line (OCI-AML3) was treated with 250, 25, or 2.5 µg/mL of BM-H. The first 2 concentrations were able to significantly decrease OCI-AML3 cell number. This reduced cell number was associated with decreased S-phase, blockade of G2/M phase of the cell cycle, and increased cell death. Similar results were obtained on all tested tumor cell lines of different origins. We found that blockade of the cell cycle was a result of upregulation of p21 protein in a p53-independent way. Increase of p21 was possibly a result of upstream upregulation of p-ERK (which stabilizes p21 protein) and downregulation of p-38 (which promotes its degradation). Regarding cell death, activation of caspase-3, but not of caspase-8 or -9, was detectable after BM-H treatment. In conclusion, these data suggest that BM-H inhibited proliferation of cell lines mainly by a p21-dependent, p53-independent mechanism and promoted apoptosis through activation of caspase-3 but not caspase-8 or -9. PMID:28110563

  20. Inhibition of Oncogenic Transcription Factor REL by the Natural Product Derivative Calafianin Monomer 101 Induces Proliferation Arrest and Apoptosis in Human B-Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Alan T. Yeo

    2015-04-01

    Full Text Available Increased activity of transcription factor NF-κB has been implicated in many B-cell lymphomas. We investigated effects of synthetic compound calafianin monomer (CM101 on biochemical and biological properties of NF-κB. In human 293 cells, CM101 selectively inhibited DNA binding by overexpressed NF-κB subunits REL (human c-Rel and p65 as compared to NF-κB p50, and inhibition of REL and p65 DNA binding by CM101 required a conserved cysteine residue. CM101 also inhibited DNA binding by REL in human B-lymphoma cell lines, and the sensitivity of several B-lymphoma cell lines to CM101-induced proliferation arrest and apoptosis correlated with levels of cellular and nuclear REL. CM101 treatment induced both phosphorylation and decreased expression of anti-apoptotic protein Bcl-XL, a REL target gene product, in sensitive B-lymphoma cell lines. Ectopic expression of Bcl-XL protected SUDHL-2 B-lymphoma cells against CM101-induced apoptosis, and overexpression of a transforming mutant of REL decreased the sensitivity of BJAB B-lymphoma cells to CM101-induced apoptosis. Lipopolysaccharide-induced activation of NF-κB signaling upstream components occurred in RAW264.7 macrophages at CM101 concentrations that blocked NF-κB DNA binding. Direct inhibitors of REL may be useful for treating B-cell lymphomas in which REL is active, and may inhibit B-lymphoma cell growth at doses that do not affect some immune-related responses in normal cells.

  1. The Hexane Fraction of Bursera microphylla A. Gray Induces p21-Mediated Anti-Proliferative and Pro-Apoptotic Effects in Human Cancer-Derived Cell Lines.

    Science.gov (United States)

    Adorisio, Sabrina; Fierabracci, Alessandra; Gigliarelli, Giulia; Muscari, Isabella; Cannarile, Lorenza; Liberati, Anna Marina; Marcotullio, Maria Carla; Riccardi, Carlo; Curini, Massimo; Robles Zepeda, Ramon Enrique; Delfino, Domenico V

    2018-03-01

    Bursera microphylla (BM), one of the common elephant trees, is widely distributed in the Sonoran Desert in Mexico. The Seri ethnic group in the Sonoran Desert uses BM as an anti-inflammatory and painkiller drug for the treatment of sore throat, herpes labialis, abscessed tooth, and wound healing. Dried stems and leaves of BM are used in a tea to relieve painful urination and to stimulate bronchial secretion. Furthermore, BM is used for fighting venereal diseases. To investigate the effects of the hexane fraction of resin methanol extract (BM-H) on cell growth, the acute myeloid cell line (OCI-AML3) was treated with 250, 25, or 2.5 µg/mL of BM-H. The first 2 concentrations were able to significantly decrease OCI-AML3 cell number. This reduced cell number was associated with decreased S-phase, blockade of the G 2 /M phase of the cell cycle, and increased cell death. Similar results were obtained on all tested tumor cell lines of different origins. We found that blockade of the cell cycle was due to upregulation of p21 protein in a p53-independent way. Increase of p21 was possibly due to upstream upregulation of p-ERK (which stabilizes p21 protein) and downregulation of p-38 (which promotes its degradation). Regarding cell death, activation of caspase-3, but not of caspase-8 or -9, was detectable after BM-H treatment. In conclusion, these data suggest that the BM's hexane fraction inhibited proliferation of cell lines mainly by a p21-dependent, p53-independent mechanism and promoted apoptosis through activation of caspase-3, but not caspase-8 or -9.

  2. The Hexane Fraction of Bursera microphylla A Gray Induces p21-Mediated Antiproliferative and Proapoptotic Effects in Human Cancer-Derived Cell Lines.

    Science.gov (United States)

    Adorisio, Sabrina; Fierabracci, Alessandra; Gigliarelli, Giulia; Muscari, Isabella; Cannarile, Lorenza; Liberati, Anna Marina; Marcotullio, Maria Carla; Riccardi, Carlo; Curini, Massimo; Robles Zepeda, Ramon Enrique; Delfino, Domenico V

    2017-09-01

    Bursera microphylla (BM), one of the common elephant trees, is widely distributed in the Sonoran desert in Mexico. The Seri ethnic group in the Sonoran desert uses BM as an anti-inflammatory and painkiller drug for the treatment of sore throat, herpes labialis, abscessed tooth, and wound healing. Dried stems and leaves of BM are used in a tea to relieve painful urination and to stimulate bronchial secretion. Furthermore, BM is used for fighting venereal diseases. To investigate the effects of the hexane fraction of resin methanol extract (BM-H) on cell growth, the acute myeloid cell line (OCI-AML3) was treated with 250, 25, or 2.5 µg/mL of BM-H. The first 2 concentrations were able to significantly decrease OCI-AML3 cell number. This reduced cell number was associated with decreased S-phase, blockade of G 2 /M phase of the cell cycle, and increased cell death. Similar results were obtained on all tested tumor cell lines of different origins. We found that blockade of the cell cycle was a result of upregulation of p21 protein in a p53-independent way. Increase of p21 was possibly a result of upstream upregulation of p-ERK (which stabilizes p21 protein) and downregulation of p-38 (which promotes its degradation). Regarding cell death, activation of caspase-3, but not of caspase-8 or -9, was detectable after BM-H treatment. In conclusion, these data suggest that BM-H inhibited proliferation of cell lines mainly by a p21-dependent, p53-independent mechanism and promoted apoptosis through activation of caspase-3 but not caspase-8 or -9.

  3. Mutational analysis of the p16 family cyclin-dependent kinase inhibitors p15INK4b and p18INK4c in tumor-derived cell lines and primary tumors.

    Science.gov (United States)

    Zariwala, M; Liu, E; Xiong, Y

    1996-01-18

    The growth suppressing activity of the retinoblastoma suspectibility gene product, pRb, is down regulated by cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) whose kinase activity is negatively regulated by CDK inhibitors of the p16 family. We have examined the genomic status of two recently isolated p16-related CDK inhibitors, p15 and p18, in 15 normal and 73 tumor-derived cell lines established from 23 different tissues, as well as 26 invasive primary breast cancers and 20 acute myelogenous leukemias. p15 was found to be homozygously deleted in 22% of the tumor derived cell lines, but no point mutations were found in either the cultured cells or the two types of primary tumors. With the exception of one breast cancer cell line, no deletions or mutations were found in the p18 gene in either cultured cell lines or primary tumors. These results indicate that mutation of the p18 gene occurs rarely in human tumors. Thus, while they share a very similar biochemical mechanism of inhibiting the kinase activity of CDK4 and CDK6, members of the p16 gene family play different roles in controlling cell proliferation and suppressing tumor growth.

  4. In vitro radiosensitivity of human leukemia cell lines

    International Nuclear Information System (INIS)

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

    1981-01-01

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

  5. Inhibition spectrum studies of microthecin and other anhydrofructose derivatives using selected strains of Gram-positive and –negative bacteria, yeast and moulds, and investigation of the cytotoxicity of microthecin to malignant blood cell lines

    DEFF Research Database (Denmark)

    Fiskesund, R.; Thomas, L.V.; Schobert, M.

    2009-01-01

    Aims: To prepare 1,5-anhydro-d-fructose (AF) derivatives, test their microbial inhibition spectrum, and to further examine the most effective AF derivative against Pseudomonas aeruginosa and malignant blood cell lines. Methods and Results: Microthecin and nine other AF derivatives were synthesized...... from AF. The 10 compounds were tested in vitro against Gram-positive (GP) and Gram-negative (GN) bacteria, yeasts and moulds using a well diffusion method and in a Bioscreen growth analyser. Of the test compounds, microthecin exhibited the most significant antibacterial activity at 100–2000 ppm against...

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

    Science.gov (United States)

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

    2009-01-01

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

  7. A Natural Triterpene Derivative from Euphorbia kansui Inhibits Cell Proliferation and Induces Apoptosis against Rat Intestinal Epithelioid Cell Line in Vitro

    Directory of Open Access Journals (Sweden)

    Fangfang Cheng

    2015-08-01

    Full Text Available Kansenone is a triterpene from the root of the traditional Chinese medicine, Euphorbia kansui. However, kansenone exerts serious toxicity, but the exact mechanism was not clear. In this work, the effects of kansenone on cell proliferation, cell cycle, cell damage, and cell apoptosis were investigated. The suppression of cell proliferation was assessed via the colorimetric MTT assay, and cell morphology was visualized via inverted microscopy after IEC-6 cells were incubated with different concentrations of kansenone. Reactive oxygen species (ROS, superoxide dismutase (SOD and malondialdehyde (MDA content were detected for evaluating cell damage. RNase/propidium iodide (PI labeling for evaluation of cell cycle distribution was performed by flow cytometry analysis. Annexin V-fluorescein isothiocyanate (FITC/PI and Hoechst 33342/Annexin V-FITC/PI staining assay for cell apoptosis detection were performed using confocal laser scanning microscopy and high content screening. Moreover, apoptosis induction was further confirmed by transmission electron microscope (TEM and JC-1 mitochondrial membrane potential, western blot and RT-PCR analysis. The results demonstrated that kansenone exerted high cytotoxicity, induced cell arrest at G0/G1 phase, and caused mitochondria damage. In addition, kansenone could up-regulate the apoptotic proteins Bax, AIF, Apaf-1, cytochrome c, caspase-3, caspase-9, caspase-8, FasR, FasL, NF-κB, and TNFR1 mRNA expression levels, and down-regulate the anti-apoptotic Bcl-2 family proteins, revealing that kansenone induces apoptosis through both the death receptor and mitochondrial pathways.

  8. Expression of osteoprotegerin, receptor activator of nuclear factor kappa-B ligand, tumor necrosis factor-related apoptosis-inducing ligand, stromal cell-derived factor-1 and their receptors in epithelial metastatic breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Labovsky Vivian

    2012-06-01

    Full Text Available Abstract Background While breast cancer (BC is the major cause of death among women worldwide, there is no guarantee of better patient survival because many of these patients develop primarily metastases, despite efforts to detect it in its early stages. Bone metastasis is a common complication that occurs in 65-80 % of patients with disseminated disease, but the molecular basis underlying dormancy, dissemination and establishment of metastasis is not understood. Our objective has been to evaluate simultaneously osteoprotegerin (OPG, receptor activator of nuclear factor kappa B ligand (RANKL, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, stromal cell-derived factor-1 (SDF-1, and their receptors (R in 2 human BC cell lines, MDA-MB-231 and MCF-7. Methods OPG, RANKL, TRAIL and SDF-1 expression and release, in addition to the expression of their receptors has been investigated using immunofluorescence, immunocytochemistry and ELISA analyses. Results MCF-7 cells released higher levels of OPG in conditioned media (CM than MDA-MB-231 cells; 100 % of both types of cell expressed OPG, RANKL, TRAIL and SDF-1. Moreover, 100 % in both lines expressed membrane RANKL and RANK, whereas only 50 % expressed CXCR4. Furthermore, 100 % expressed TRAIL-R1 and R4, 30-50 % TRAIL-R2, and 40-55 % TRAIL-R3. Conclusions MCF-7 and MDA-MB-231 cells not only released OPG, but expressed RANKL, TRAIL and SDF-1. The majority of the cells also expressed RANK, CXCR4 and TRAIL-R. Since these ligands and their receptors are implicated in the regulation of proliferation, survival, migration and future bone metastasis during breast tumor progression, assessment of these molecules in tumor biopsies of BC patients could be useful in identifying patients with more aggressive tumors that are also at risk of bone metastasis, which may thus improve the available options for therapeutic intervention.

  9. Derivation, Characterization, and Neural Differentiation of Integration-Free Induced Pluripotent Stem Cell Lines from Parkinson's Disease Patients Carrying SNCA, LRRK2, PARK2, and GBA Mutations

    DEFF Research Database (Denmark)

    Momcilovic, Olga; Sivapatham, Renuka; Oron, Tal Ronnen

    2016-01-01

    We report generation of induced pluripotent stem cell (iPSC) lines from ten Parkinson's disease (PD) patients carrying SNCA, PARK2, LRRK2, and GBA mutations, and one age-matched control. After validation of pluripotency, long-term genome stability, and integration-free reprogramming, eight...... not be sufficient to determine the cause or mechanism of the disease, and highlights the need to use more focused strategies for large-scale data analysis........ We further examined gene expression in a stress model (MPTP-induced dopaminergic neuronal death) using two clones from the SNCA triplication line, and detected changes in genes associated with mitophagy. Our data suggested that even a well-characterized line of a monogenic disease may...

  10. Thyroid cell lines in research on goitrogenesis.

    Science.gov (United States)

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

    1991-12-01

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

  11. Derivation and characterisation of hESC lines from supernumerary embryos, experience from Odense, Denmark

    DEFF Research Database (Denmark)

    Harkness, Linda; Rasmussen, Iben Anne; Erb, Karin

    2010-01-01

    The derivation and characterisation of human embryonic stem cells provides a source of pluripotent stem cells with potential for clinical applications. Utilising locally sourced embryos from two IVF clinics, we derived and characterised five new cell lines for use in a non-clinical setting. Analy...

  12. Synthesis of (3S,4S,5S)-trihydroxylpiperidine derivatives as enzyme stabilizers to improve therapeutic enzyme activity in Fabry patient cell lines.

    Science.gov (United States)

    Li, Huang-Yi; Lee, Jay-Der; Chen, Chiao-Wen; Sun, Ying-Chieh; Cheng, Wei-Chieh

    2018-01-20

    A series of 3S,4S,5S-trihydroxylated piperidines bearing structural diversity at C-2 or C-6 positions has been synthesized and tested to determine their ability to stabilize the activity of recombinant human α-Galactosidase A (rh-α-Gal A). Hit molecules were identified by rapid inhibitory activity screening, and then further investigated for their ability to protect this enzyme from thermo-induced denaturation and enhance its activity in Fabry patient cell lines. Our study resulted in the identification of a new class of small molecules as enzyme stabilizers for the potential treatment of Fabry disease. Of these, stabilizer 21 was the most effective, showing a 12-fold increase in rh-α-Gal A activity in Fabry disease cell lines. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  13. Generation of a KLF15 homozygous knockout human embryonic stem cell line using paired CRISPR/Cas9n, and human cardiomyocytes derivation

    Directory of Open Access Journals (Sweden)

    Claudia Noack

    2017-08-01

    Full Text Available Krueppel-like factor 15 (KLF15 is abundantly expressed in liver, kidney, and muscle, including myocardium. In the adult heart KLF15 is important to maintain homeostasis and to repress hypertrophic remodeling. We generated a homozygous hESC KLF15 knockout (KO line using paired CRISPR/Cas9n. KLF15-KO cells maintained full pluripotency and differentiation potential as well as genomic integrity. We demonstrated that KLF15-KO cells can be differentiated into morphologically normal cardiomyocytes turning them into a valuable tool for studying human KLF15-mediated mechanisms resulting in human cardiac dysfunction.

  14. Influence of vitamin D on cisplatin sensitivity in testicular germ cell cancer-derived cell lines and in a NTera2 xenograft model

    DEFF Research Database (Denmark)

    Jørgensen, Anne; Blomberg Jensen, Martin; Nielsen, John Erik

    2013-01-01

    . Future studies are needed to investigate potential beneficial effects of vitamin D with lower cisplatin doses, and to determine whether 1,25(OH)(2)D(3) may increase cisplatin sensitivity in chemotherapy-resistant TGCTs. This article is part of a Special Issue entitled 'Vitamin D Workshop'.......The active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) has anti-proliferative, pro-apoptotic, and pro-differentiating effects in somatic cancer cells in vitro and in vivo. 1,25(OH)(2)D(3) also augments the anti-tumor effects of several chemotherapeutic agents, including...... cisplatin, which may have clinical relevance. Given the pro-differentiation effect of vitamin D recently demonstrated in testicular germ cell tumors (TGCTs), we hypothesized that 1,25(OH)(2)D(3) could be a beneficial adjunctive to existing chemotherapy regime used to treat these tumors. In this study, cell...

  15. Visual exploratory analysis of integrated chromosome 19 proteomic data derived from glioma cancer stem-cell lines based on novel nonlinear dimensional data reduction techniques

    Science.gov (United States)

    Lespinats, Sylvain; Pinker-Domenig, Katja; Meyer-Bäse, Uwe; Meyer-Bäse, Anke

    2015-05-01

    Chromosome 19 is known to be linked to neurodegeneration and many cancers. Glioma-derived cancer stem cells (GSCs) are tumor-initiating cells and may be refractory to radiation and chemotherapy and thus have important implications for tumor biology and therapeutics. The analysis and interpretation of large proteomic data sets requires the development of new data mining and visualization approaches. Traditional techniques are insufficient to interpret and visualize these resulting experimental data. The emphasis of this paper lies in the presentation of novel approaches for the visualization, clustering and projection representation to unveil hidden data structures relevant for the accurate interpretation of biological experiments. These qualitative and quantitative methods are applied to the proteomic analysis of data sets derived from the GSCs. The achieved clustering and visualization results provide a more detailed insight into the expression patterns for chromosome 19 proteins.

  16. Study of heat and radiation response of a malignant, melanin-producing cell line derived from C3H 10T1/2 cells transformed in culture by radiation

    Energy Technology Data Exchange (ETDEWEB)

    Raaphorst, G.P.; Vadasz, J.; Azzam, E.I.

    1986-12-01

    The mouse C3H 10T1/2 cell line was transformed to the malignant state using ionizing radiation. One of the transformed lines (R25) that was isolated, displayed some properties similar to malignant melanoma cells. The cells became dark and pigmented after prolonged time in culture and this cell line produced tumors in C3H mice. The radiation survival curve of R25 had a large shoulder which was also observed for human melanoma cell lines. R25 was more resistant to heating at 45.0 degrees C than the normal cell line. Heating at 45.0 degrees C before irradiation resulted in a reduction of the survival curve shoulder. The heat and radiation sensitivity of R25 did not appear to be related to the melanin content of these cells.

  17. Immune characterization of the Lewis rats inoculated with K2 sarcoma cell line and newly derived R5-28 malignant cells

    Czech Academy of Sciences Publication Activity Database

    Morávková, Alena; Málek, Ondřej; Pokorná, Eva; Strnádel, Ján; Hradecký, Jan; Horák, Vratislav

    2005-01-01

    Roč. 51, - (2005), s. 159-165 ISSN 0015-5500 R&D Projects: GA ČR GA524/04/0102 Grant - others:Ústav živočišné fyziologie a genetiky AV ČR(CZ) IAPG/05/16 Institutional research plan: CEZ:AV0Z50450515 Keywords : rat sarcoma * malignancy * myeloid cells Subject RIV: EC - Immunology Impact factor: 0.719, year: 2005

  18. Further studies on the intermediary metabolism of bone in vitro and a monoclonal cell line (MMB-1) derived from bone: effects of parathyroid hormone and acetazolamide

    International Nuclear Information System (INIS)

    Nichols, F.C.

    1981-01-01

    The mechanism/mechanisms by which PTH affects Ca homeostasis between blood and bone have not been clearly established. Most studies of metabolic acid production in bone took place during the late 1950s and early 1960s. Since that time, assay techniques for metabolic acid production have been improved for greater sensitivity, more has been learned as to how PTH mediates its cellular responses, and techniques for cell isolation and culture have dramatically improved. These improvements have made possible new approaches to the study of short term effects of PTH on metabolic acid production in bone. Chapter 1 explores the potential of bone to utilize substrates other than glucose for metabolic energy transfer. Chapter 2 characterizes glucose metabolism in mouse calvaria in vitro and explores effects of PTH, acetazolamide, and C1 13,850 on calvaria oxidative metabolism. Chapter 3 describes PTH effects on glucose metabolism of MMB-1 cells, a monoclonal cell line reported to possess osteoblast-like characteristics

  19. Characterization of arsenic trioxide resistant clones derived from Jurkat leukemia T cell line: focus on PI3K/Akt signaling pathway.

    Science.gov (United States)

    Roszak, Joanna; Smok-Pieniążek, Anna; Nocuń, Marek; Stępnik, Maciej

    2013-10-05

    In this study the role of PI3K/Akt signaling pathway in arsenic trioxide (ATO)-treated parental Jurkat cells and also in derived ATO-resistant clones grown in the presence of given ATO concentration was investigated. ATO-resistant clones (cultured for 8-12weeks in the presence of 1, 2.5 and 5μM ATO) were characterized by high viability in the presence of ATO but slower growth rate compared to the parental cells. Morphological and functional characterization of derived ATO-resistant clones revealed that they did not differ fundamentally from parental Jurkat cells in terms of cell size, level of GSH, the lysosomal fluorescence or CD95/Fas surface antigen expression. However, a slight increase in the mitochondrial potential (JC-1 staining) was detected in the clones compared to parental Jurkat cells. Side population analysis (Vybrant DyeCycle Violet™ staining) in ATO resistant clones did not indicate any enrichment withcancer stem cells. Akt1/2, AktV or wortmannin inhibitors decreased viability of ATO-resistant clones grown in the presence of ATO, with no effect on ATO-treated parental cells. Flow cytometry analysis showed that ATO decreased the level of p-Akt in ATO-treated parental cells, while the resistant clones exhibited higher levels of p-Akt immunostaining than parental Jurkat cells. Expression analysis of 84 genes involved in the PI3K/Akt pathway revealed that this pathway was predominantly active in ATO-resistant clones. c-JUN seems to play a key role in the induction of cell death in ATO-treated parental Jurkat cells, as dose-dependent strong up-regulation of JUN was specific for the ATO-treated parental Jurkat cells. On the other hand, changes in expression of cyclin D1 (CCND1), insulin receptor substrate 1 (IRS1) and protein kinase C isoforms (PRKCZ,PRKCB and PRKCA) may be responsible for the induction of resistance to ATO. The changes in expression of growth factor receptor-bound protein 10 (GRB10) observed in ATO-resistant clones suggest a

  20. Generation of GZKHQi001-A and GZWWTi001-A, two induced pluripotent stem cell lines derived from peripheral blood mononuclear cells of Duchenne muscular dystrophy patients

    Directory of Open Access Journals (Sweden)

    Xie Yuhuan

    2018-04-01

    Full Text Available Duchenne muscular dystrophy (DMD is an X-linked disease caused by mutations in the DMD gene, which spans ~2.4 Mb of genomic sequence at locus Xp21. This mutation results in the loss of the protein dystrophin. DMD patients die in their second or third decade due to either respiratory failure or cardiomyopathy, as the absence of dystrophin leads to myofiber membrane fragility and necrosis, eventually resulting in muscle atrophy and contractures. Currently, there is no effective treatment for DMD, therefore induced pluripotent stem cells from DMD patients would be a powerful tool for studying disease mechanisms.

  1. Epithelial-to-Mesenchymal Transition in Pancreatic Ductal Adenocarcinoma and Pancreatic Tumor Cell Lines: The Role of Neutrophils and Neutrophil-Derived Elastase

    Directory of Open Access Journals (Sweden)

    Thomas Große-Steffen

    2012-01-01

    Full Text Available Pancreatic ductal adenocarcinoma (PDAC is frequently associated with fibrosis and a prominent inflammatory infiltrate in the desmoplastic stroma. Moreover, in PDAC, an epithelial-to-mesenchymal transition (EMT is observed. To explore a possible connection between the infiltrating cells, particularly the polymorphonuclear neutrophils (PMN and the tumor cell transition, biopsies of patients with PDAC (n=115 were analysed with regard to PMN infiltration and nuclear expression of β-catenin and of ZEB1, well-established indicators of EMT. In biopsies with a dense PMN infiltrate, a nuclear accumulation of β-catenin and of ZEB1 was observed. To address the question whether PMN could induce EMT, they were isolated from healthy donors and were cocultivated with pancreatic tumor cells grown as monolayers. Rapid dyshesion of the tumor cells was seen, most likely due to an elastase-mediated degradation of E-cadherin. In parallel, the transcription factor TWIST was upregulated, β-catenin translocated into the nucleus, ZEB1 appeared in the nucleus, and keratins were downregulated. EMT was also induced when the tumor cells were grown under conditions preventing attachment to the culture plates. Here, also in the absence of elastase, E-cadherin was downmodulated. PMN as well as prevention of adhesion induced EMT also in liver cancer cell line. In conclusion, PMN via elastase induce EMT in vitro, most likely due to the loss of cell-to-cell contact. Because in pancreatic cancers the transition to a mesenchymal phenotype coincides with the PMN infiltrate, a contribution of the inflammatory response to the induction of EMT and—by implication—to tumor progression is possible.

  2. Cell line and patient-derived xenograft models reveal elevated CDCP1 as a target in high-grade serous ovarian cancer.

    Science.gov (United States)

    Harrington, Brittney S; He, Yaowu; Davies, Claire M; Wallace, Sarah J; Adams, Mark N; Beaven, Elizabeth A; Roche, Deborah K; Kennedy, Catherine; Chetty, Naven P; Crandon, Alexander J; Flatley, Christopher; Oliveira, Niara B; Shannon, Catherine M; deFazio, Anna; Tinker, Anna V; Gilks, C Blake; Gabrielli, Brian; Brennan, Donal J; Coward, Jermaine I; Armes, Jane E; Perrin, Lewis C; Hooper, John D

    2016-02-16

    Development of targeted therapies for high-grade serous ovarian cancer (HGSC) remains challenging, as contributing molecular pathways are poorly defined or expressed heterogeneously. CUB-domain containing protein 1 (CDCP1) is a cell-surface protein elevated in lung, colorectal, pancreas, renal and clear cell ovarian cancer. CUB-domain containing protein 1 was examined by immunohistochemistry in HGSC and fallopian tube. The impact of targeting CDCP1 on cell growth and migration in vitro, and intraperitoneal xenograft growth in mice was examined. Three patient-derived xenograft (PDX) mouse models were developed and characterised for CDCP1 expression. The effect of a monoclonal anti-CDCP1 antibody on PDX growth was examined. Src activation was assessed by western blot analysis. Elevated CDCP1 was observed in 77% of HGSC cases. Silencing of CDCP1 reduced migration and non-adherent cell growth in vitro and tumour burden in vivo. Expression of CDCP1 in patient samples was maintained in PDX models. Antibody blockade of CDCP1 significantly reduced growth of an HGSC PDX. The CDCP1-mediated activation of Src was observed in cultured cells and mouse xenografts. CUB-domain containing protein 1 is over-expressed by the majority of HGSCs. In vitro and mouse model data indicate that CDCP1 has a role in HGSC and that it can be targeted to inhibit progression of this cancer.

  3. Cell line and patient-derived xenograft models reveal elevated CDCP1 as a target in high-grade serous ovarian cancer

    Science.gov (United States)

    Harrington, Brittney S; He, Yaowu; Davies, Claire M; Wallace, Sarah J; Adams, Mark N; Beaven, Elizabeth A; Roche, Deborah K; Kennedy, Catherine; Chetty, Naven P; Crandon, Alexander J; Flatley, Christopher; Oliveira, Niara B; Shannon, Catherine M; deFazio, Anna; Tinker, Anna V; Gilks, C Blake; Gabrielli, Brian; Brennan, Donal J; Coward, Jermaine I; Armes, Jane E; Perrin, Lewis C; Hooper, John D

    2016-01-01

    Background: Development of targeted therapies for high-grade serous ovarian cancer (HGSC) remains challenging, as contributing molecular pathways are poorly defined or expressed heterogeneously. CUB-domain containing protein 1 (CDCP1) is a cell-surface protein elevated in lung, colorectal, pancreas, renal and clear cell ovarian cancer. Methods: CUB-domain containing protein 1 was examined by immunohistochemistry in HGSC and fallopian tube. The impact of targeting CDCP1 on cell growth and migration in vitro, and intraperitoneal xenograft growth in mice was examined. Three patient-derived xenograft (PDX) mouse models were developed and characterised for CDCP1 expression. The effect of a monoclonal anti-CDCP1 antibody on PDX growth was examined. Src activation was assessed by western blot analysis. Results: Elevated CDCP1 was observed in 77% of HGSC cases. Silencing of CDCP1 reduced migration and non-adherent cell growth in vitro and tumour burden in vivo. Expression of CDCP1 in patient samples was maintained in PDX models. Antibody blockade of CDCP1 significantly reduced growth of an HGSC PDX. The CDCP1-mediated activation of Src was observed in cultured cells and mouse xenografts. Conclusions: CUB-domain containing protein 1 is over-expressed by the majority of HGSCs. In vitro and mouse model data indicate that CDCP1 has a role in HGSC and that it can be targeted to inhibit progression of this cancer. PMID:26882065

  4. Induced mutant lines derived from irradiated mungbean varieties

    International Nuclear Information System (INIS)

    Sumanggono, A.M.R.

    1991-01-01

    The mungbean cultivars Manyar and Walet were irradiated with several doses of gamma rays and Nuri with fast neutrons. Selection for desired characters, such as synchronized maturity and more pods per plant than the control, were carried out in the M 2 generation. In the M 5 generation, about 164 mungbean mutant lines were selected. In 1988, a preliminary yield trial was carried out on 46 selected M 5 homogenous lines and, in 1989, an advanced yield trial on selected M 6 lines. From these observations, it was shown that some promising mutant lines had been recovered, i.e. four high yielding mutant lines derived from the gamma irradiation of Walet, three lines which showed synchronized maturity as well as larger pods and a greater number of seeds derived from the gamma irradiation of Manyar, and a high seed protein content in mutant lines derived from the fast neutron irradiation of Nuri. (author). 2 refs, 2 tabs

  5. Effect of tamoxifen and fulvestrant long-term treatments on ROS production and (pro/anti)-oxidant enzymes mRNA levels in a MCF-7-derived breast cancer cell line.

    Science.gov (United States)

    Badia, Eric; Morena, Marion; Lauret, Céline; Boulahtouf, Abdelhay; Boulle, Nathalie; Cavaillès, Vincent; Balaguer, Patrick; Cristol, Jean Paul

    2016-09-01

    Reactive oxygen species (ROS) are key players in the apoptotic effects induced by short-term tamoxifen treatment of breast cancer cells, but also in acquired resistance following long-term treatment. Whereas the use of the selective estrogen receptor down-regulator fulvestrant is promising, especially in patients who develop tamoxifen resistance, only few studies addressed its implication in the modulation of cellular redox status. The regulation of (pro/anti)-oxidant players were first investigated at the mRNA level in a MCF-7-derived cell line after short-term (24 h) estradiol treatment. Long-term anti-estrogen treated MCF-7 derived cell lines were also developed: 3 months of 4-hydroxytamoxifen alone (MCF7L-OHTLT) or followed by 3 months of fulvestrant (MCF7L-ICILT). Growth properties, hormone sensitivity, receptor content, ROS production and relative mRNA expression of pro or antioxidant enzymes were evaluated in these long-term treated cell lines. Short-term estradiol treatment showed a hormone sensitivity of Nox2, GPx1, GPx2 and SOD1 mRNA levels. The long-term fulvestrant treatment (3 months) of MCF7L-OHTLT led to a reduced level of ROS production accompanied with a drastic drop of the accessory protein p22(phox) mRNA. This ROS reduction, although not clearly related to antioxidant enzymes level, seems to be involved in fulvestrant sensitivity of long-term anti-estrogen treated cells, as suggested by the effects of antiradical tempol treatment. When compared to long-term 4-hydroxytamoxifen-treated breast cancer cells, addition of fulvestrant treatment was able to diminish ROS production and p22(phox) mRNA level, and made cells more sensitive to growth inhibition induced by tempol. These effects may be a valuable asset of the fulvestrant treatment.

  6. The radiosensitizing effect of a N(4)-tolyl-2-acetylpyridine derived thiosemicarbazone and its metallic complex against a glioblastoma cell line

    International Nuclear Information System (INIS)

    Vilas Boas, Fabricio A.S.; Hudson, Luiza O.; Santos, Raquel G. dos; Mendes, Isolda C.; Beraldo, Heloisa O.

    2009-01-01

    Cancer is one of the most prevalent and difficult diseases to be treated. Despite the efforts at improving diagnose and treatment, the success is still very limited. One of the factors implicated in such limitation is the inherent radioresistance of most tumors, specially the cerebral ones. They are poorly vascularized due to the rapid growth of cells and disorganized angiogenesis that leads to hypoxic tissue that increases radioresistance. Also another issue is the side effects of exposition to high levels of radiation and chemicals. Combined approaches using both chemo and radiotherapy are one of the most effective strategies applied to maximize the results and decrease the side effects of the treatment to the patient. One of the drugs that are commonly used is cisplatin that has some, yet limited result. Given this context, our group has been testing several synthetic compounds of the thiosemicarbazone class. These chemicals have broad pharmacologic profile including antitumoral effect. We have shown in previous works the effective reduction of cell viability and proliferation using very low concentrations of thiosemicarbazones both in free form and complexed with metals like copper. In this work we present another application of this compound that can also be used as a radiosensitization agent in glioblastoma multiform cell line RT-2 present that the combined approach increases de effect of gamma radiation. Also, that the coordination to copper apparently does not increase this activity. (author)

  7. Synthesis of 9,9-dialkyl-4,5-diazafluorene derivatives and their structure-activity relationships toward human carcinoma cell lines.

    Science.gov (United States)

    Wang, Qiwei; Yuen, Marcus Chun-Wah; Lu, Guo-Liang; Ho, Cheuk-Lam; Zhou, Gui-Jiang; Keung, Oi-Mei; Lam, Kim-Hung; Gambari, Roberto; Tao, Xiao-Ming; Wong, Raymond Siu-Ming; Tong, See-Wai; Chan, Kit-Wah; Lau, Fung-Yi; Cheung, Filly; Cheng, Gregory Yin-Ming; Chui, Chung-Hin; Wong, Wai-Yeung

    2010-04-06

    A homologous set of 9,9-dialkyl-4,5-diazafluorene compounds were prepared by alkylation of 4,5-diazafluorene with the appropriate alkyl bromide and under basic conditions. The structures of these simple organic compounds were confirmed by spectroscopic techniques (FTIR, NMR, and FABMS). Their biological effects toward a panel of human carcinoma cells, including Hep3B hepatocellular carcinoma, MDAMB-231 breast carcinoma, and SKHep-1 hepatoma cells, were investigated; a structure-activity correlation was established with respect to the length of the alkyl chain and the fluorene ring structure. The relationship between the mean potency [log(1/IC(50))] and alkyl chain length was systematically studied. The results show that compounds with butyl, hexyl, and octyl chains exhibit good growth inhibitory effects toward these three human carcinoma cell lines, and the 9,9-dihexyl-4,5-diazafluorene further exhibits antitumor activity in athymic nude mice Hep3B xenograft models. For the structurally related dialkylfluorenes that lack the diaza functionality, in vitro cytotoxicity was not observed at clinically relevant concentrations.

  8. In vitro anticancer activity of Betulinic acid and derivatives thereof on equine melanoma cell lines from grey horses and in vivo safety assessment of the compound NVX-207 in two horses.

    Science.gov (United States)

    Liebscher, G; Vanchangiri, K; Mueller, Th; Feige, K; Cavalleri, J-M V; Paschke, R

    2016-02-25

    Betulinic acid, a pentacyclic triterpene, and its derivatives are promising compounds for cancer treatment in humans. Melanoma is not only a problem for humans but also for grey horses as they have a high potential of developing melanoma lesions coupled to the mutation causing their phenotype. Current chemotherapeutic treatment carries the risk of adverse health effects for the horse owner or the treating veterinarian by exposure to antineoplastic compounds. Most treatments have low prospects for systemic tumor regression. Thus, a new therapy is needed. In this in vitro study, Betulinic acid and its two derivatives B10 and NVX-207, both with an improved water solubility compared to Betulinic acid, were tested on two equine melanoma cell lines (MelDuWi and MellJess/HoMelZh) and human melanoma (A375) cell line. We could demonstrate that all three compounds especially NVX-207 show high cytotoxicity on both equine melanoma cell lines. The treatment with these compounds lead to externalization of phosphatidylserines on the cell membrane (AnnexinV-staining), DNA-fragmentation (cell cycle analysis) and activation of initiator and effector caspases (Caspase assays). Our results indicate that the apoptosis is induced in the equine melanoma cells by all three compounds. Furthermore, we succeed in encapsulating the most active compound NVX-207 in 2-Hydroxyprolyl-β-cyclodextrine without a loss of its activity. This formulation can be used as a promising antitumor agent for treating grey horse melanoma. In a first tolerability evaluation in vivo the formulation was administered every one week for 19 consecutive weeks and well tolerated in two adult melanoma affected horses. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  9. Comparative analyses of volatile organic compounds (VOCs) from patients, tumors and transformed cell lines for the validation of lung cancer-derived breath markers.

    Science.gov (United States)

    Filipiak, Wojciech; Filipiak, Anna; Sponring, Andreas; Schmid, Thomas; Zelger, Bettina; Ager, Clemens; Klodzinska, Ewa; Denz, Hubert; Pizzini, Alex; Lucciarini, Paolo; Jamnig, Herbert; Troppmair, Jakob; Amann, Anton

    2014-06-01

    Breath analysis for the purpose of non-invasive diagnosis of lung cancer has yielded numerous candidate compounds with still questionable clinical relevance. To arrive at suitable volatile organic compounds our approach combined the analysis of different sources: isolated tumor samples compared to healthy lung tissues, and exhaled breath from lung cancer patients and healthy controls. Candidate compounds were further compared to substances previously identified in the comparison of transformed and normal lung epithelial cell lines. For human studies, a breath sampling device was developed enabling automated and CO2-controlled collection of the end-tidal air. All samples were first preconcentrated on multibed sorption tubes and analyzed with gas chromatography mass spectrometry (GC-MS). Significantly (p < 0.05) higher concentrations in all three types of cancer samples studied were observed for ethanol and n-octane. Additional metabolites (inter alia 2-methylpentane, n-hexane) significantly released by lung cancer cells were observed at higher levels in cancer lung tissues and breath samples (compared to respective healthy controls) with statistical significance (p < 0.05) only in breath samples. The results obtained confirmed the cancer-related origin of volatile metabolites, e.g. ethanol and octane that were both detected at significantly (p < 0.05) elevated concentrations in all three kinds of cancer samples studied. This work is an important step towards identification of volatile breath markers of lung cancer through the demonstration of cancer-related origin of certain volatile metabolites.

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

    Science.gov (United States)

    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.

  11. A One Line Derivation of EGARCH

    Directory of Open Access Journals (Sweden)

    Michael McAleer

    2014-06-01

    Full Text Available One of the most popular univariate asymmetric conditional volatility models is the exponential GARCH (or EGARCH specification. In addition to asymmetry, which captures the different effects on conditional volatility of positive and negative effects of equal magnitude, EGARCH can also accommodate leverage, which is the negative correlation between returns shocks and subsequent shocks to volatility. However, the statistical properties of the (quasi- maximum likelihood estimator of the EGARCH parameters are not available under general conditions, but rather only for special cases under highly restrictive and unverifiable conditions. It is often argued heuristically that the reason for the lack of general statistical properties arises from the presence in the model of an absolute value of a function of the parameters, which does not permit analytical derivatives, and hence does not permit (quasi- maximum likelihood estimation. It is shown in this paper for the non-leverage case that: (1 the EGARCH model can be derived from a random coefficient complex nonlinear moving average (RCCNMA process; and (2 the reason for the lack of statistical properties of the estimators of EGARCH under general conditions is that the stationarity and invertibility conditions for the RCCNMA process are not known.

  12. VOLIN and KJON-Two novel hyperdiploid myeloma cell lines.

    Science.gov (United States)

    Våtsveen, Thea Kristin; Børset, Magne; Dikic, Aida; Tian, Erming; Micci, Francesca; Lid, Ana H B; Meza-Zepeda, Leonardo A; Coward, Eivind; Waage, Anders; Sundan, Anders; Kuehl, W Michael; Holien, Toril

    2016-11-01

    Multiple myeloma can be divided into two distinct genetic subgroups: hyperdiploid (HRD) or nonhyperdiploid (NHRD) myeloma. Myeloma cell lines are important tools to study myeloma cell biology and are commonly used for preclinical screening and testing of new drugs. With few exceptions human myeloma cell lines are derived from NHRD patients, even though about half of the patients have HRD myeloma. Thus, there is a need for cell lines of HRD origin to enable more representative preclinical studies. Here, we present two novel myeloma cell lines, VOLIN and KJON. Both of them were derived from patients with HRD disease and shared the same genotype as their corresponding primary tumors. The cell lines' chromosomal content, genetic aberrations, gene expression, immunophenotype as well as some of their growth characteristics are described. Neither of the cell lines was found to harbor immunoglobulin heavy chain translocations. The VOLIN cell line was established from a bone marrow aspirate and KJON from peripheral blood. We propose that these unique cell lines may be used as tools to increase our understanding of myeloma cell biology. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Derivation of the Duchenne muscular dystrophy patient-derived induced pluripotent stem cell line lacking DMD exons 49 and 50 (CCMi001DMD-A-3, ∆49, ∆50).

    Science.gov (United States)

    Spaltro, Gabriella; Vigorelli, Vera; Casalnuovo, Federica; Spinelli, Pietro; Castiglioni, Elisa; Rovina, Davide; Paganini, Stefania; Di Segni, Marina; Nigro, Patrizia; Gervasini, Cristina; Pompilio, Giulio; Gowran, Aoife

    2017-12-01

    Duchenne muscular dystrophy (DMD) is caused by abnormalities in the dystrophin gene and is clinically characterised by childhood muscle degeneration and cardiomyopathy. We produced an induced pluripotent stem cell line from a DMD patient's dermal fibroblasts by electroporation with episomal vectors containing: hL-MYC, hLIN28, hSOX2, hKLF4, hOCT3/4. The resultant DMD iPSC line (CCMi001DMD-A-3) displayed iPSC morphology, expressed pluripotency markers, possessed trilineage differentiation potential and was karyotypically normal. MLPA analyses performed on DNA extracted from CCMi001DMD-A-3 showed a deletion of exons 49 and 50 (CCMi001DMD-A-3, ∆49, ∆50). Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  14. Derivation of the Duchenne muscular dystrophy patient-derived induced pluripotent stem cell line lacking DMD exons 49 and 50 (CCMi001DMD-A-3, ∆49, ∆50

    Directory of Open Access Journals (Sweden)

    Gabriella Spaltro

    2017-12-01

    Full Text Available Duchenne muscular dystrophy (DMD is caused by abnormalities in the dystrophin gene and is clinically characterised by childhood muscle degeneration and cardiomyopathy. We produced an induced pluripotent stem cell line from a DMD patient's dermal fibroblasts by electroporation with episomal vectors containing: hL-MYC, hLIN28, hSOX2, hKLF4, hOCT3/4. The resultant DMD iPSC line (CCMi001DMD-A-3 displayed iPSC morphology, expressed pluripotency markers, possessed trilineage differentiation potential and was karyotypically normal. MLPA analyses performed on DNA extracted from CCMi001DMD-A-3 showed a deletion of exons 49 and 50 (CCMi001DMD-A-3, ∆49, ∆50.

  15. D-1 dopaminergic and beta-adrenergic stimulation of adenylate cyclase in a clone derived from the human astrocytoma cell line G-CCM.

    Science.gov (United States)

    Balmforth, A J; Ball, S G; Freshney, R I; Graham, D I; McNamee, H B; Vaughan, P F

    1986-09-01

    Clones have been isolated from the human astrocytoma cell line G-CCM. Homogenates of clone D384 contain an adenylate cyclase that is stimulated by 3,4-dihydroxyphenylethylamine (dopamine), noradrenaline, and isoprenaline with Ka apparent values of 4, 56, and 2.7 microM, respectively. The Ka apparent value for dopamine was increased by the D-1 antagonist cis-flupenthixol, 25 and 100 nM, to 23 and 190 microM, respectively, but was unaffected by propranolol (1 microM). Noradrenaline stimulation of adenylate cyclase was only partially inhibited by either propranolol (10 microM) or cis-flupenthixol (1 microM). Propranolol (10 microM), but not cis-flupenthixol (1 microM), prevented stimulation by isoprenaline. The stimulation of adenylate cyclase by dopamine and noradrenaline remained unchanged in the presence of phentolamine (1 microM) and sulpiride (1 microM). These results suggest that clone D384 contains both D-1 dopaminergic and beta-adrenergic receptors coupled to adenylate cyclase. Dopamine stimulates D384 adenylate cyclase through D-1 receptors, isoprenaline via beta-receptors, and noradrenaline through both receptors.

  16. Synthesis, characterization of 1,2,4-triazole Schiff base derived 3d-metal complexes: Induces cytotoxicity in HepG2, MCF-7 cell line, BSA binding fluorescence and DFT study

    Science.gov (United States)

    Tyagi, Prateek; Tyagi, Monika; Agrawal, Swati; Chandra, Sulekh; Ojha, Himanshu; Pathak, Mallika

    2017-01-01

    Two novel Schiff base ligands H2L1 and H2L2 have been synthesized by condensation reaction of amine derivative of 1,2,4-triazole moiety with 2-hydroxy-4-methoxybenzaldehyde. Co(II), Ni(II), Cu(II) and Zn(II) of the synthesized Schiff bases were prepared by using a molar ratio of ligand:metal as 1:1. The structure of the Schiff bases and synthesized metal complexes were established by 1H NMR, UV-Vis, IR, Mass spectrometry and molar conductivity. The thermal stability of the complexes was study by TGA. Fluorescence quenching mechanism of metal complexes 1-4 show that Zn(II) and Cu(II) complex binds more strongly to BSA. In DFT studies the geometries of Schiff bases and metal complexes were fully optimized with respect to the energy using the 6-31 + g(d,p) basis set. The spectral data shows that the ligands behaves as binegative tridentate. On the basis of the spectral studies, TGA and DFT data an octahedral geometry has been assigned for Co(II), Ni(II), square planar for Cu(II) and tetrahedral for Zn(II) complexes. The anticancer activity were screened against human breast cancer cell line (MCF-7) and human hepatocellular liver carcinoma cell line (Hep-G2). Result indicates that metal complexes shows increase cytotoxicity in proliferation to cell lines as compared to free ligand.

  17. Mouse ES cell-derived hematopoietic progenitor cells.

    Science.gov (United States)

    Kim, Eun-Mi; Manzar, Gohar; Zavazava, Nicholas

    2013-01-01

    Future stem cell-based therapies will benefit from the new discoveries being made on pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (IPS) cells. Understanding the genes regulating pluripotency has opened new opportunities to generate patient-tailored therapies. However, protocols for deriving progenitor cells of therapeutic grade from these pluripotent stem cells are not yet worked out. In particular the potential of these cells in treating diseases when compared to their adult progenitor counterparts is unknown. This is crucial work that needs to be studied in detail because we will need to determine engraftment potential of these cells and their ability for multi-lineage engraftment in the in vivo setting before any clinical applications. The ability of these cells to engraft is dependent on their expression of cell surface markers which guide their homing patterns. In this review, I discuss murine hematopoietic progenitor cells derived from mouse ES cells. Stem cells in the bone marrow are found in the bone marrow niches. Our knowledge of the bone marrow niches is growing and will ultimately lead to improved clinical transplantation of bone marrow cells. We are, however, a long way in appreciating how hematopoietic progenitor cells migrate and populate lymphoid tissues. One of the variables in generating hematopoietic progenitor cells is that different labs use different approaches in generating progenitor cells. In some cases, the ES cell lines used show some variability as well. The cell culture media used by the different investigators highly influence the maturation level of the cells and their homing patterns. Here, mouse ES cell-derived progenitor cells are discussed.

  18. Brain-Derived Neurotrophic Factor Facilitates Functional Recovery from ALS-Cerebral Spinal Fluid-Induced Neurodegenerative Changes in the NSC-34 Motor Neuron Cell Line.

    Science.gov (United States)

    Shruthi, Shanmukha; Sumitha, R; Varghese, Anu Mary; Ashok, S; Chandrasekhar Sagar, B K; Sathyaprabha, T N; Nalini, A; Kramer, Boris W; Raju, Trichur R; Vijayalakshmi, K; Alladi, Phalguni Anand

    2017-01-01

    The survival of motor neurons is dependent upon neurotrophic factors both during childhood and adolescence and during adult life. In disease conditions, such as in patients with amyotrophic lateral sclerosis (ALS), the mRNA levels of trophic factors like brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor are downregulated. This was replicated in our in vivo experimental system following the injection of cerebral spinal fluid (CSF) of sporadic ALS (ALS-CSF) patients. To evaluate the protective role of BDNF in a model of sporadic ALS patients. The expressions of endogenous BDNF, its receptor TrkB, the enzyme choline acetyl transferase (ChAT), and phosphorylated neurofilaments were studied in NSC-34 cells. The calcium-buffering and proapoptotic effects were assessed by calbindin-D28K and caspase-3 expression, respectively. ALS-CSF considerably depleted the endogenous BDNF protein, while its effect on IGF-1 and FGF-2 was inconsequential; this indirectly indicates a key role for BDNF in supporting motor neuronal survival. The exogenous supplementation of BDNF reversed autocrine expression; however, it may not be completely receptor mediated, as the TrkB levels were not restored. BDNF completely revived ChAT expression. It may inhibit apoptosis by restoring Ca2+ homeostasis, since caspase-3 and calbindin-D28K expression was back to normal. The organellar ultrastructural changes were only partially reversed. Our study provides evidence that BDNF supplementation ameliorates most but not all degenerative changes. The incomplete revival at the ultrastructural level signifies the requirement of factors other than BDNF for near-total protection of motor neurons, and, to an extent, it explains why only a partial success is achieved in clinical trials with BDNF in ALS patients. © 2016 S. Karger AG, Basel.

  19. Genome wide profiling of human embryonic stem cells (hESCs, their derivatives and embryonal carcinoma cells to develop base profiles of U.S. Federal government approved hESC lines

    Directory of Open Access Journals (Sweden)

    Baker Shawn C

    2006-05-01

    Full Text Available Abstract Background In order to compare the gene expression profiles of human embryonic stem cell (hESC lines and their differentiated progeny and to monitor feeder contaminations, we have examined gene expression in seven hESC lines and human fibroblast feeder cells using Illumina® bead arrays that contain probes for 24,131 transcript probes. Results A total of 48 different samples (including duplicates grown in multiple laboratories under different conditions were analyzed and pairwise comparisons were performed in all groups. Hierarchical clustering showed that blinded duplicates were correctly identified as the closest related samples. hESC lines clustered together irrespective of the laboratory in which they were maintained. hESCs could be readily distinguished from embryoid bodies (EB differentiated from them and the karyotypically abnormal hESC line BG01V. The embryonal carcinoma (EC line NTera2 is a useful model for evaluating characteristics of hESCs. Expression of subsets of individual genes was validated by comparing with published databases, MPSS (Massively Parallel Signature Sequencing libraries, and parallel analysis by microarray and RT-PCR. Conclusion we show that Illumina's bead array platform is a reliable, reproducible and robust method for developing base global profiles of cells and identifying similarities and differences in large number of samples.

  20. Genome wide profiling of human embryonic stem cells (hESCs), their derivatives and embryonal carcinoma cells to develop base profiles of U.S. Federal government approved hESC lines.

    Science.gov (United States)

    Liu, Ying; Shin, Soojung; Zeng, Xianmin; Zhan, Ming; Gonzalez, Rodolfo; Mueller, Franz-Josef; Schwartz, Catherine M; Xue, Haipeng; Li, Huai; Baker, Shawn C; Chudin, Eugene; Barker, David L; McDaniel, Timothy K; Oeser, Steffen; Loring, Jeanne F; Mattson, Mark P; Rao, Mahendra S

    2006-05-03

    In order to compare the gene expression profiles of human embryonic stem cell (hESC) lines and their differentiated progeny and to monitor feeder contaminations, we have examined gene expression in seven hESC lines and human fibroblast feeder cells using Illumina bead arrays that contain probes for 24,131 transcript probes. A total of 48 different samples (including duplicates) grown in multiple laboratories under different conditions were analyzed and pairwise comparisons were performed in all groups. Hierarchical clustering showed that blinded duplicates were correctly identified as the closest related samples. hESC lines clustered together irrespective of the laboratory in which they were maintained. hESCs could be readily distinguished from embryoid bodies (EB) differentiated from them and the karyotypically abnormal hESC line BG01V. The embryonal carcinoma (EC) line NTera2 is a useful model for evaluating characteristics of hESCs. Expression of subsets of individual genes was validated by comparing with published databases, MPSS (Massively Parallel Signature Sequencing) libraries, and parallel analysis by microarray and RT-PCR. we show that Illumina's bead array platform is a reliable, reproducible and robust method for developing base global profiles of cells and identifying similarities and differences in large number of samples.

  1. Adipose-Derived Stem Cells

    DEFF Research Database (Denmark)

    Toyserkani, Navid Mohamadpour; Quaade, Marlene Louise; Sheikh, Søren Paludan

    2015-01-01

    Emerging evidence has shown that adipose tissue is the richest and most accessible source of mesenchymal stem cells. Many different therapies for chronic wounds exist with varying success rates. The capacity of adipose-derived stem cells (ASCs) to promote angiogenesis, secrete growth factors......, regulate the inflammatory process, and differentiate into multiple cell types makes them a potential ideal therapy for chronic wounds. The aim of this article was to review all preclinical trials using ASCs in problem wound models. A systematic search was performed and 12 studies were found where different...

  2. Comparative study on the effect of fish-oil derived docosahexaenoic and ecosapantaenoic acid on proliferation of colorectal cancer cell line: a breif report

    Directory of Open Access Journals (Sweden)

    Parinaz Ahangar

    2014-03-01

    Results: Viability analysis showed that 150 µM/L PUFAs decreased significantly the proliferation of treated cells, as compared to untreated cells. In this regard, cell viabil-ities were found to be %31±%5.1 and %30±%2.6 for DHA and EPA respectively. Moreover, treatment of cells with increasing concentrations of EPA and DHA signifi-cantly decreased growth rates in a dose-and time-dependent manner. Following 72 hours treatments with 150 µM/L PUFAs, growth rates were found to be %19±%5.5 and %20±%5 for DHA and EPA relative to untreated cells respectively. Conclusion: The results of this study indicate that n3-PUFAs decrease cell proliferation and could provide new approaches in malignant tumor therapeutic strategies.

  3. Exposure to Nickel, Chromium, or Cadmium Causes Distinct Changes in the Gene Expression Patterns of a Rat Liver Derived Cell Line

    Science.gov (United States)

    2011-11-16

    Saccharomyces cerevisiae : gene expression and identification of novel essential genes. Mol Biol Cell 15: 5295–5305. 14. Boyack KW, Wylie BN, Davidson GS (2002...major roles in postreplication mutation avoidance in Saccharomyces cerevisiae . Mol Cell Biol 19: 2000–2007. 37. Kawakami H, Katayama T (2010) DnaA, ORC...cells (ATCC, Manassas, VA) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Lonza, Walkers- ville, MD) containing 10% fetal bovine serum

  4. Resolving traceability issues of product derivation for software product lines

    OpenAIRE

    Abid, Saad bin

    2009-01-01

    peer-reviewed Dealing with traceability management issues during model based product derivation in large complex industrial SPL is error prone due to the lack of tool support. As a result traceability management between connected models emerges as an important research topic. In this position paper, we discuss research challenges as scenarios from developed example product line and give recommendations on resolving traceability issues during product derivation. We also discuss initial idea...

  5. PrPC displays an essential protective role from oxidative stress in an astrocyte cell line derived from PrPC knockout mice

    International Nuclear Information System (INIS)

    Bertuchi, Fernanda R.; Bourgeon, Dominique M.G.; Landemberger, Michele C.; Martins, Vilma R.; Cerchiaro, Giselle

    2012-01-01

    Highlights: ► PrP C in solution acts as a radical scavenger. ► PrP C reduces hydrogen peroxide toxicity in astrocytes. ► Increase in ROS disrupted the cell cycle in the PrP C -knockout astrocytes. ► PrP C prevents the cell death independently of an SOD-like activity. -- Abstract: The PrP C protein, which is especially present in the cellular membrane of nervous system cells, has been extensively studied for its controversial antioxidant activity. In this study, we elucidated the free radical scavenger activity of purified murine PrP C in solution and its participation as a cell protector in astrocytes that were subjected to treatment with an oxidant. In vitro and using an EPR spin-trapping technique, we observed that PrP C decreased the oxidation of the DMPO trap in a Fenton reaction system (Cu 2+ /ascorbate/H 2 O 2 ), which was demonstrated by approximately 70% less DMPO/OH · . In cultured PrP C -knockout astrocytes from mice, the absence of PrP C caused an increase in intracellular ROS (reactive oxygen species) generation during the first 3 h of H 2 O 2 treatment. This rapid increase in ROS disrupted the cell cycle in the PrP C -knockout astrocytes, which increased the population of cells in the sub-G1 phase when compared with cultured wild-type astrocytes. We conclude that PrP C in solution acts as a radical scavenger, and in astrocytes, it is essential for protection from oxidative stress caused by an external chemical agent, which is a likely condition in human neurodegenerative CNS disorders and pathological conditions such as ischemia.

  6. PrP{sup C} displays an essential protective role from oxidative stress in an astrocyte cell line derived from PrP{sup C} knockout mice

    Energy Technology Data Exchange (ETDEWEB)

    Bertuchi, Fernanda R. [Center for Natural Sciences and Humanities, Federal University of ABC - UFABC, Avenida dos Estados, 5001, Bloco B, 09210-170, Santo Andre, SP (Brazil); Bourgeon, Dominique M.G.; Landemberger, Michele C.; Martins, Vilma R. [International Center for Research and Education, A.C. Camargo Hospital, Rua Tagua 440, 01505-010 Sao Paulo, SP (Brazil); Cerchiaro, Giselle, E-mail: giselle.cerchiaro@ufabc.edu.br [Center for Natural Sciences and Humanities, Federal University of ABC - UFABC, Avenida dos Estados, 5001, Bloco B, 09210-170, Santo Andre, SP (Brazil)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer PrP{sup C} in solution acts as a radical scavenger. Black-Right-Pointing-Pointer PrP{sup C} reduces hydrogen peroxide toxicity in astrocytes. Black-Right-Pointing-Pointer Increase in ROS disrupted the cell cycle in the PrP{sup C}-knockout astrocytes. Black-Right-Pointing-Pointer PrP{sup C} prevents the cell death independently of an SOD-like activity. -- Abstract: The PrP{sup C} protein, which is especially present in the cellular membrane of nervous system cells, has been extensively studied for its controversial antioxidant activity. In this study, we elucidated the free radical scavenger activity of purified murine PrP{sup C} in solution and its participation as a cell protector in astrocytes that were subjected to treatment with an oxidant. In vitro and using an EPR spin-trapping technique, we observed that PrP{sup C} decreased the oxidation of the DMPO trap in a Fenton reaction system (Cu{sup 2+}/ascorbate/H{sub 2}O{sub 2}), which was demonstrated by approximately 70% less DMPO/OH{sup {center_dot}}. In cultured PrP{sup C}-knockout astrocytes from mice, the absence of PrP{sup C} caused an increase in intracellular ROS (reactive oxygen species) generation during the first 3 h of H{sub 2}O{sub 2} treatment. This rapid increase in ROS disrupted the cell cycle in the PrP{sup C}-knockout astrocytes, which increased the population of cells in the sub-G1 phase when compared with cultured wild-type astrocytes. We conclude that PrP{sup C} in solution acts as a radical scavenger, and in astrocytes, it is essential for protection from oxidative stress caused by an external chemical agent, which is a likely condition in human neurodegenerative CNS disorders and pathological conditions such as ischemia.

  7. Inhibition of Oncogenic Transcription Factor REL by the Natural Product Derivative Calafianin Monomer 101 Induces Proliferation Arrest and Apoptosis in Human B-Lymphoma Cell Lines

    OpenAIRE

    Yeo, Alan; Chennamadhavuni, Spandan; Whitty, Adrian; Porco, John; Gilmore, Thomas

    2015-01-01

    Increased activity of transcription factor NF-κB has been implicated in many B-cell lymphomas. We investigated effects of synthetic compound calafianin monomer (CM101) on biochemical and biological properties of NF-κB. In human 293 cells, CM101 selectively inhibited DNA binding by overexpressed NF-κB subunits REL (human c-Rel) and p65 as compared to NF-κB p50, and inhibition of REL and p65 DNA binding by CM101 required a conserved cysteine residue. CM101 also inhibited DNA binding by REL in ...

  8. Comparison of the effect of cortisol on aromatase activity and androgen metabolism in two human fibroblast cell lines derived from the same individual

    DEFF Research Database (Denmark)

    Svenstrup, B; Brünner, N; Dombernowsky, P

    1990-01-01

    The effect of preincubation with cortisol on estrogen and androgen metabolism was investigated in human fibroblast monolayers grown from biopsies of genital and non-genital skin of the same person. The activity in the cells of aromatase, 5 alpha-reductase, 17 beta-hydroxysteroid oxidoreductase...... and 3 alpha-hydroxysteroid oxidoreductase was investigated by isolating estrone, estradiol, estriol, dihydrotestosterone, androstanedione, androsterone, 3 alpha-androstanediol, testosterone and androstenedione after incubation of the cells with [14C]testosterone or [14C]androstenedione. For experiments...

  9. Establishment and characterization of BHD-F59RSVT, an immortalized cell line derived from a renal cell carcinoma in a patient with Birt-Hogg-Dubé syndrome.

    Science.gov (United States)

    Furuya, Mitsuko; Hasumi, Hisashi; Baba, Masaya; Tanaka, Reiko; Iribe, Yasuhiro; Onishi, Takahiro; Nagashima, Yoji; Nakatani, Yukio; Isono, Yasuhiro; Yao, Masahiro

    2017-03-01

    Hereditary renal cell carcinomas (RCCs) are life-threatening disorders not only for the patients but also for their relatives. Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder caused by germline mutations in the folliculin gene (FLCN). The protein product, FLCN, functions as a tumor suppressor, and the affected patients have high risks of developing multiple RCCs. The carcinogenic mechanisms stemming from FLCN dysfunction have been investigated using rodent models and human RCC tissues. However, very limited information has been available about in vitro signaling of human renal cells with genetically mutant FLCN. Herein, we established a new cell line, BHD-F59RSVT, from a BHD patient's chromophobe RCC by transfecting SV40 large T antigen. We investigated FLCN mutations, chromosome profiles, and cytopathologic characteristics of the cell line. BHD-F59RSVT reflected the patient's FLCN germline mutation, a 3-nt deletion in exon 13 (c.1528_1530delGAG). Neither somatic mutation nor loss of heterozygosity of FLCN was detectable. Chromosome 17p11.2 of the FLCN proximal region demonstrated a trimodal pattern. Genome-wide chromosomal analysis revealed a loss of chromosome 16 and mosaic segmental gains in chromosome 7. BHD-F59RSVT cells were positive when immunostained for cytokeratin 7, supporting their origin from distal convoluted tubules. Western blotting analysis demonstrated severely suppressed FLCN expression at the protein level. The collective findings indicate that the established cell line will be suitable for functional analysis of the typical phenotype of BHD-associated RCC with suppressed FLCN expression.

  10. Comparison of the effect of cortisol on aromatase activity and androgen metabolism in two human fibroblast cell lines derived from the same individual

    DEFF Research Database (Denmark)

    Svenstrup, B; Brünner, N; Dombernowsky, P

    1990-01-01

    with 14C-labeled substrate the cells were incubated in medium, charcoal stripped of steroids without Phenol Red. Preincubation from 6 to 36 h with cortisol in concentrations of 10(-8) - 10(-6) M showed maximal stimulation of aromatase activity after 12 h preincubation with cortisol in concentrations of 0...

  11. Derivation of human embryonic stem cells in defined conditions.

    Science.gov (United States)

    Ludwig, Tenneille E; Levenstein, Mark E; Jones, Jeffrey M; Berggren, W Travis; Mitchen, Erika R; Frane, Jennifer L; Crandall, Leann J; Daigh, Christine A; Conard, Kevin R; Piekarczyk, Marian S; Llanas, Rachel A; Thomson, James A

    2006-02-01

    We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.

  12. Fraction against Human Cancer Cell Lines

    African Journals Online (AJOL)

    kidney carcinoma cell lines of hamsters (BSR). [11]. While, in another article, A. sieberia unrefined extract exhibited dose dependent antiproliferative activity against several cancer cell lines (human bladder carcinoma RT112, human laryngeal carcinoma and human myelogenous leukaemia K562), with IC50. = 81.59, 59.05 ...

  13. Difference in membrane repair capacity between cancer cell lines and a normal cell line

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; McNeil, Anna K.; Novak, Ivana

    2016-01-01

    repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique......, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. Systematic Derivation of Static Analyses for Software Product Lines

    DEFF Research Database (Denmark)

    Midtgaard, Jan; Brabrand, Claus; Wasowski, Andrzej

    2014-01-01

    A recent line of work lifts particular verification and analysis methods to Software Product Lines (SPL). In an effort to generalize such case-by-case approaches, we develop a systematic methodology for lifting program analyses to SPLs using abstract interpretation. Abstract interpretation...... is a classical framework for deriving static analyses in a compositional, step-by-step manner. We show how to take an analysis expressed as an abstract interpretation and lift each of the abstract interpretation steps to a family of programs. This includes schemes for how to lift domain types, and combinators...... analysis for a simple imperative language....

  16. Structure-activity relationships of dimethylsphingosine (DMS) derivatives and their effects on intracellular pH and Ca2+ in the U937 monocyte cell line.

    Science.gov (United States)

    Chang, Young-Ja; Lee, Yun-Kyung; Lee, Eun-Hee; Park, Jeong-Ju; Chung, Sung-Kee; Im, Dong-Soon

    2006-08-01

    We recently reported that dimethylsphingosine (DMS), a metabolite of sphingolipids, increased intracellular pH and Ca2+ concentration in U937 human monocytes. In the present study, we found that dimethylphytosphingosine (DMPH) induced the above responses more robustly than DMS. However, phytosphingosine, monomethylphytosphingosine or trimethylsphingosine showed little or no activity. Synthetic C3 deoxy analogues of sphingosine did show similar activities, with the C16 analogue more so than C18. The following structure-activity relationships were observed between DMS derivatives and the intracellular pH and Ca2+ concentrations in U937 monocytes; 1) dimethyl modification is important for the DMS-induced increase of intracellular pH and Ca2+, 2) the addition of an OH group on C4 enhances both activities, 3) the deletion of the OH group on C3 has a negligible effect on the activities, and 4) C16 appears to be more effective than C18. We also found that W-7, a calmodulin inhibitor, blocked the DMS-induced pH increase, whereas, KN-62, ML9, and MMPX, specific inhibitors for calmodulin-dependent kinase II, myosin light chain kinase, and Ca(2+)-calmodulin-dependent phosphodiesterase, respectively, did not affect DMS-induced increases of pH in the U937 monocytes.

  17. Stemness is derived from thyroid cancer cells

    Directory of Open Access Journals (Sweden)

    Risheng eMa

    2014-07-01

    Full Text Available Background: One hypothesis for thyroid cancer development is its derivation from thyroid cancer stem cells (CSCs. Such cells could arise via different paths including from mutated resident stem cells within the thyroid gland or via epithelial to mesenchymal transition (EMT from malignant cells since EMT is known to confer stem-like characteristics. Methods: To examine the status of stemness in thyroid papillary cancer we employed a murine model of thyroid papillary carcinoma and examined the expression of stemness and EMT using qPCR and histochemistry in mice with a thyroid-specific knock-in of oncogenic Braf (LSL-Braf(V600E/TPO-Cre. This construct is only activated at the time of thyroid peroxidase (TPO expression in differentiating thyroid cells and cannot be activated by undifferentiated stem cells which do not express TPO.Results: There was decreased expression of thyroid specific genes such as Tg and NIS and increased expression of stemness markers such as Oct4, Rex1, CD15 and Sox2 in the thyroid carcinoma tissue from 6 week old BRAFV600E mice. The decreased expression of the epithelial marker E-cadherin and increased EMT regulators including Snail, Slug, and TGF-β1 and TGF-β3, and the mesenchymal marker vimentin demonstrated the simultaneous progression of EMT and the CSC-like phenotype. Stemness was also found in a derived cancer thyroid cell line in which overexpression of Snail caused up-regulation of vimentin expression and up regulation of stemness markers Oct4, Rex1, CD15 with enhanced migration ability of the cells. Conclusions: Our findings support our earlier hypothesis that stemness in thyroid cancer is derived via EMT rather than from resident thyroid stem cells. In mice with a thyroid-specific knock-in of oncogenic Braf (LSL-Braf(V600E/TPO-Cre the neoplastic changes were dependent on thyroid cell differentiation and the onset of stemness must have been derived from differentiated thyroid epithelial cells.

  18. Establishment of cell lines with rat spermatogonial stem cell characteristics

    NARCIS (Netherlands)

    van Pelt, Ans M. M.; Roepers-Gajadien, Hermien L.; Gademan, Iris S.; Creemers, Laura B.; de Rooij, Dirk G.; van Dissel-Emiliani, Federica M. F.

    2002-01-01

    Spermatogonial cell lines were established by transfecting a mixed population of purified rat A(s) (stem cells), A(pr) and A(al) spermatogonia with SV40 large T antigen. Two cell lines were characterized and found to express Hsp90alpha and oct-4, specific markers for germ cells and A spermatogonia,

  19. Plants Regeneration Derived From Various on Peanut on Mutant Lines

    International Nuclear Information System (INIS)

    Dewi, Kumala; Masrizal; Mugiono

    1998-01-01

    The study of calli, greenspot formation and shoot regeneration on peanut mutant lines has ben conducted by MS media. Three explants derived from shoot tips, embryo and seeding root of two mutant lines a/20/3 and D/25/3/2 were used in this experiment. the explants were cultured on modified MS media enriched by vitamins, growth regulation, amino acids for fourth teen calli were transferred on regeneration media. The ability of calli formation and plant regeneration of each explant and genotypes of plants was varied. Greenspot and shoot formation were observed seventh days after the calli transferred on regeneration media. It is shown that the ability of calli, greenspot and shoot formation of each explants and genotypes was varied. the high ability of calli, greenspot and shoot formation were found in explant derived from shoot tip and embryo. Seedling root explant has lower ability in calli formation, while greenspot and shoot was formatted. The ability of calli, greenspot and shoot formation on A/20/3 mutant line was better than D/25/3/2 mutant line. (author)

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

    Directory of Open Access Journals (Sweden)

    Julian Biau

    2016-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Michel Fausther

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

  2. Dose-Exposure Proportionality of a Novel Recombinant Follicle-Stimulating Hormone (rFSH), FE 999049, Derived from a Human Cell Line, with Comparison Between Caucasian and Japanese Women After Subcutaneous Administration

    OpenAIRE

    Olsson, H?kan; Sandstr?m, Rikard; Bagger, Yu

    2015-01-01

    Background and Objectives FE 999049 is a novel recombinant follicle-stimulating hormone (rFSH) preparation expressed by a human cell line (PER.C6?), in contrast to existing rFSH preparations expressed by Chinese hamster ovary (CHO) cell lines. Since the individual dose of rFSH may be altered depending on the response in women undergoing assisted reproductive technologies, knowledge on the dose-exposure linearity and proportionality is important. The purpose of these studies was to investigate...

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

    Science.gov (United States)

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

    2014-09-09

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

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

    Science.gov (United States)

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

    2014-01-01

    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

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

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

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

  6. Differential CCR4 Expression And Function in Cutaneous T-Cell Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Chieh-Shan Wu

    2008-11-01

    Full Text Available Cutaneous T cell lymphoma (CTCL is a clonal epidermotropic malignancy of memory T cells primarily involving the skin. However, the mechanisms governing migration of CTCL cells have not been fully clarified. It has been shown that certain chemokine receptors are upregulated in CTCL cells, but it remains unanswered whether these chemokine receptors play a critical role in the migration dynamics of CTCL. Using cell lines originally derived from patients with different subtypes of CTCL, we have shown higher CCR4 expression in the line derived from the mycosis fungoides (MJ, compared with the line derived from Sézary syndrome (Hut78. In specific responses to CCL22 (a CCR4 ligand treatments, MJ cells showed significant chemotactic migration, enhanced activation and adhesion of certain integrins (CD49d and CD29 in vitro, while the control cells (Hut78, CD4+CD45RO+ memory T cells, and Jurkat cells did not. Furthermore, compared with Hut78 cells, MJ cells manifested significantly more transendothelial migration in responses to treatments with either CCL22 or conditioned medium from dendritic cells in vitro. These results provide further dynamic evidence, in line with the multistep cascade paradigm for leukocyte transendothelial migration, to support a critical role for CCR4 in CTCL migration.

  7. Pharmacological Activation Gi/o Protein Increases Glial Cell Line-Derived Neurotrophic Factor Production through Fibroblast Growth Factor Receptor and Extracellular Signal-Regulated Kinase Pathway in Primary Cultured Rat Cortical Astrocytes.

    Science.gov (United States)

    Hisaoka-Nakashima, Kazue; Matsumoto, Chie; Azuma, Honami; Taki, Sayaka; Takebayashi, Minoru; Nakata, Yoshihiro; Morioka, Norimitsu

    2017-01-01

    A significant reduction of glial cell line-derived neurotrophic factor (GDNF) has been identified in the pathophysiology of neurodegenerative and neuropsychiatric disorders. Thus, clarification of the mechanism of GDNF production, and modulating brain GDNF levels could be a novel therapeutic approach. A previous study demonstrated that antidepressant amitriptyline-induced GDNF production was significantly inhibited by pertussis toxin (PTX), a Gi/o protein inhibitor in astrocytes, the main source of GDNF in the brain. However, it is not known whether direct activation of Gi/o protein might induce GDNF expression, and what mechanisms might be involved after Gi/o protein activation. The current study investigated Gi/o protein-initiated GDNF production in rat cortical astrocytes using activators that directly activate Gi/o protein, mastoparan and compound48/80. Treatment of astrocytes with either mastoparan or compound48/80 increased GDNF mRNA expression at 3 and 6 h, and GDNF protein release at 24 h. Treatment of astrocyte with either mastoparan or compound48/80 increased brain-derived neurotrophic factor (BDNF) mRNA expression as well as GDNF. Mastoparan and compound48/80-induced GDNF mRNA expression were significantly inhibited by not only PTX, but also fibroblast growth factor receptor (FGFR) inhibitors, and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor. In fact, both FGFR substrate2α (FRS2α) and ERK phosphorylation were increased by treatment with either mastoparan or compound48/80, and these were significantly blocked by PTX. Thus, direct, receptor-independent Gi/o protein activation increases GDNF production through FGFR/ERK signaling pathway. The current results indicate a critical role of Gi/o signaling in the regulation of GDNF expression in astrocytes.

  8. Genomic characterisation of acral melanoma cell lines.

    Science.gov (United States)

    Furney, Simon J; Turajlic, Samra; Fenwick, Kerry; Lambros, Maryou B; MacKay, Alan; Ricken, Gerda; Mitsopoulos, Costas; Kozarewa, Iwanka; Hakas, Jarle; Zvelebil, Marketa; Lord, Christopher J; Ashworth, Alan; Reis-Filho, Jorge S; Herlyn, Meenhard; Murata, Hiroshi; Marais, Richard

    2012-07-01

    Acral melanoma is a rare melanoma subtype with distinct epidemiological, clinical and genetic features. To determine if acral melanoma cell lines are representative of this melanoma subtype, six lines were analysed by whole-exome sequencing and array comparative genomic hybridisation. We demonstrate that the cell lines display a mutation rate that is comparable to that of published primary and metastatic acral melanomas and observe a mutational signature suggestive of UV-induced mutagenesis in two of the cell lines. Mutations were identified in oncogenes and tumour suppressors previously linked to melanoma including BRAF, NRAS, KIT, PTEN and TP53, in cancer genes not previously linked to melanoma and in genes linked to DNA repair such as BRCA1 and BRCA2. Our findings provide strong circumstantial evidence to suggest that acral melanoma cell lines and acral tumours share genetic features in common and that these cells are therefore valuable tools to investigate the biology of this aggressive melanoma subtype. Data are available at: http://rock.icr.ac.uk/collaborations/Furney_et_al_2012/. © 2012 John Wiley & Sons A/S.

  9. Distinct metabolic responses of an ovarian cancer stem cell line.

    Science.gov (United States)

    Vermeersch, Kathleen A; Wang, Lijuan; McDonald, John F; Styczynski, Mark P

    2014-12-18

    Cancer metabolism is emerging as an important focus area in cancer research. However, the in vitro cell culture conditions under which much cellular metabolism research is performed differ drastically from in vivo tumor conditions, which are characterized by variations in the levels of oxygen, nutrients like glucose, and other molecules like chemotherapeutics. Moreover, it is important to know how the diverse cell types in a tumor, including cancer stem cells that are believed to be a major cause of cancer recurrence, respond to these variations. Here, in vitro environmental perturbations designed to mimic different aspects of the in vivo environment were used to characterize how an ovarian cancer cell line and its derived, isogenic cancer stem cells metabolically respond to environmental cues. Mass spectrometry was used to profile metabolite levels in response to in vitro environmental perturbations. Docetaxel, the chemotherapeutic used for this experiment, caused significant metabolic changes in amino acid and carbohydrate metabolism in ovarian cancer cells, but had virtually no metabolic effect on isogenic ovarian cancer stem cells. Glucose deprivation, hypoxia, and the combination thereof altered ovarian cancer cell and cancer stem cell metabolism to varying extents for the two cell types. Hypoxia had a much larger effect on ovarian cancer cell metabolism, while glucose deprivation had a greater effect on ovarian cancer stem cell metabolism. Core metabolites and pathways affected by these perturbations were identified, along with pathways that were unique to cell types or perturbations. The metabolic responses of an ovarian cancer cell line and its derived isogenic cancer stem cells differ greatly under most conditions, suggesting that these two cell types may behave quite differently in an in vivo tumor microenvironment. While cancer metabolism and cancer stem cells are each promising potential therapeutic targets, such varied behaviors in vivo would need to

  10. Metronidazole affects breast cancer cell lines.

    Science.gov (United States)

    Sadowska, A; Prokopiuk, S; Miltyk, W; Surażyński, A; Konończuk, J; Sawicka, D; Car, H

    2013-01-01

    The aim of our study was to evaluate the impact of metronidazole (MTZ) on cytotoxicity and DNA synthesis in MCF-7 (estrogen receptor positive) and MDA-MB-231 (estrogen receptor negative) breast cancer cell lines. Toxicity of MTZ was determined by MTT test. MCF-7 and MDA-MB-231 cells were incubated with metronidazole used in different concentrations for 24, 48 and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. We showed that MTZ in concentration 250 μg/ml significantly increases the growth of MCF-7 cell lines after 24 hours of incubation, but it reduces cell viability in concentrations 1 and 10 μg/ml 72 hours after the drug application. Significant increase of MDA-MB-231 cell viability was obtained in MTZ concentration of 250 μg/ml after 24 and 72 hours. The increase of [3H]-thymidine incorporation in MCF-7 cell line treated with MTZ in concentration 250 μg/ml was statistically significant after 24 hours. Great suppression of cell proliferation was obtained in MDA-MB-231 breast cell line after application of the following concentrations of MTZ: 0.1 μg/ml (after 24 hours) and 0.1, 10, 50, 250 μg/ml (after 72h). We found that metronidazole exerts different dose- and time- dependent effects on human breast cancer cell lines characterized by presence or absence of estrogen receptors. We suggest that these discrepancies may be influenced by the estrogen signaling.

  11. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

    DEFF Research Database (Denmark)

    Xu, Xun; Pan, Shengkai; Liu, Xin

    2011-01-01

    Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most...

  12. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

    DEFF Research Database (Denmark)

    Xu, Xun; Pan, Shengkai; Liu, Xin

    2011-01-01

    Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of...

  13. Effect of New Water-Soluble Dendritic Phthalocyanines on Human Colorectal and Liver Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Ebru YABAŞ

    2017-08-01

    Full Text Available Human hepatocellular carcinoma (HepG2 cells and colorectal adenocarcinoma (DLD-1 cells were treated with the synthesized water soluble phthalocyanine derivatives to understand the effect of the compounds both on colorectal and liver cancer cells. The compounds inhibited cell proliferation and displayed cytotoxic effect on these cancer cell lines however; the effect of the compounds on healthy control fibroblast cell line was comparatively lower. The compounds can be employed for cancer treatment as anticancer agents.

  14. Proteomics Applications in Dental Derived Stem Cells.

    Science.gov (United States)

    Li, Jie; Tian, Weidong; Song, Jinlin

    2017-07-01

    At present, the existence of a variety of dental derived stem cells has been documented. These cells displayed promising clinical application potential not only for teeth and its surrounding tissue regeneration, but also for other tissues, such as nerve and bone regeneration. Proteomics is an unbiased, global informatics tool that provides information on all protein expression levels as well as post-translational modification in cells or tissues and is applicable to dental derived stem cells research. Over the last decade, considerable progress has been made to study the global proteome, secrotome, and membrane proteome of dental derived stem cells. Here, we present an overview of the proteomics studies in the context of stem cell research. Particular attention is given to dental derived stem cell types as well as current challenges and opportunities. J. Cell. Physiol. 232: 1602-1610, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. Subcloning of ovarian cancer cell lines.

    Science.gov (United States)

    Grunt, T W

    2001-01-01

    Cellular heterogeneity of malignant tissues is a well-known phenomenon (1). Intralineal/intraclonal diversity may be explained in part by proposing the concept of a hierarchically ordered, differentiating and self-renewing stem cell system for transformed cell populations (2). However, in many solid tumors, the stem cells are not easily accessible to phenotypic identification. In the past, density gradient centrifugation was successfully used to separate cells from tumors and from cell lines into distinct subpopulations (3-5). Using Percoll density gradients, we isolated undifferentiated clonogenic tumor stem-cell fractions from HOC-7 human ovarian adenocarcinoma cells. In addition, we also identified a low-density cell subpopulation formed by large, vacuolated, slowly growing, adenoid differentiated cells with very low clonogenic activity (6-11). Further characterization of these cell fractions in terms of stability of the isolated phenotypes is essential for the assessment of their biological significance. Subcloning of the isolated cell fractions by limiting dilution culture (12) followed by long-term culture yielded three permanent monoclonal sublines, which reveal a stable adenoid differentiated phenotype, and three subclones representing undifferentiated, clonogenic tumor stem cells (13). These data demonstrate that the isolated phenotypes represent distinct cell entities reflecting specific stages of ovarian surface epithelial cell differentiation.

  16. Embryonic liver cells and permanent lines as models for hepatocyte and bile duct cell differentiation.

    Science.gov (United States)

    Strick-Marchand, Hélène; Weiss, Mary C

    2003-01-01

    Analysis of liver cells during development is facilitated by the possibility of complementing in vivo analysis with experiments on cultured cells. In this review, we discuss results from several laboratories concerning bipotential hepatic stem cells from mouse (HBC-3, H-CFU-C, MMH and BMEL), rat (rhe14321) and primate (IPFLS) embryos. Several groups have used fluorescence-activated cell sorting to identify clonogenic bipotential cells; others have derived bipotential cell lines by plating liver cell suspensions and cloning. The bipotential cells, which probably originate from hepatoblasts, can differentiate as hepatocytes or bile duct cells, and undergo morphogenesis in culture. Disparities in differentiation can be explained by distinct medium compositions, extracellular matrix coated culture surfaces, and gene expression detection methods. Potential applications of these cell lines are discussed.

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

    Science.gov (United States)

    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.

  18. A PCR-aided transcript titration assay revealing very low expression of a gene at band 3p21 in 33 cells lines derived from all types of lung cancer

    NARCIS (Netherlands)

    Kok, K; Buchhagen, D L; Carritt, B; Buys, C H; van den Berg, Anke

    1993-01-01

    We have developed a general PCR-based method to quantify the amount of a specific mRNA present in a given cell line or tissue. We applied this quantitative PCR to analyse the expression of D8, a human gene which we recently identified in the chromosomal region 3p21, the common deletion region of

  19. LINE DERIVED INFRARED EXTINCTION TOWARD THE GALACTIC CENTER

    International Nuclear Information System (INIS)

    Fritz, T. K.; Gillessen, S.; Dodds-Eden, K.; Lutz, D.; Genzel, R.; Raab, W.; Ott, T.; Pfuhl, O.; Eisenhauer, F.; Yusef-Zadeh, F.

    2011-01-01

    We derive the extinction curve toward the Galactic center (GC) from 1 to 19 μm. We use hydrogen emission lines of the minispiral observed by ISO-SWS and SINFONI. The extinction-free flux reference is the 2 cm continuum emission observed by the Very Large Array. Toward the inner 14'' x 20'', we find an extinction of A 2.166μm = 2.62 ± 0.11, with a power-law slope of α = -2.11 ± 0.06 shortward of 2.8 μm, consistent with the average near-infrared slope from the recent literature. At longer wavelengths, however, we find that the extinction is grayer than shortward of 2.8 μm. We find that it is not possible to fit the observed extinction curve with a dust model consisting of pure carbonaceous and silicate grains only, and the addition of composite particles, including ices, is needed to explain the observations. Combining a distance-dependent extinction with our distance-independent extinction, we derive the distance to the GC to be R 0 = 7.94 ± 0.65 kpc. Toward Sgr A* (r H = 4.21 ± 0.10, A Ks = 2.42 ± 0.10, and A L' = 1.09 ± 0.13.

  20. (Asteraceae) Fraction against Human Cancer Cell Lines

    African Journals Online (AJOL)

    Purpose: To investigate the anti-proliferative and apoptotic activity of crude and dichloromethane fraction of A. sieberi against seven cancer cell lines (Colo20, HCT116, DLD, MCF7, Jurkat, HepG2 and L929). Methods: A. sieberi was extracted with methanol and further purification was carried out using liquidliquid extraction ...

  1. Breast cancer cell lines: friend or foe?

    International Nuclear Information System (INIS)

    Burdall, Sarah E; Hanby, Andrew M; Lansdown, Mark RJ; Speirs, Valerie

    2003-01-01

    The majority of breast cancer research is conducted using established breast cancer cell lines as in vitro models. An alternative is to use cultures established from primary breast tumours. Here, we discuss the pros and cons of using both of these models in translational breast cancer research

  2. Establishment of human cell lines showing circadian rhythms of bioluminescence.

    Science.gov (United States)

    Yoshikawa, Aki; Shimada, Hiroko; Numazawa, Kahori; Sasaki, Tsukasa; Ikeda, Masaaki; Kawashima, Minae; Kato, Nobumasa; Tokunaga, Katsushi; Ebisawa, Takashi

    2008-11-28

    We have established human retinal pigment epithelial cell lines stably expressing the luciferase gene, driven by the human Bmal1 promoter, to obtain human-derived cells that show circadian rhythms of bioluminescence after dexamethasone treatment. The average circadian period of bioluminescence for the obtained clones was 24.07+/-0.48 h. Lithium (10 mM) in the medium significantly lengthened the circadian period of bioluminescence, which is consistent with previous reports, while 2 mM or 5 mM lithium had no effect. This is the first report on the establishment of human-derived cell lines that proliferate infinitely and show circadian rhythms of bioluminescence, and also the first to investigate the effects of low-dose lithium on the circadian rhythms of human-derived cells in vitro. The established cells will be useful for various in vitro studies of human circadian rhythms and for the development of new therapies for human disorders related to circadian rhythm disturbances.

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

    Science.gov (United States)

    Kniss, Douglas A; Summerfield, Taryn L

    2014-08-01

    Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research. © The Author(s) 2014.

  4. Assessment of tumor characteristic gene expression in cell lines using a tissue similarity index (TSI).

    Science.gov (United States)

    Sandberg, Rickard; Ernberg, Ingemar

    2005-02-08

    The gene expression profiles of 60 cell lines, derived from nine different tissues, were compared with their corresponding in vivo tumors and tissues. Cell lines expressed few tissue-specific (2%) or tumor-specific (5%) genes when analyzed group-wise. A tissue similarity index (TSI) was designed based upon singular value decomposition that measured in vivo tumor characteristic gene expression in each cell line independently. Only 34 of the 60 cell lines received the highest TSI toward its tumor of origin. In addition, we identified the most appropriate cell lines to be used as model systems for different in vivo tumors. Seven cell lines were identified as being of another origin than the originally presumed one. The proposed TSI will likely become an important tool for the selection of the most appropriate cell lines in pharmaceutical screening programs and experimental and biomedical research.

  5. Changes in Chromosome Counts and Patterns in CHO Cell Lines upon Generation of Recombinant Cell Lines and Subcloning.

    Science.gov (United States)

    Vcelar, Sabine; Melcher, Michael; Auer, Norbert; Hrdina, Astrid; Puklowski, Anja; Leisch, Friedrich; Jadhav, Vaibhav; Wenger, Till; Baumann, Martina; Borth, Nicole

    2018-03-01

    Chinese hamster ovary (CHO) cells are the number one production system for therapeutic proteins. A pre-requirement for their use in industrial production of biopharmaceuticals is to be clonal, thus originating from a single cell in order to be phenotypically and genomically identical. In the present study it was evaluated whether standard procedures, such as the generation of a recombinant cell line in combination with selection for a specific and stable phenotype (expression of the recombinant product) or subcloning have any impact on karyotype stability or homogeneity in CHO cells. Analyses used were the distribution of chromosome counts per cell as well as chromosome painting to identify specific karyotype patterns within a population. Results indicate that subclones both of the host and the recombinant cell line are of comparable heterogeneity and (in)stability as the original pool. In contrast, the rigorous selection for a stably expressing phenotype generated cell lines with fewer variation and more stable karyotypes, both at the level of the sorted pool and derivative subclones. We conclude that the process of subcloning itself does not contribute to an improved karyotypic homogeneity of a population, while the selection for a specific cell property inherently can provide evolutionary pressure that may lead to improved chromosomal stability as well as to a more homogenous population. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Radiation-induced apoptosis and cell cycle checkpoints in human colorectal tumour cell lines

    International Nuclear Information System (INIS)

    Playle, L.C.

    2001-03-01

    The p53 tumour suppressor gene is mutated in 75% of colorectal carcinomas and is critical for DNA damage-induced G1 cell cycle arrest. Data presented in this thesis demonstrate that after treatment with Ionizing Radiation (IR), colorectal tumour cell lines with mutant p53 are unable to arrest at G1 and undergo cell cycle arrest at G2. The staurosporine derivative, UCN-01, was shown to abrogate the IR-induced G2 checkpoint in colorectal tumour cell lines. Furthermore, in some cell lines, abrogation of the G2 checkpoint was associated with radiosensitisation. Data presented in this study demonstrate that 2 out of 5 cell lines with mutant p53 were sensitised to IR by UCN-01. In order to determine whether radiosensitisation correlated with lack of functional p53, transfected derivatives of an adenoma-derived cell line were studied, in which endogenous wild type p53 was disrupted by expression of a dominant negative p53 mutant protein (and with a vector control). In both these cell lines UCN-01 abrogated the G2 arrest however this was not associated with radiosensitisation, indicating that radiosensitisation is a cell type-specific phenomenon. Although 2 colorectal carcinoma cell lines, with mutant p53, were sensitised to IR by UCN-01, the mechanisms of p53-independent IR-induced apoptosis in the colon are essentially unknown. The mitogen-activated protein kinase (MAPK) pathways (that is the JNK, p38 and ERK pathways) have been implicated in apoptosis in a range of cell systems and in IR-induced apoptosis in some cell types. Data presented in this study show that, although the MAPKs can be activated by the known activator anisomycin, there is no evidence of a role for MAPKs in IR-induced apoptosis in colorectal tumour cell lines, regardless of p53 status. In summary, some colorectal tumour cell lines with mutant p53 can be sensitised to IR-induced cell death by G2 checkpoint abrogation and this may be an important treatment strategy, however mechanisms of IR-induced p53

  7. Procedures for Derivation and Characterisation of Human Embryonic Stem Cells from Odense, Denmark

    DEFF Research Database (Denmark)

    Harkness, Linda; Kassem, Moustapha

    2012-01-01

    In 1998, a development occurred in stem cell biology with the fi rst report of the derivation of a human embryonic stem cell (hESC) line. Since then a number of techniques have been used to derive and characterise hESCs. Here, we describe the derivation methods used by our laboratory for isolation...

  8. Gene expression profiling in chemoresistant variants of three cell lines of different origin

    DEFF Research Database (Denmark)

    Johnsson, Anders; Vallon-Christensson, Johan; Strand, Carina

    2005-01-01

    . Several genes encoding ABC transporters were highly up-regulated, most notably ABCB1 (MDR1) and ABCB4 in several cell lines and ABCG2 (MXR) specifically in MX-resistant cell lines. A pronounced down-regulation of several histones was noted in the MCF-7-derived resistant sublines. Altered expression...

  9. Abnormal A-type lamin organization in a human lung carcinoma cell line

    NARCIS (Netherlands)

    Machiels, BM; Broers, JL; Raymond, Y; de Leij, Louis; Kuijpers, HJH; Caberg, NEH; Ramaekers, Frans C. S.

    We have studied the expression of lamins A and C (A-type lamins) in a lung carcinoma cell line using type-specific monoclonal antibodies, Using immunofluorescence and immunoblotting studies it was noted that several irregularities in lamin expression exist in the cell line GLC-A1, derived from an

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

    Science.gov (United States)

    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.

  11. Glucocorticoid inhibition of cellular proliferation in rat hepatoma cell lines

    International Nuclear Information System (INIS)

    Cook, P.W.

    1987-01-01

    Glucocorticoids were shown to inhibit the growth rate of Fu5 rat hepatoma cells cultured in the presence or absence of serum and thus, induced a more stringent dependence on serum for growth in this cell line. Fu5 cells, made quiescent at low cell density by continuous exposure to glucocorticoid in the absence of serum, were induced with serum and insulin, which subsequently caused a rapid reinitiation of cellular proliferation. Analysis of total RNA isolated from hormone treated Fu5 cells undergoing serum/insulin induction of DNA synthesis revealed a sequential expression of cellular proto-oncogene products in the absence of any immediate changes in intracellular Ca ++ levels. Introduction of functional glucocorticoid receptor genes into both classes of dexamethasone resistant variants restored glucocorticoid responsiveness and suppression of cell growth. The BDS1 rat hepatoma cell line, an Fu5 derived subclone hypersensitive to the antiprofliferation effects of glucocorticoid, was observed to externalize a glucocorticoid suppressible mitogen (GSM) activity capable of mimicking EGF and insulin induced stimulation of [ 3 H]thymidine incorporation into serum starved, competant Balb/c 3T3 cells

  12. Establishment of clinically relevant radioresistant cell lines and their characteristics

    International Nuclear Information System (INIS)

    Fukumoto, Manabu; Kuwahara, Yoshikazu; Suzuki, Masatoshi

    2014-01-01

    Although radiotherapy is one of the major therapeutic modalities for eradicating malignant tumors, the existence of radioresistant cells remains one of the most critical obstacles. Standard radiotherapy consists of fractionated radiation (FR) of 2-Gy X-rays once a day, 5 days a week, over 60 Gy in total. To understand the characteristics of radioresistant cells and to develop more effective radiotherapy, we have established novel radioresistant cell lines by long-term (> 5 years) exposure to moderate doses of fractionated X-rays. While all the parental human cancer cells ceased, their radioresistant derivatives continue to proliferate with daily exposure to 2-Gy FR for more than 30 days. We have coined those cells as 'clinically relevant radioresistant' (CRR) cells. Transplanted tumors into nude mice were also CRR, indicating that CRR cell lines are powerful tools to improve cancer radiotherapy. We have shown that the suppression of autophagic cell death but not apoptosis was mainly involved in cellular radioresistance. An inhibitor of the mTOR pathway which enhances autophagy was effective to overcome CRR tumors induced in nude mice. But the underlined mechanism was not through the inhibition of autophagy. Guanine nucleotide-binding protein 1 (GBP1) over expression was necessary for maintaining the CRR phenotype, but radioresistant cells were not necessarily cancer stem cells (CSCs). Targeting GBP1 positive cancer cells may be a more efficient method in conquering cancer than targeting CSCs. Slight but significant radioresistance was acquired by 0.5 Gy/12 hrs of long-term FR exposures to parental cells for more than 31 days in accordance with cyclinD1 over expression. This acquired radioresistance (ARR) was stably maintained in the tumor cells even on 31 days after the cessation of 0.5-Gy FR. Present observations give a mechanistic insight for ARR of tumor cells through long-term FR exposure, and provide novel therapeutic targets for radiosensitization

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

    DEFF Research Database (Denmark)

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

    1997-01-01

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

  14. Rhodacyanine derivative selectively targets cancer cells and overcomes tamoxifen resistance.

    Directory of Open Access Journals (Sweden)

    John Koren

    Full Text Available MKT-077, a rhodacyanine dye, was shown to produce cancer specific cell death. However, complications prevented the use of this compound beyond clinical trials. Here we describe YM-1, a derivative of MKT-077. We found that YM-1 was more cytotoxic and localized differently than MKT-077. YM-1 demonstrated this cytotoxicity across multiple cancer cell lines. This toxicity was limited to cancer cell lines; immortalized cell models were unaffected. Brief applications of YM-1 were found to be non-toxic. Brief treatment with YM-1 restored tamoxifen sensitivity to a refractory tamoxifen-resistant MCF7 cell model. This effect is potentially due to altered estrogen receptor alpha phosphorylation, an outcome precipitated by selective reductions in Akt levels (Akt/PKB. Thus, modifications to the rhodocyanine scaffold could potentially be made to improve efficacy and pharmacokinetic properties. Moreover, the impact on tamoxifen sensitivity could be a new utility for this compound family.

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

    DEFF Research Database (Denmark)

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

    1997-01-01

    PURPOSE: The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based...... on the construction of continuous exponential growth curves. METHODS AND MATERIALS: Fifteen SCLC cell lines were studied, applying a slightly modified clonogenic assay and a growth extrapolation method. A dose-survival curve was obtained for each experiment and used for calculating several survival parameters...... to calculate the surviving fraction after 2-Gy irradiation (SF2). RESULTS: In our investigation, the extrapolation method proved to be inappropriate for the study of in vitro cellular radiosensitivity due to lack of reproducibility. The results obtained by the clonogenic assay showed that the cell lines...

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

    Directory of Open Access Journals (Sweden)

    Syed Mohmad Shah

    2015-11-01

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

  17. Adipose-Derived Stem Cells

    NARCIS (Netherlands)

    Gathier, WA; Türktas, Z; Duckers, HJ

    2015-01-01

    Until recently bone marrow was perceived to be the only significant reservoir of stem cells in the body. However, it is now recognized that there are other and perhaps even more abundant sources, which include adipose tissue. Subcutaneous fat is readily available in most patients, and can easily be

  18. Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them.

    Directory of Open Access Journals (Sweden)

    Sushil Kumar Mohapatra

    Full Text Available Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT has had a limited applicability due to very low (>1% live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF and Hand-made cloning (TE-HMC, and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF.

  19. Comparative Analysis of Osteogenic/Chondrogenic Differentiation Potential in Primary Limb Bud-Derived and C3H10T1/2 Cell Line-Based Mouse Micromass Cultures

    Directory of Open Access Journals (Sweden)

    Róza Zákány

    2013-08-01

    Full Text Available Murine micromass models have been extensively applied to study chondrogenesis and osteogenesis to elucidate pathways of endochondral bone formation. Here we provide a detailed comparative analysis of the differentiation potential of micromass cultures established from either BMP-2 overexpressing C3H10T1/2 cells or mouse embryonic limb bud-derived chondroprogenitor cells, using micromass cultures from untransfected C3H10T1/2 cells as controls. Although the BMP-2 overexpressing C3H10T1/2 cells failed to form chondrogenic nodules, cells of both models expressed mRNA transcripts for major cartilage-specific marker genes including Sox9, Acan, Col2a1, Snorc, and Hapln1 at similar temporal sequence, while notable lubricin expression was only detected in primary cultures. Furthermore, mRNA transcripts for markers of osteogenic differentiation including Runx2, Osterix, alkaline phosphatase, osteopontin and osteocalcin were detected in both models, along with matrix calcification. Although the adipogenic lineage-specific marker gene FABP4 was also expressed in micromass cultures, Oil Red O-positive cells along with PPARγ2 transcripts were only detected in C3H10T1/2-derived micromass cultures. Apart from lineage-specific marker genes, pluripotency factors (Nanog and Sox2 were also expressed in these models, reflecting on the presence of various mesenchymal lineages as well as undifferentiated cells. This cellular heterogeneity has to be taken into consideration for the interpretation of data obtained by using these models.

  20. Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos

    Directory of Open Access Journals (Sweden)

    Daniela Ávila-González

    2015-09-01

    Full Text Available Data from the literature suggest that human embryonic stem cell (hESC lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1 from poor-quality (PQ embryos derived and maintained on human amniotic epithelial cells (hAEC. This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers.

  1. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  2. The Importance of Physiologically Relevant Cell Lines for Studying Virus–Host Interactions

    Directory of Open Access Journals (Sweden)

    David Hare

    2016-11-01

    Full Text Available Viruses interact intimately with the host cell at nearly every stage of replication, and the cell model that is chosen to study virus infection is critically important. Although primary cells reflect the phenotype of healthy cells in vivo better than cell lines, their limited lifespan makes experimental manipulation challenging. However, many tumor-derived and artificially immortalized cell lines have defects in induction of interferon-stimulated genes and other antiviral defenses. These defects can affect virus replication, especially when cells are infected at lower, more physiologically relevant, multiplicities of infection. Understanding the selective pressures and mechanisms underlying the loss of innate signaling pathways is helpful to choose immortalized cell lines without impaired antiviral defense. We describe the trials and tribulations we encountered while searching for an immortalized cell line with intact innate signaling, and how directed immortalization of primary cells avoids many of the pitfalls of spontaneous immortalization.

  3. Using Program Transformations to Derive Line-Drawing Algorithms

    Science.gov (United States)

    1981-04-01

    Using Program Transformations to I)erime I ine-I)ra ing Algorithms 1. Introduction Many computer graphics devices use "line-drawing algorithms" to...that should be illuminated to display a line. Simplicity and speed the the key design criteria for line-draAing algorithms, because the computations are...ing algorithm is to enumerate those pixels that lie closest to the true linc, the mathenatical line from (0.0) to (dx. d.v). Figurc I illustrites a

  4. Anti-Inflammatory and Cytoprotective Effects of TMC-256C1 from Marine-Derived Fungus Aspergillus sp. SF-6354 via up-Regulation of Heme Oxygenase-1 in Murine Hippocampal and Microglial Cell Lines

    Directory of Open Access Journals (Sweden)

    Dong-Cheol Kim

    2016-04-01

    Full Text Available In the course of searching for bioactive secondary metabolites from marine fungi, TMC-256C1 was isolated from an ethyl acetate extract of the marine-derived fungus Aspergillus sp. SF6354. TMC-256C1 displayed anti-neuroinflammatory effect in BV2 microglial cells induced by lipopolysaccharides (LPS as well as neuroprotective effect against glutamate-stimulated neurotoxicity in mouse hippocampal HT22 cells. TMC-256C1 was shown to develop a cellular resistance to oxidative damage caused by glutamate-induced cytotoxicity and reactive oxygen species (ROS generation in HT22 cells, and suppress the inflammation process in LPS-stimulated BV2 cells. Furthermore, the neuroprotective and anti-neuroinflammatory activities of TMC-256C1 were associated with upregulated expression of heme oxygenase (HO-1 and nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2 in HT22 and BV2 cells. We also found that TMC-256C1 activated p38 mitogen-activated protein kinases (MAPK and phosphatidylinositol 3-kinase (PI3K/Akt signaling pathways in HT22 and BV2 cells. These results demonstrated that TMC-256C1 activates HO-1 protein expression, probably by increasing nuclear Nrf2 levels via the activation of the p38 MAPK and PI3K/Akt pathways.

  5. Engineered cell lines for fish health research.

    Science.gov (United States)

    Collet, Bertrand; Collins, Catherine; Lester, Katherine

    2018-03-01

    As fish farming continues to increase worldwide, the related research areas of fish disease and immunology are also expanding, aided by the revolution in access to genomic information and molecular technology. The genomes of most fish species of economic importance are now available and annotation based on sequence homology with characterised genomes is underway. However, while useful, functional homology is more difficult to determine, there being a lack of widely distributed and well characterised reagents such as monoclonal antibodies, traditionally used in mammalian studies, to help with confirming functions and cellular interactions of fish molecules. In this context, fish cell lines and the possibility of their genetic engineering offer good prospects for studying functional genomics with respect to fish diseases. In this review, we will give an overview of available permanently genetically engineered fish cell lines, as cell-based reporter systems or platforms for expression of endogenous immune or pathogen genes, to investigate interactions and function. The advantages of such systems and the technical challenge for their development will be discussed. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

  6. Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis).

    Science.gov (United States)

    Jager, Martine J; Magner, J Antonio Bermudez; Ksander, Bruce R; Dubovy, Sander R

    2016-08-01

    To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines.

  7. Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis)

    Science.gov (United States)

    Jager, Martine J.; Magner, J. Antonio Bermudez; Ksander, Bruce R.; Dubovy, Sander R.

    2016-01-01

    Purpose To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Methods Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Results Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. Conclusions All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines. PMID:28018010

  8. Tangent Lines without Derivatives for Quadratic and Cubic Equations

    Science.gov (United States)

    Carroll, William J.

    2009-01-01

    In the quadratic equation, y = ax[superscript 2] + bx + c, the equation y = bx + c is identified as the equation of the line tangent to the parabola at its y-intercept. This is extended to give a convenient method of graphing tangent lines at any point on the graph of a quadratic or a cubic equation. (Contains 5 figures.)

  9. Tricyclic antidepressant-induced lipidosis in human peripheral monocytes in vitro, as well as in a monocyte-derived cell line, as monitored by spectrofluorimetry and flow cytometry after staining with Nile red.

    Science.gov (United States)

    Xia, Z; Appelkvist, E L; DePierre, J W; Nässberger, L

    1997-05-15

    Human mono- and lymphocytes from peripheral blood and the monoblastoid cell line U-937 were used in this in vitro study of drug-induced lipidosis. Mono- and lymphocytes were exposed for 4 days to three different tricyclic antidepressants (TCAs), imipramine (25 microM), clomipramine (10 microM) and citalopram (80 microM). The lipophilic fluorophore Nile red, which stains intracellular lipid structures selectively, was used as a lipid probe. Fluorescence microscopy, spectrofluorimetry and flow cytometry were used to detect cellular lipidosis, as verified by electron microscopy. Our results demonstrate that imipramine, clomipramine and citalopram induce lipidosis in monocytes and U-937 cells, but not in lymphocytes. An accurate quantitation of induced intracellular lipidosis can be achieved by spectrofluorimetric and flow cytometric analysis.

  10. Tumor-derived exosomes induce CD8+T cell suppressors.

    Science.gov (United States)

    Maybruck, Brian T; Pfannenstiel, Lukas W; Diaz-Montero, Marcela; Gastman, Brian R

    2017-08-15

    The suppressive nature of immune cells in the tumor microenvironment plays a major role in regulating anti-tumor immune responses. Our previous work demonstrated that a soluble factor from tumor cells is able to induce a suppressor phenotype (SP) in human CD8 + T cells typified by loss of CD27/CD28 expression and acquisition of a potent suppressor function. The present study hypothesized that the soluble mechanism that is inducing the SP in CD8 + T cells are tumor-derived exosomes (TDEs). Membrane vesicles and TDEs from multiple head and neck cancer cell line's conditioned growth media were isolated by ultracentrifugation and precipitation, respectively. Human purified CD3 + CD8 + T cells were assessed for their induction of the T cell SP by flow cytometry identifying loss of CD27/CD28 expression and in vitro suppression assays. Furthermore, the T cell SP was characterized for the attenuation of IFN-γ production. To delineate exosomal proteins contributing to T cell SP, mass spectrometry was used to identify unique proteins that were present in TDEs. CRISPR/Cas9 knockout constructs were used to examine the role of one of these proteins, galectin-1. To assess the role of exosomal RNA, RNA purified from TDEs was nucleofected into CD8 + T cells followed by suppression analysis. Using fractionated conditioned growth media, factors >200 kDa induced CD8 + T cell SP, which was determined to be an exosome by mass spectrometry analysis. Multiple head and neck cancer-derived cell lines were found to secrete T cell SP-inducing exosomes. Mass spectrometry analysis revealed that an immunoregulatory protein, galectin-1 (Gal-1), was expressed in those exosomes, but not in TDEs unable to induce T cell SP. Galectin-1 knockout cells were found to be less able to induce T cell SP. Furthermore, RNA purified from the T cell SP-inducing exosomes were found to partially induce the SP when transfected into normal CD8 + T cells. For the first-time, TDEs have been identified to induce a

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

    Directory of Open Access Journals (Sweden)

    Rajvi H Mehta

    2014-01-01

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

  12. Sourcing human embryos for embryonic stem cell lines: problems & perspectives.

    Science.gov (United States)

    Mehta, Rajvi H

    2014-11-01

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

  13. Presence of dopamine D-2 receptors in human tumoral cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Sokoloff, P.; Riou, J.F.; Martres, M.P.; Schwartz, J.C. (Centre Paul Broca, Paris (France))

    1989-07-31

    ({sup 125}I) Iodosulpride binding was examined on eight human cell lines derived from lung, breast and digestive tract carcinomas, neuroblastomas and leukemia. Specific binding was detected in five of these cell lines. In the richest cell line N417, derived from small cell lung carcinoma, ({sup 125}I) iodosulpride bound with a high affinity (Kd = 1.3 nM) to an apparently homogeneous population of binding site (Bmax = 1,606 sites per cell). These sites displayed a typical D-2 specificity, established with several dopaminergic agonists and antagonists selective of either D-1 or D-2 receptor subtypes. In addition, dopamine, apomorphine and RU 24926 distinguished high- and low-affinity sites, suggesting that the binding sites are associated with a G-protein. The biological significance and the possible diagnostic implication of the presence of D-2 receptors on these cell lines are discussed.

  14. Radiosensitivity of Human Melanoma Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-07-01

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

  15. Histone signature of metanephric mesenchyme cell lines.

    Science.gov (United States)

    McLaughlin, Nathan; Yao, Xiao; Li, Yuwen; Saifudeen, Zubaida; El-Dahr, Samir S

    2013-09-01

    The metanephric mesenchyme (MM) gives rise to nephrons, the filtering units of the mature kidney. The MM is composed of uninduced (Six2(high)/Lhx1(low)) and induced (Wnt-stimulated, Six2(low)/Lhx1(high)) cells. The global epigenetic state of MM cells is unknown, partly due to technical difficulty in isolating sufficient numbers of homogenous cell populations. We therefore took advantage of two mouse clonal cell lines representing the uninduced (mK3) and induced (mK4) metanephric mesenchyme (based on gene expression profiles and ability to induce branching of ureteric bud). ChIP-Seq revealed that whereas H3K4me3 active region "peaks" are enriched in metabolic genes, H3K27me3 peaks decorate mesenchyme and epithelial cell fate commitment genes. In uninduced mK3 cells, promoters of "stemness" genes (e.g., Six2, Osr1) are enriched with H3K4me3 peaks; these are lost in induced mK4 cells. ChIP-qPCR confirmed this finding and further demonstrated that G9a/H3K9me2 occupy the promoter region of Six2 in induced cells, consistent with the inactive state of transcription. Conversely, genes that mark the induced epithelialized state (e.g., Lhx1, Pax8), transition from a non-permissive to an active chromatin signature in mK3 vs. mK4 cells, respectively. Importantly, stimulation of Wnt signaling in uninduced mK3 cells provokes an active chromatin state (high H3K4me3, low H3K27me3), recruitment of β-catenin, and loss of pre-bound histone methyltransferase Ezh2 in silent induced genes followed by activation of transcription. We conclude that the chromatin signature of uninduced and induced cells correlates strongly with their gene expression states, suggesting a role of chromatin-based mechanisms in MM cell fate.

  16. Transplantability of human lymphoid cell line, lymphoma, and leukemia in splenectomized and/or irradiated nude mice

    International Nuclear Information System (INIS)

    Watanabe, S.; Shimosato, Y.; Kuroki, M.; Sato, Y.; Nakajima, T.

    1980-01-01

    The effects of splenectomy and/or whole-body irradiation of nude mice before xenotransplantation of lymphoid cell lines, lymphoma, and leukemia were studied. Transplantation after whole-body irradiation resulted in the increased ''take'' rate of three cultured cell lines (two of T-cell-derived acute lymphocytic leukemia and one of B-cell derived acute lymphocytic leukemia) and in the tumorous growth of Burkitt-derived Raji and spontaneously transformed lymphoblastoid cell lines. With splenectomy plus irradiation as a pretreatment, tumorous growth occurred in four other cell lines which were not transplantable after irradiation only (two cell lines of Epstein-Barr virus-transformed cord blood cells and one each of null acute lymphocytic leukemia and nodular lymphoma-derived cell lines). Direct transplantation of leukemia and lymphoma cells into the pretreated mice was successful in 7 of 24 cases (29%). B-cell-derived diffuse large lymphoid lymphoma was transplantable in three of seven cases (43%). However, lymphoma and leukemia of peripheral T-cell origin was difficult to transplant even with pretreatment, and only one pleomorphic T-cell lymphoma grew to a significant size (2 cm). One tumor each of B-cell-derived diffuse large lymphoid and T-cell diffuse lymphoblastic lymphoma became transplantable

  17. Menadione inhibits MIBG uptake in two neuroendocrine cell lines

    NARCIS (Netherlands)

    Cornelissen, J.; Tytgat, G. A.; van den Brug, M.; van Kuilenburg, A. B.; Voûte, P. A.; van Gennip, A. H.

    1997-01-01

    In this paper we report on our studies of the effect of menadione on the uptake of MIBG in the neuroendocrine cell lines PC12 and SK-N-SH. Menadione inhibits the uptake of MIBG in both cell lines in a dose-dependent manner. Inhibition of MIBG uptake is most pronounced in the PC12 cell line.

  18. 77 FR 5489 - Identification of Human Cell Lines Project

    Science.gov (United States)

    2012-02-03

    ... selection of this technology over other possible candidates for this project include: (i) The ability to... cell line, whether the cell line is misidentified, cross- contaminated, or genetically unstable... database. Submission Process: Submitters should contact Margaret Kline with a list of proposed cell lines...

  19. Novel stable HBV producing cell line systems for expression and screening antiviral inhibitor of hepatitis B virus in human hepatoma cell line.

    Science.gov (United States)

    Ogura, Naoki; Ogawa, Kazuya; Watashi, Koichi; Ito, Takayoshi; Wakita, Takaji

    2018-03-25

    Chronic hepatitis B virus (HBV) infection is currently a major public health burden. Therefore, there is an urgent need for the development of novel antiviral inhibitors. The stable HBV-producing cell lines of genotype D are widely used to investigate the HBV life cycle and to evaluate antiviral agents. However, stable HBV-producing cell lines of different genotypes do not exist. To construct more convenient and efficient novel cell systems, stable cell lines of genotypes A, B, and C were established using a full-length HBV genome sequence isolated from chronic HBV patients in human hepatoma HepG2 cells. Novel HBV clones were identified and stable HBV-producing cell lines derived from these clones were constructed. HBV replication activities demonstrated time-dependent expression, and the novel cell lines were susceptible to several antiviral inhibitors with no cytotoxicity. Furthermore, infectious viruses were produced from these cell lines. In conclusion, we have established novel stable HBV-producing cell line systems of genotypes A, B, and C. These systems can provide valuable tools for screening antiviral agents and analyzing viral phenotypes in vitro. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Trophoblast lineage cells derived from human induced pluripotent stem cells

    International Nuclear Information System (INIS)

    Chen, Ying; Wang, Kai; Chandramouli, Gadisetti V.R.; Knott, Jason G.; Leach, Richard

    2013-01-01

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro

  1. Trophoblast lineage cells derived from human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying, E-mail: ying.chen@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Wang, Kai; Chandramouli, Gadisetti V.R. [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Knott, Jason G. [Developmental Epigenetics Laboratory, Department of Animal Science, Michigan State University (United States); Leach, Richard, E-mail: Richard.leach@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Department of Obstetrics, Gynecology and Women’s Health, Spectrum Health Medical Group (United States)

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  2. S100 protein expression in human melanoma cells: Comparison of levels of expression among different cell lines and individual cells in different phases of the cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Marks, A.; O' Hanlon, D.; Dunn, R. (Univ. of Toronto, Ontario (Canada)); Petsche, D.; Baumal, R. (Hospital for Sick Children, Toronto, Ontario (Canada)); Kwong, P.C.; Stead, R. (McMaster Univ., Hamilton, Ontario (Canada)); Liao, S.K. (McMaster Univ., Hamilton, Ontario (Canada) Biotherapeutics, Inc., Franklin, TN (United States))

    1990-03-01

    The synthesis of S100 protein in cultured human melanoma cells was examined using metabolic labeling with ({sup 35}S)methionine, immunoprecipitation with anti-S100 protein antiserum, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Six of seven cell lines derived from melanomas synthesized relatively large amounts of S100 protein, whereas three cell lines derived from normal melanocytes synthesized lesser amounts. Synthesis of S100 protein was not detected in 10 human cell lines of non-neuroectodermal origin. Analysis of poly(A{sup +}) RNA form one melanoma cell line by Northern blot hybridization with a probe specific for the {beta} subunit of rat S100 protein revealed a single mRNA species of 1.0 kb coding for the human protein. Flow cytometric analysis of individual cells of two melanoma cell lines and the rat glioma cell line C6 indicated that G0/G1 cells were heterogeneous with respect to S100 protein expression, while almost all the cells in S+G2+M expressed S100 protein. These results suggest that expression of S100 protein in G0/G1 could be a prerequisite for progression of the cells through the cell cycle.

  3. Low antigenicity of hematopoietic progenitor cells derived from human ES cells

    Directory of Open Access Journals (Sweden)

    Eun-Mi Kim

    2010-02-01

    Full Text Available Eun-Mi Kim1, Nicholas Zavazava1,21Department of Internal Medicine, University of Iowa and Veterans Affairs Medical Center, Iowa City, Iowa, USA; 2Immunology Graduate Program, University of Iowa, Iowa City, Iowa, USAAbstract: Human embryonic stem (hES cells are essential for improved understanding of diseases and our ability to probe new therapies for use in humans. Currently, bone marrow cells and cord blood cells are used for transplantation into patients with hematopoietic malignancies, immunodeficiencies and in some cases for the treatment of autoimmune diseases. However, due to the high immunogenicity of these hematopoietic cells, toxic regimens of drugs are required for preconditioning and prevention of rejection. Here, we investigated the efficiency of deriving hematopoietic progenitor cells (HPCs from the hES cell line H13, after co-culturing with the murine stromal cell line OP9. We show that HPCs derived from the H13 ES cells poorly express major histocompatibility complex (MHC class I and no detectable class II antigens (HLA-DR. These characteristics make hES cell-derived hematopoietic cells (HPCs ideal candidates for transplantation across MHC barriers under minimal immunosuppression.Keywords: human embryonic stem cells, H13, hematopoiesis, OP9 stromal cells, immunogenicity

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    UV light excites aromatic residues, causing these to disrupt nearby disulphide bridges. The EGF receptor is rich in aromatic residues near the disulphide bridges. Herein we show that laser-pulsed UV illumination of two different skin-derived cancer cell lines i.e. Cal-39 and A431, which both...... antibodies. There was a threshold level, below which the receptor could not be blocked. In addition, illumination caused the cells to upregulate the cyclin-dependent kinase inhibitor p21WAF1, irrespective of the p53 status. Since the EGF receptor is often overexpressed in cancers and other proliferative skin...... disorders, it might be possible to significantly reduce the proliferative potential of these cells making them good targets for laser-pulsed UV light treatment....

  5. Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines.

    Directory of Open Access Journals (Sweden)

    Olivier Féraud

    Full Text Available Hematopoiesis generated from human embryonic stem cells (ES and induced pluripotent stem cells (iPS are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process.

  6. Interaction between x-irradiated plateau-phase bone marrow stromal cell lines and co-cultivated factor-dependent cell lines leading to leukemogenesis in vitro

    International Nuclear Information System (INIS)

    Naparstek, E.; Anklesaria, P.; FitzGerald, T.J.; Sakakeeny, M.A.; Greenberger, J.S.

    1987-01-01

    Plateau-phase mouse clonal bone marrow stromal cell lines D2XRII and C3H cl 11 produce decreasing levels of M-CSF (CSF-1), a specific macrophage progenitor cell humoral regulator, following X-irradiation in vitro. The decrease did not go below 40% of control levels, even after irradiation doses of 50,000 rad (500 Gy). In contrast, a distinct humoral regulator stimulating growth of GM-CSF/IL-3 factor-dependent (FD) hematopoietic progenitor cell lines was detected following radiation to doses above 2000 rad. This humoral factor was not detectable in conditioned medium from irradiated cells, weakly detected using factor-dependent target cell populations in agar overlay, and was prominently detected by liquid co-cultivation of factor-dependent cells with irradiated stromal cell cultures. Subclonal lines of FD cells, derived after co-cultivation revealed karyotypic abnormalities and induced myeloblastic tumors in syngeneic mice. Five-eight weeks co-cultivation was required for induction of factor independence and malignancy and was associated with dense cell to cell contact between FD cells and stromal cells demonstrated by light and electron microscopy. Increases in hematopoietic to stromal cell surface area, total number of adherent cells per flask, total non-adherent cell colonies per flask, and cumulative non-adherent cell production were observed after irradiation. The present data may prove very relevant to an understanding of the cell to cell interactions during X-irradiation-induced leukemia

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

    Directory of Open Access Journals (Sweden)

    Anja M Oelschlegel

    Full Text Available Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice. We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases.

  8. Paulomycin G, a New Natural Product with Cytotoxic Activity against Tumor Cell Lines Produced by Deep-Sea Sediment Derived Micromonospora matsumotoense M-412 from the Avilés Canyon in the Cantabrian Sea.

    Science.gov (United States)

    Sarmiento-Vizcaíno, Aida; Braña, Alfredo F; Pérez-Victoria, Ignacio; Martín, Jesús; de Pedro, Nuria; Cruz, Mercedes de la; Díaz, Caridad; Vicente, Francisca; Acuña, José L; Reyes, Fernando; García, Luis A; Blanco, Gloria

    2017-08-28

    The present article describes a structurally novel natural product of the paulomycin family, designated as paulomycin G ( 1 ), obtained from the marine strain Micromonospora matsumotoense M-412, isolated from Cantabrian Sea sediments collected at 2000 m depth during an oceanographic expedition to the submarine Avilés Canyon. Paulomycin G is structurally unique since-to our knowledge-it is the first member of the paulomycin family of antibiotics lacking the paulomycose moiety. It is also the smallest bioactive paulomycin reported. Its structure was determined using HRMS and 1D and 2D NMR spectroscopy. This novel natural product displays strong cytotoxic activities against different human tumour cell lines, such as pancreatic adenocarcinoma (MiaPaca_2), breast adenocarcinoma (MCF-7), and hepatocellular carcinoma (HepG2). The compound did not show any significant bioactivity when tested against a panel of bacterial and fungal pathogens.

  9. Characterization of human follicular thyroid cancer cell lines in preclinical mouse models

    Directory of Open Access Journals (Sweden)

    Ashley N Reeb

    2016-04-01

    Full Text Available Follicular thyroid cancer (FTC is the second most common type of thyroid cancers. In order to develop more effective personalized therapies, it is necessary to thoroughly evaluate patient-derived cell lines in in vivo preclinical models before using them to test new, targeted therapies. This study evaluates the tumorigenic and metastatic potential of a panel of three human FTC cell lines (WRO, FTC-238, and TT1609-CO2 with defined genetic mutations in two in vivo murine models: an orthotopic thyroid cancer model to study tumor progression and a tail vein injection model to study metastasis. All cell lines developed tumors in the orthotopic model, with take rates of 100%. Notably, WRO-derived tumors grew two to four times faster than tumors arising from the FTC-238 and TT2609-CO2 cell lines. These results mirrored those of a tail vein injection model for lung metastasis: one hundred percent of mice injected with WRO cells in the tail vein exhibited aggressive growth of bilateral lung metastases within 35 days. In contrast, tail vein injection of FTC-238 or TT2609-CO2 cells did not result in lung metastasis. Together, our work demonstrates that these human FTC cell lines display highly varied tumorigenic and metastatic potential in vivo with WRO being the most aggressive cell line in both orthotopic and lung metastasis models. This information will be valuable when selecting cell lines for preclinical drug testing.

  10. Familial Follicular-Cell Derived Carcinoma

    Directory of Open Access Journals (Sweden)

    Eun Ju eSon

    2012-05-01

    Full Text Available Follicular cell-derived well-differentiated thyroid cancer, papillary (PTC and follicular thyroid carcinomas (FTC compose 95% of all thyroid malignancies. Familial follicular cell-derived well-differentiated thyroid cancers contribute to 5% of those cases. These familial follicular cell derived carcinomas or non-medullary thyroid carcinomas (NMTC divide into two clinical-pathological groups. One group, syndromic-associated, composed by predominately non-thyroidal tumors, is comprised of Pendred syndrome, Warner syndrome, Carney complex type 1, PTEN-hamartoma tumor syndrome (Cowden disease; PHTS, familial adenomatous polyposis (FAP/Gardner syndrome. Additionally other less established links correlated to the development of follicular cell-derived tumors have also included Ataxia-teleangiectasia syndrome, McCune Albright syndrome, and Peutz-Jeghers syndrome. The subsequent group encompasses syndromes typified by non-medullary thyroid carcinomas or NMTC, as well as, pure familial (f PTC with or without oxyphilia, fPTC with multinodular goiter and fPTC with papillary renal cell carcinoma. This heterogeneous group of diseases has not a established genotype-phenotype correlation as the well-known genetic events identified in the familial C-cell-derived tumors or medullary thyroid carcinomas (MTC. Clinicians should be have the knowledge to identify the likelihood of a patient presenting with thyroid cancer having an additional underlying familial syndrome stemming from characteristics through morphological findings that would alert the pathologist to have the patient undergo subsequent molecular genetics evaluations. This review will discuss the clinical and pathological findings of the patients with familial papillary thyroid carcinoma, such as familial adenomatous polyposis, Carney complex, Werner syndrome, and Pendred syndrome and the heterogeneous group of familial papillary thyroid carcinoma.

  11. Rigorous theoretical derivation of lumped models to transmission line systems

    International Nuclear Information System (INIS)

    Zhao Jixiang

    2012-01-01

    By virtue of the negative electric parameter concept, i.e. negative lumped resistance, inductance, conductance and capacitance (N-RLGC), the lumped equivalent models of transmission line systems, including the circuit model, two-port π-network and T-network, are given. We start from the N-segment-ladder-like equivalent networks composed distributed parameters, and achieve the input impedance in the form of a continued fraction. Utilizing the continued fraction theory, the expressions of input impedance are obtained under three kinds of extreme cases, i.e. the load impedances are equal to zero, infinity and characteristic impedance, respectively. When the number of segment N is limited to infinity, they are transformed to lumped elements. Comparison between the distributed model and lumped model of transmission lines, the expression of tanh γd, which is the key term in the transmission line equations, are obtained by RLGC, furthermore, according to input admittance, admittance matrix and ABCD matrix of transmission lines, the lumped equivalent circuit models, π-networks and T-networks have been given. The models are verified in the frequency and time domain, respectively, showing that the models are accurate and efficient. (semiconductor integrated circuits)

  12. 'Fluorescent Cell Chip' for immunotoxicity testing: Development of the c-fos expression reporter cell lines

    International Nuclear Information System (INIS)

    Trzaska, Dominika; Zembek, Patrycja; Olszewski, Maciej; Adamczewska, Violetta; Ulleras, Erik; Dastych, JarosIaw

    2005-01-01

    The Fluorescent Cell Chip for in vitro immunotoxicity testing employs cell lines derived from lymphocytes, mast cells, and monocytes-macrophages transfected with various EGFP cytokine reporter gene constructs. While cytokine expression is a valid endpoint for in vitro immunotoxicity screening, additional marker for the immediate-early response gene expression level could be of interest for further development and refinement of the Fluorescent Cell Chip. We have used BW.5147.3 murine thymoma transfected with c-fos reporter constructs to obtain reporter cell lines expressing ECFP under the control of murine c-fos promoter. These cells upon serum withdrawal and readdition and incubation with heavy metal compounds showed paralleled induction of c-Fos expression as evidenced by Real-Time PCR and ECFP fluorescence as evidenced by computer-supported fluorescence microscopy. In conclusion, we developed fluorescent reporter cell lines that could be employed in a simple and time-efficient screening assay for possible action of chemicals on c-Fos expression in lymphocytes. The evaluation of usefulness of these cells for the Fluorescent Cell Chip-based detection of immunotoxicity will require additional testing with a larger number of chemicals

  13. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Itoigawa, Yoshiaki [Tohoku University School of Medicine, Sendai (Japan); Juntendo University School of Medicine, Tokyo (Japan); Kishimoto, Koshi N., E-mail: kishimoto@med.tohoku.ac.jp [Tohoku University School of Medicine, Sendai (Japan); Okuno, Hiroshi; Sano, Hirotaka [Tohoku University School of Medicine, Sendai (Japan); Kaneko, Kazuo [Juntendo University School of Medicine, Tokyo (Japan); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan)

    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.

  14. CD3 receptor modulation in Jurkat leukemic cell line.

    Directory of Open Access Journals (Sweden)

    Jacek M Witkowski

    2004-03-01

    Full Text Available CD3 antigen is a crucial molecule in T cell signal transduction. Although its expression on cell surface is constitutive, dynamic regulation of TCR-CD3 level is probably the most important mechanism allowing T cells to calibrate their response to different levels of stimuli. In our study we examined the role of two main T cell signal transduction pathways in controlling the surface level of CD3 antigen, one based on protein kinase C activity and the other dependent on calcineurin. As an experimental model we used three clones derived from Jurkat cell line, expressing different levels of CD3 antigen surface expression: CD3(low (217.6, CD3+(217.9 or CD3(low (217.7. The cells were stimulated with PMA or ionomycin, acting directly on PKC and calcineurin, respectively. Prior to the stimulation cells were incubated with PKC inhibitor--chelerythrine or calcineurin blocker--cyclosporine A. Changes in CD3 surface expression were measured by flow cytometry. Only PMA and chelerythrine were able to change CD3 expression suggesting important involvement of PKC in the regulation of its expression. To confirm these findings, PKC activity was estimated in Jurkat clones. Our data demonstrated that Jurkat clones with different CD3 expression showed also different PKC activities, so we conclude that PKC-dependent pathway is the main way of controlling CD3 level on Jurkat clones.

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

    International Nuclear Information System (INIS)

    Krarup, Marianne; Poulsen, Hans Skovgaard; Spang-Thomsen, Mogens

    1997-01-01

    Purpose: The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based on the construction of continuous exponential growth curves. Methods and Materials: Fifteen SCLC cell lines were studied, applying a slightly modified clonogenic assay and a growth extrapolation method. A dose-survival curve was obtained for each experiment and used for calculating several survival parameters. The multitarget single hit model was applied to calculate the cellular radiosensitivity (D 0 ), the capacity for sublethal damage repair (D q ), and the extrapolation number (n). Values for α and β were determined from best-fit curves according to the linear-quadratic model and these values were applied to calculate the surviving fraction after 2-Gy irradiation (SF 2 ). Results: In our investigation, the extrapolation method proved to be inappropriate for the study of in vitro cellular radiosensitivity due to lack of reproducibility. The results obtained by the clonogenic assay showed that the cell lines studied were radiobiologically heterogeneous with no discrete features of the examined parameters including the repair capacity. Conclusion: The results indicate that SCLC tumors per se are not generally candidates for hyperfractionated radiotherapy

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

    Directory of Open Access Journals (Sweden)

    Zhang Ping

    2006-09-01

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

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

    International Nuclear Information System (INIS)

    Zhang, Ping; Zhang, Zhiyuan; Zhou, Xiaojian; Qiu, Weiliu; Chen, Fangan; Chen, Wantao

    2006-01-01

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

  18. Curcumin Modulates Pancreatic Adenocarcinoma Cell-Derived Exosomal Function

    Science.gov (United States)

    Osterman, Carlos J. Diaz; Lynch, James C.; Leaf, Patrick; Gonda, Amber; Ferguson Bennit, Heather R.; Griffiths, Duncan; Wall, Nathan R.

    2015-01-01

    Pancreatic cancer has the highest mortality rates of all cancer types. One potential explanation for the aggressiveness of this disease is that cancer cells have been found to communicate with one another using membrane-bound vesicles known as exosomes. These exosomes carry pro-survival molecules and increase the proliferation, survival, and metastatic potential of recipient cells, suggesting that tumor-derived exosomes are powerful drivers of tumor progression. Thus, to successfully address and eradicate pancreatic cancer, it is imperative to develop therapeutic strategies that neutralize cancer cells and exosomes simultaneously. Curcumin, a turmeric root derivative, has been shown to have potent anti-cancer and anti-inflammatory effects in vitro and in vivo. Recent studies have suggested that exosomal curcumin exerts anti-inflammatory properties on recipient cells. However, curcumin’s effects on exosomal pro-tumor function have yet to be determined. We hypothesize that curcumin will alter the pro-survival role of exosomes from pancreatic cancer cells toward a pro-death role, resulting in reduced cell viability of recipient pancreatic cancer cells. The main objective of this study was to determine the functional alterations of exosomes released by pancreatic cancer cells exposed to curcumin compared to exosomes from untreated pancreatic cancer cells. We demonstrate, using an in vitro cell culture model involving pancreatic adenocarcinoma cell lines PANC-1 and MIA PaCa-2, that curcumin is incorporated into exosomes isolated from curcumin-treated pancreatic cancer cells as observed by spectral studies and fluorescence microscopy. Furthermore, curcumin is delivered to recipient pancreatic cancer cells via exosomes, promoting cytotoxicity as demonstrated by Hoffman modulation contrast microscopy as well as AlamarBlue and Trypan blue exclusion assays. Collectively, these data suggest that the efficacy of curcumin may be enhanced in pancreatic cancer cells through

  19. Cell sex affects extracellular matrix protein expression and proliferation of smooth muscle progenitor cells derived from human pluripotent stem cells.

    Science.gov (United States)

    Li, Yanhui; Wen, Yan; Green, Morgaine; Cabral, Elise K; Wani, Prachi; Zhang, Fan; Wei, Yi; Baer, Thomas M; Chen, Bertha

    2017-07-04

    Smooth muscle progenitor cells (pSMCs) differentiated from human pluripotent stem cells (hPSCs) hold great promise for treating diseases or degenerative conditions involving smooth muscle pathologies. However, the therapeutic potential of pSMCs derived from men and women may be very different. Cell sex can exert a profound impact on the differentiation process of stem cells into somatic cells. In spite of advances in translation of stem cell technologies, the role of cell sex and the effect of sex hormones on the differentiation towards mesenchymal lineage pSMCs remain largely unexplored. Using a standard differentiation protocol, two human embryonic stem cell lines (one male line and one female line) and three induced pluripotent stem cell lines (one male line and two female lines) were differentiated into pSMCs. We examined differences in the differentiation of male and female hPSCs into pSMCs, and investigated the effect of 17β-estradiol (E2) on the extracellular matrix (ECM) metabolisms and cell proliferation rates of the pSMCs. Statistical analyses were performed by using Student's t test or two-way ANOVA, p cell proliferation rates. E2 treatment induced the expression of myogenic gene markers and suppressed ECM degradation activities through reduction of MMP activity and increased expression of TIMP-1 in female pSMCs, but not in male pSMCs. hPSC-derived pSMCs from different sexes show differential expression of ECM proteins and proliferation rates. Estrogen appears to promote maturation and ECM protein expression in female pSMCs, but not in male pSMCs. These data suggest that intrinsic cell-sex differences may influence progenitor cell biology.

  20. A protocol for embryonic stem cell derivation by somatic cell nuclear transfer into human oocytes

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Dieter Egli & Gloryn Chia ### Abstract Here we describe detailed methods that allowed us to derive embryonic stem cell lines by nuclear transfer of fibroblasts from a newborn and from a type 1 diabetic adult. The protocol is based on the insight that 1) agents for cell fusion can act as potent mediators of oocyte activation by compromising maintaining plasma membrane integrity; minimizing the concentration at which they are used, and at least transiently remove calcium from ...

  1. Dihydrochalcone Compounds Isolated from Crabapple Leaves Showed Anticancer Effects on Human Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Xiaoxiao Qin

    2015-11-01

    Full Text Available Seven dihydrochalcone compounds were isolated from the leaves of Malus crabapples, cv. “Radiant”, and their chemical structures were elucidated by UV, IR, ESI-MS, 1H-NMR and 13C-NMR analyses. These compounds, which include trilobatin (A1, phloretin (A2, 3-hydroxyphloretin (A3, phloretin rutinoside (A4, phlorizin (A5, 6′′-O-coumaroyl-4′-O-glucopyranosylphloretin (A6, and 3′′′-methoxy-6′′-O-feruloy-4′-O-glucopyranosyl-phloretin (A7, all belong to the phloretin class and its derivatives. Compounds A6 and A7 are two new rare dihydrochalcone compounds. The results of a MTT cancer cell growth inhibition assay demonstrated that phloretin and these derivatives showed significant positive anticancer activities against several human cancer cell lines, including the A549 human lung cancer cell line, Bel 7402 liver cancer cell line, HepG2 human ileocecal cancer cell line, and HT-29 human colon cancer cell line. A7 had significant effects on all cancer cell lines, suggesting potential applications for phloretin and its derivatives. Adding a methoxyl group to phloretin dramatically increases phloretin’s anticancer activity.

  2. Extracellular vesicles from a muscle cell line (C2C12) enhance cell survival and neurite outgrowth of a motor neuron cell line (NSC-34).

    Science.gov (United States)

    Madison, Roger D; McGee, Christopher; Rawson, Renee; Robinson, Grant A

    2014-01-01

    There is renewed interest in extracellular vesicles over the past decade or 2 after initially being thought of as simple cellular garbage cans to rid cells of unwanted components. Although there has been intense research into the role of extracellular vesicles in the fields of tumour and stem cell biology, the possible role of extracellular vesicles in nerve regeneration is just in its infancy. When a peripheral nerve is damaged, the communication between spinal cord motor neurons and their target muscles is disrupted and the result can be the loss of coordinated muscle movement. Despite state-of-the-art surgical procedures only approximately 10% of adults will recover full function after peripheral nerve repair. To improve upon such results will require a better understanding of the basic mechanisms that influence axon outgrowth and the interplay between the parent motor neuron and the distal end organ of muscle. It has previously been shown that extracellular vesicles are immunologically tolerated, display targeting ligands on their surface, and can be delivered in vivo to selected cell populations. All of these characteristics suggest that extracellular vesicles could play a significant role in nerve regeneration. We have carried out studies using 2 very well characterized cell lines, the C2C12 muscle cell line and the motor neuron cell line NSC-34 to ask the question: Do extracellular vesicles from muscle influence cell survival and/or neurite outgrowth of motor neurons? Our results show striking effects of extracellular vesicles derived from the muscle cell line on the motor neuron cell line in terms of neurite outgrowth and survival.

  3. Expression and function of β-adrenergic receptors in human hematopoietic cell lines

    International Nuclear Information System (INIS)

    Maeki, T.; Andersson, L.C.; Kontula, K.K.

    1992-01-01

    We investigated the expression and functional characteristics of β-adrenoceptors in a panel of 10 phenotypically different human hematopoietic cell lines. A binding assay with [ 125 I]iodocyanopindolol as the ligand revealed that cell lines of myelomonocytic or histiocytic derivation (HL-60, ML-2, RC-2A, U-937) expressed high numbers of β-adrenoceptors. An intermediate density of receptors was found in a non-T, non-B cell leukemia line (Nall-1), whereas T-cell (JM, CCRF-CEM), B-cell (Raji) or erythroleukemic cell lines (K-562, HEL) displayed minimala or undetectable binding of the radioligand. Isoprenaline-stimulated cAMP production by the cells correlated to their extent of β-adrenoceptor expression. Southern blot hybridization analysis of genomic DNA from the cell lines with a 32 P-labelled β 2 -adrenoceptor cDNA probe revealed no evidence for major rearrangement or amplification of the receptor gene. Incubation with isoprenaline in vitro suppressed the proliferation of the receptor-rich RC-2A cells but did not affect the growth rate of the receptor-deficient K-562 cells. Treatment with propranolol slightly enhanced the proliferation of the RC-2A cells but did not markedly alter the growth rate of two other cell lines, regardless of their β-adrenoceptor status. These findings indicate a regulatory influence by the sympathoadrenergic system on selected cells of the myelomonocytic lineage. (au)

  4. Check your cultures! A list of cross-contaminated or misidentified cell lines.

    Science.gov (United States)

    Capes-Davis, Amanda; Theodosopoulos, George; Atkin, Isobel; Drexler, Hans G; Kohara, Arihiro; MacLeod, Roderick A F; Masters, John R; Nakamura, Yukio; Reid, Yvonne A; Reddel, Roger R; Freshney, R Ian

    2010-07-01

    Continuous cell lines consist of cultured cells derived from a specific donor and tissue of origin that have acquired the ability to proliferate indefinitely. These cell lines are well-recognized models for the study of health and disease, particularly for cancer. However, there are cautions to be aware of when using continuous cell lines, including the possibility of contamination, in which a foreign cell line or microorganism is introduced without the handler's knowledge. Cross-contamination, in which the contaminant is another cell line, was first recognized in the 1950s but, disturbingly, remains a serious issue today. Many cell lines become cross-contaminated early, so that subsequent experimental work has been performed only on the contaminant, masquerading under a different name. What can be done in response-how can a researcher know if their own cell lines are cross-contaminated? Two practical responses are suggested here. First, it is important to check the literature, looking for previous work on cross-contamination. Some reports may be difficult to find and to make these more accessible, we have compiled a list of known cross-contaminated cell lines. The list currently contains 360 cell lines, drawn from 68 references. Most contaminants arise within the same species, with HeLa still the most frequently encountered (29%, 106/360) among human cell lines, but interspecies contaminants account for a small but substantial minority of cases (9%, 33/360). Second, even if there are no previous publications on cross-contamination for that cell line, it is essential to check the sample itself by performing authentication testing.

  5. Whole-cell biosensor for label-free detection of GPCR-mediated drug responses in personal cell lines

    NARCIS (Netherlands)

    Hillger, J.M.; Schoop, J.; Boomsma, D.I.; Slagboom, P.E.; IJzerman, A.P.; Heitman, L.H.

    2015-01-01

    Deciphering how genetic variation in drug targets such as G protein-coupled receptors (GPCRs) affects drug response is essential for precision medicine. GPCR signaling is traditionally investigated in artificial cell lines which do not provide sufficient physiological context. Patient-derived cell

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

    NARCIS (Netherlands)

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

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

  7. Induced pluripotent stem cells derived from rabbits exhibit some characteristics of naïve pluripotency

    OpenAIRE

    Osteil, Pierre; Tapponnier, Yann; Markossian, Suzy; Godet, Murielle; Panneau, Barbara; Jouneau, Luc; Cabau, Cédric; Joly, Thierry; Blachère, Thierry; Gócza, Elen; Bernat, Agnieszka; Yerle, Martine; ACLOQUE, HERVE; Hidot, Sullivan; Bosze, Zsuzsanna

    2013-01-01

    Summary Not much is known about the molecular and functional features of pluripotent stem cells (PSCs) in rabbits. To address this, we derived and characterized 2 types of rabbit PSCs from the same breed of New Zealand White rabbits: 4 lines of embryonic stem cells (rbESCs), and 3 lines of induced PSCs (rbiPSCs) that were obtained by reprogramming adult skin fibroblasts. All cell lines required fibroblast growth factor 2 for their growth and proliferation. All rbESC lines showed molecular and...

  8. Derivation of keratinocytes from chicken embryonic stem cells: Establishment and characterization of differentiated proliferative cell populations

    Directory of Open Access Journals (Sweden)

    Mathilde Couteaudier

    2015-03-01

    Full Text Available A common challenge in avian cell biology is the generation of differentiated cell-lines, especially in the keratinocyte lineage. Only a few avian cell-lines are available and very few of them show an interesting differentiation profile. During the last decade, mammalian embryonic stem cell-lines were shown to differentiate into almost all lineages, including keratinocytes. Although chicken embryonic stem cells had been obtained in the 1990s, few differentiation studies toward the ectodermal lineage were reported. Consequently, we explored the differentiation of chicken embryonic stem cells toward the keratinocyte lineage by using a combination of stromal induction, ascorbic acid, BMP4 and chicken serum. During the induction period, we observed a downregulation of pluripotency markers and an upregulation of epidermal markers. Three homogenous cell populations were derived, which were morphologically similar to chicken primary keratinocytes, displaying intracellular lipid droplets in almost every pavimentous cell. These cells could be serially passaged without alteration of their morphology and showed gene and protein expression profiles of epidermal markers similar to chicken primary keratinocytes. These cells represent an alternative to the isolation of chicken primary keratinocytes, being less cumbersome to handle and reducing the number of experimental animals used for the preparation of primary cells.

  9. Characterization of UV radiation sensitive frog cell lines

    International Nuclear Information System (INIS)

    Smith-Stein, A.C.

    1983-01-01

    Twenty-one subclones of nine frog cell isolates were tested for sensitivity to a panel of DNA damaging agents. Two clones were identified which had a greater than wild type level of sensitivity to UV radiation but had a wild type level of sensitivity to the other agents. These clones were the haploid RRP602-7 and the diploid RRP802-1. RRP802-1 was found to be unstable with respect to UV sensitivity. The line was cloned in order to isolate stable sensitive and wild type derivatives. RRP802-1-16, a UV sensitive clone and RRP802-1-13, a clone with a wild type level of sensitivity to UV radiation, were isolated. The UV radiation sensitivity of RRP602-7, RRP802-1 and RRP802-1-16 did not correlate with cell size, cell shape, cell cycle distribution or ploidy. The cell cycle distribution after UV irradiation, the rate of DNA synthesis after UV-irradiation, the DNA polymerase α activity and the sister chromatid exchange frequency were all measured in RRP602-7, RRP802-1 and RRP802-1-16 in order to examine the DNA repair capacity. The presence of DNA repair pathways was examined directly in RRP602-7, RRP802-1 and RRP802-1-16. All were found to be proficient in photo-reactivation repair and postreplication repair of UV elicited DNA damage

  10. Modeling Adenovirus Latency in Human Lymphocyte Cell Lines ▿ †

    OpenAIRE

    Zhang, Yange; Huang, Wen; Ornelles, David A.; Gooding, Linda R.

    2010-01-01

    Species C adenovirus establishes a latent infection in lymphocytes of the tonsils and adenoids. To understand how this lytic virus is maintained in these cells, four human lymphocytic cell lines that support the entire virus life cycle were examined. The T-cell line Jurkat ceased proliferation and died shortly after virus infection. BJAB, Ramos (B cells), and KE37 (T cells) continued to divide at nearly normal rates while replicating the virus genome. Viral genome numbers peaked and then decl...

  11. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay.

    Science.gov (United States)

    Alley, M C; Scudiero, D A; Monks, A; Hursey, M L; Czerwinski, M J; Fine, D L; Abbott, B J; Mayo, J G; Shoemaker, R H; Boyd, M R

    1988-02-01

    For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.

  12. Preparing for Automated Derivation of Products in a Software Product Line

    National Research Council Canada - National Science Library

    McGregor, John D

    2005-01-01

    ... to bring a product to market, or through other production improvements. Business goals such as these make automated product derivation an appealing strategy to a software product line organization...

  13. Isolation of a Wheat Cell Line with Altered Membrane Properties

    Science.gov (United States)

    Erdei, László; Vigh, László; Dudits, Dénes

    1982-01-01

    A spontaneous dimethylsulfoxide (DMSO)-tolerant cell line was isolated from a cell culture of wheat (Triticum monococcum L.). The tolerant cells were able to grow in the presence of 4% DMSO. Cells formed from protoplasts of the tolerant line required DMSO for division in culture medium of high osmotic value. Fatty acid composition and the molar ratio of phospholipids/sterols suggest a more ordered membrane structure in the tolerant line. Accordingly, a lower K+ influx rate was detected in the tolerant cells in comparison with the original line. These characteristics were maintained after 6 months' cultivation of the cells in DMSO-free growth medium. This suggested that genetic changes could be responsible for differences between the two cell lines. PMID:16662251

  14. Immune suppressor factor confers stromal cell line with enhanced supporting activity for hematopoietic stem cells

    International Nuclear Information System (INIS)

    Nakajima, Hideaki; Shibata, Fumi; Fukuchi, Yumi; Goto-Koshino, Yuko; Ito, Miyuki; Urano, Atsushi; Nakahata, Tatsutoshi; Aburatani, Hiroyuki; Kitamura, Toshio

    2006-01-01

    Immune suppressor factor (ISF) is a subunit of the vacuolar ATPase proton pump. We earlier identified a short form of ISF (ShIF) as a stroma-derived factor that supports cytokine-independent growth of mutant Ba/F3 cells. Here, we report that ISF/ShIF supports self-renewal and expansion of primary hematopoietic stem cells (HSCs). Co-culture of murine bone marrow cells with a stromal cell line overexpressing ISF or ShIF (MS10/ISF or MS10/ShIF) not only enhanced their colony-forming activity and the numbers of long-term culture initiating cells, but also maintained the competitive repopulating activity of HSC. This stem cell supporting activity depended on the proton-transfer function of ISF/ShIF. Gene expression analysis of ISF/ShIF-transfected cell lines revealed down-regulation of secreted frizzled-related protein-1 and tissue inhibitor of metalloproteinase-3, and the restoration of their expressions in MS10/ISF cells partially reversed its enhanced LTC-IC supporting activity to a normal level. These results suggest that ISF/ShIF confers stromal cells with enhanced supporting activities for HSCs by modulating Wnt-activity and the extracellular matrix

  15. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

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

    2011-01-01

    incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size......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 mu M were...

  16. Survival curves of neoplastic and non transformed human cell lines: statistical analysis using different models

    International Nuclear Information System (INIS)

    Fertil, B.; Deschavanne, P.; Malaise, E.P.; Lachet, B.

    1978-01-01

    The 'in vitro' radiosensitivity to γ rays of 6 human cell lines is studied by the colony method: 3 cell lines are derived from colorectal carcinomas, one from a squamous cell carcinoma, one from a Melanoma and the last one is a non-transformed fibroblast cell line. We have attempted to get the lowest percentage of surviving cells as possible. We have used four statistical models to fit the different survival curves: the multitarget single hit, the multitarget two hits, the 'Wideroe', and the quadratic models. The main conclusions drawn from these analyses are the following: -the discrimination between the models could be done by the shape of the curve and the accuracy of the experimental results, but also by the determination of the cell survival after high doses. The quadratic model fits in best with the suvival curves of the 6 cell lines. It allows an accurate description of the shoulder of the survival curves and thus provides information for the comprehension of results in the radiotherapy of human cancers. The quadratic dependence of the surviving fraction on the dose is precisely determined; -significative differences in radiosensitivity show up among the cell lines. The biological interpretation of this finding is discussed: -concerning the fibroblast cell line, it appears that: as opposed to most results published till now, the interpretation of their survival curves must be based upon a quadratic radiation action. Despite their radiosensitivity higher than most other mammalian cells in vitro, fibroblast cells seem to have a comparable survival curve

  17. Induced Pluripotent Stem Cell-Derived Natural Killer Cells for Treatment of Ovarian Cancer.

    Science.gov (United States)

    Hermanson, David L; Bendzick, Laura; Pribyl, Lee; McCullar, Valarie; Vogel, Rachel Isaksson; Miller, Jeff S; Geller, Melissa A; Kaufman, Dan S

    2016-01-01

    Natural killer (NK) cells can provide effective immunotherapy for ovarian cancer. Here, we evaluated the ability of NK cells isolated from peripheral blood (PB) and NK cells derived from induced pluripotent stem cell (iPSC) to mediate killing of ovarian cancer cells in a mouse xenograft model. A mouse xenograft model was used to evaluate the intraperitoneal delivery of three different NK cell populations: iPSC-derived NK cells, PB-NK cells that had been activated and expanded in long-term culture, and overnight activated PB-NK cells that were isolated through CD3/CD19 depletion of PB B and T cells. Bioluminescent imaging was used to monitor tumor burden of luciferase expressing tumor lines. Tumors were allowed to establish prior to administering NK cells via intraperitoneal injection. These studies demonstrate a single dose of any of the three NK cell populations significantly reduced tumor burden. When mice were given three doses of either iPSC-NK cells or expanded PB-NK cells, the median survival improved from 73 days in mice untreated to 98 and 97 days for treated mice, respectively. From these studies, we conclude iPSC-derived NK cells mediate antiovarian cancer killing at least as well as PB-NK cells, making these cells a viable resource for immunotherapy for ovarian cancer. Due to their ability to be easily differentiated into NK cells and their long-term expansion potential, iPSCs can be used to produce large numbers of well-defined NK cells that can be banked and used to treat a large number of patients including treatment with multiple doses if necessary. © 2015 AlphaMed Press.

  18. The pursuit of ES cell lines of domesticated ungulates

    Science.gov (United States)

    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...

  19. Adipose-derived mesenchymal stem cells promote cell proliferation and invasion of epithelial ovarian cancer

    Energy Technology Data Exchange (ETDEWEB)

    Chu, Yijing; Tang, Huijuan; Guo, Yan; Guo, Jing; Huang, Bangxing; Fang, Fang; Cai, Jing, E-mail: caijingmmm@hotmail.com; Wang, Zehua, E-mail: zehuawang@163.net

    2015-09-10

    Adipose-derived mesenchymal stem cell (ADSC) is an important component of tumor microenvironment. However, whether ADSCs have a hand in ovarian cancer progression remains unclear. In this study, we investigated the impact of human ADSCs derived from the omentum of normal donors on human epithelial ovarian cancer (EOC) cells in vitro and in vivo. Direct and indirect co-culture models including ADSCs and human EOC cell lines were established and the effects of ADSCs on EOC cell proliferation were evaluated by EdU incorporation and flow cytometry. Transwell migration assays and detection of MMPs were performed to assess the invasion activity of EOC cells in vitro. Mouse models were established by intraperitoneal injection of EOC cells with or without concomitant ADSCs to investigate the role of ADSCs in tumor progression in vivo. We found that ADSCs significantly promoted proliferation and invasion of EOC cells in both direct and indirect co-culture assays. In addition, after co-culture with ADSCs, EOC cells secreted higher levels of matrix metalloproteinases (MMPs), and inhibition of MMP2 and MMP9 partially relieved the tumor-promoting effects of ADSCs in vitro. In mouse xenograft models, we confirmed that ADSCs promoted EOC growth and metastasis and elevated the expression of MMP2 and MMP9. Our findings indicate that omental ADSCs play a promotive role during ovarian cancer progression. - Highlights: • Omental adipose derived stem cells enhanced growth and invasion properties of ovarian cancer cells. • Adipose derived stem cells promoted the growth and metastasis of ovarian cancer in mice models. • Adipose derived stem cells promoted MMPs expression and secretion of ovarian cancer cells. • Elevated MMPs mediated the tumor promoting effects of ADSCs.

  20. Generation of induced pluripotent stem cell-derived mice by reprogramming of a mature NKT cell.

    Science.gov (United States)

    Ren, Yue; Dashtsoodol, Nyambayar; Watarai, Hiroshi; Koseki, Haruhiko; Quan, Chengshi; Taniguchi, Masaru

    2014-10-01

    NKT cells are characterized by their expression of an NKT-cell-specific invariant antigen-receptor α chain encoded by Vα14Jα18 gene segments. These NKT cells bridge the innate and acquired immune systems to mediate effective and augmented responses; however, the limited number of NKT cells in vivo hampers their analysis. Here, two lines of induced pluripotent stem cell-derived mice (NKT-iPSC-derived mice) were generated by reprogramming of mature NKT cells, where one harbors both rearranged Vα14Jα18 and Vβ7 genes and the other carries rearranged Vα14Jα18 on both alleles but germline Vβ loci. The analysis of NKT-iPSC-derived mice showed a significant increase in NKT cell numbers with relatively normal frequencies of functional subsets, but significantly enhanced in some cases, and acquired functional NKT cell maturation in