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Sample records for subclonal cell line

  1. Isolation of TPO-dependent subclones from the multipotent 32D cell line.

    Science.gov (United States)

    Amabile, Giovanni; Di Noia, Antonella; Alfani, Elena; Vannucchi, Alessandro Maria; Sanchez, Massimo; Bosco, Domenico; Migliaccio, Anna Rita; Migliaccio, Giovanni

    2005-01-01

    Using thrombopoietin (TPO), as selective pressure, several TPO-dependent clones were isolated from the murine multipotential IL-3-dependent cell line 32D. Four of them were fully characterized. They depended on TPO for survival and proliferation and, although retaining the capacity to grow in IL-3, did not respond to either EPO, G-CSF or GM-CSF. 32D TPO cells were heterogeneous in morphology and ranged from small cells, with a DNA content nearly tetraploid and a modal chromosome no. 66, to cells 50-75 microm in diameter containing multiple (up to 5-6) interconnected nuclei with a clear megakaryocyte (Mk) morphology by electron microscopy. Cell sorter isolation and single cell cloning experiments indicated that the small cells were those capable to proliferate in TPO and to generate the larger ones over time. 32D TPO cells expressed Mk-specific markers by FACS (CD41, CD61 and 2D5) and RT-PCR (acetyl cholinesterase E and platelet factor 4) and their unique profile, by gene array analysis, included expression of urokinase plasminogen activator surface receptor (CD87 or uPAR), plasminogen activator inhibitor and coagulation factor II (thrombin) receptor (Cf2r). In addition, by quantitative RT-PCR, 32D TPO clones expressed levels of Gata1 similar to those expressed by freshly isolated Mks (DeltaCt approximately 4.7 in both cases). In conclusion, the 32D TPO subclones described here are among the few pure Mk cell lines isolated so far and, for their unique properties, may prove themselves as a useful model to study Mk differentiation.

  2. 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.

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    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.

  3. Dominant expansion of a cryptic subclone with an abnormal karyotype in B lymphoblastoid cell lines during culture.

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    Danjoh, I; Shirota, R; Hiroyama, T; Nakamura, Y

    2013-01-01

    Although B lymphoblastoid cell lines (B-LCLs) are thought to maintain their original genomic structures during long-term culture, there has been considerable disagreement on the actual genomic stability of these cells. This study was initiated to determine whether B-LCLs develop cell populations with abnormal genomes during culture and to search for factors important to the maintenance of the original genome. We established continuous cultures of B-LCLs for more than 6 months and analyzed the cells using array-based comparative genome hybridization (CGH) analysis, conventional karyotyping and analysis of V(D)J recombination in the immunoglobulin (Ig) gene. We found that one B-LCL acquired an extra chromosome 4 without any other genomic rearrangements at passage 16 of continuous culture. At the Ig light- and heavy-chain loci, analysis of the major cell population showed a difference between cultures at early and later passages. Another aneuploid line was detected among B-LCLs established elsewhere and deposited previously into the RIKEN Cell Bank. Our findings indicate that some of the genomic rearrangements in B-LCLs are not caused by gradual accumulation of mutations and rearrangements during the B-LCL establishment processes, but rather as a result of a change in the cell population from clones with a normal genome to clones with de novo rearrangements. It is therefore feasible to maintain B-LCLs with a normal genomic structure by cell cloning or similar treatment. Copyright © 2012 S. Karger AG, Basel.

  4. Quantitative proteomics as a tool to identify resistance mechanisms in erlotinib-resistant subclones of the non-small cell lung cancer cell line HCC827

    DEFF Research Database (Denmark)

    Jacobsen, Kirstine

    Background: Erlotinib (Tarceva®, Roche) has significantly changed the treatment of non-small cell lung cancer (NSCLC) as approximately 70% of patients show significant tumor regression when treated (Santarpia et. al., 2013). However, all patients relapse due to development of acquired resistance......, which in 43-50% of cases are caused by a secondary mutation (T790M) in EGFR. Importantly, a majority of resistance cases are still unexplained (Lin & Bivona, 2012). Our aim is to identify novel resistance mechanisms – and potentially new drug targets - in erlotinib-resistant subclones of the NSCLC cell...... was downregulated, indicating a bypass signaling mechanism to achieve resistance. More specifically, mitogen-activated protein kinase 1 (MAPK1) and synovial apoptosis inhibitor 1 (SYNV1) were upregulated. Conclusions: In conclusion, cancer-related networks such as proliferation and apoptosis were found...

  5. Genetic subclone architecture of tumor clone-initiating cells in colorectal cancer.

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    Giessler, Klara M; Kleinheinz, Kortine; Huebschmann, Daniel; Balasubramanian, Gnana Prakash; Dubash, Taronish D; Dieter, Sebastian M; Siegl, Christine; Herbst, Friederike; Weber, Sarah; Hoffmann, Christopher M; Fronza, Raffaele; Buchhalter, Ivo; Paramasivam, Nagarajan; Eils, Roland; Schmidt, Manfred; von Kalle, Christof; Schneider, Martin; Ulrich, Alexis; Scholl, Claudia; Fröhling, Stefan; Weichert, Wilko; Brors, Benedikt; Schlesner, Matthias; Ball, Claudia R; Glimm, Hanno

    2017-07-03

    A hierarchically organized cell compartment drives colorectal cancer (CRC) progression. Genetic barcoding allows monitoring of the clonal output of tumorigenic cells without prospective isolation. In this study, we asked whether tumor clone-initiating cells (TcICs) were genetically heterogeneous and whether differences in self-renewal and activation reflected differential kinetics among individual subclones or functional hierarchies within subclones. Monitoring genomic subclone kinetics in three patient tumors and corresponding serial xenografts and spheroids by high-coverage whole-genome sequencing, clustering of genetic aberrations, subclone combinatorics, and mutational signature analysis revealed at least two to four genetic subclones per sample. Long-term growth in serial xenografts and spheroids was driven by multiple genomic subclones with profoundly differing growth dynamics and hence different quantitative contributions over time. Strikingly, genetic barcoding demonstrated stable functional heterogeneity of CRC TcICs during serial xenografting despite near-complete changes in genomic subclone contribution. This demonstrates that functional heterogeneity is, at least frequently, present within genomic subclones and independent of mutational subclone differences. © 2017 Giessler et al.

  6. Ultra-deep sequencing reveals the subclonal structure and genomic evolution of oral squamous cell carcinoma

    DEFF Research Database (Denmark)

    Tabatabaeifar, Siavosh; Thomassen, Mads; Larsen, Martin Jakob

    complex subclonal architectures comprising distinct subclones only found in geographically distinct regions of the tumors. The metastatic potential of the tumor is acquired early in the tumor evolution, as indicated by the lymph node sharing the majority of the mutations with the tumor biopsies, while...... rarely acquiring novel mutations that are specific for the metastasis. Conclusion: Ultra-deep sequencing of multiple biopsies from OSCC and metastasis enables detection of subclonal structure and genomic evolution. The metastatic potential of OSCC is acquired early in the tumor evolution, and our results...... structure remains unexplored due to lack of sampling multiple tumor biopsies from each patient. Materials and methods: To examine the clonal structure and describe the genomic cancer evolution we applied whole-exome sequencing combined with targeted ultra-deep targeted sequencing on biopsies from 5stage IV...

  7. The subclonal structure and genomic evolution of oral squamous cell carcinoma revealed by ultra-deep sequencing

    DEFF Research Database (Denmark)

    Tabatabaeifar, Siavosh; Thomassen, Mads; Larsen, Martin J

    2017-01-01

    Recent studies suggest that head and neck squamous cell carcinomas are very heterogeneous between patients; however the subclonal structure remains unexplored mainly due to studies using only a single biopsy per patient. To deconvolutethe clonal structure and describe the genomic cancer evolution...... tumor that are not present in the lymph node metastasis. We conclude that metastatic potential in our population is acquired early in tumor evolution as evident by the ongoing parallel evolution in several primary tumors.......Recent studies suggest that head and neck squamous cell carcinomas are very heterogeneous between patients; however the subclonal structure remains unexplored mainly due to studies using only a single biopsy per patient. To deconvolutethe clonal structure and describe the genomic cancer evolution...

  8. Riesling Italico subclones

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    Ivanišević Dragoslav

    2012-01-01

    Full Text Available Clone selection in viticulture represents a way of eliminating the negative influence of mutation changes in the future vineyard, as well as preventing the virus-infected plants and plants infected with virus-related diseases from further propagation. Riesling Italico variety is an old variety. It has been grown in Vojvodina for centuries. The clone selection of Riesling Italico variety in Sremski Karlovci was started in 1975. In January 1991 three clones were recognized. Shortly after, the subclone selection of these three clones was initiated. This paper presents the resultants of subclone selection of Riesling Italico. Subclones SK 54-4 and SK 54-10 are characterized by good quality and the grapes of these subclones can be used for the production of high quality wines in most of the years.

  9. The Subclonal Structure and Genomic Evolution of Oral Squamous Cell Carcinoma Revealed by Ultra-deep Sequencing

    DEFF Research Database (Denmark)

    Tabatabaeifar, Siavosh; Thomassen, Mads; Larsen, Martin Jakob

    complex subclonal architectures comprising distinct subclones only found in geographically distinct regions of the tumors. The metastatic potential of the tumor is acquired early in the tumor evolution, as indicated by the lymph node sharing the majority of the mutations with the tumor biopsies, while...... rarely acquiring novel mutations that are specific for the metastasis. Conclusion: Ultra-deep sequencing of multiple biopsies from OSCC and metastasis enables detection of subclonal structure and genomic evolution. The metastatic potential of OSCC is acquired early in the tumor evolution, and our results...... structure remains unexplored due to lack of sampling multiple tumor biopsies from each patient. Materials and methods: To examine the clonal structure and describe the genomic cancer evolution we applied whole-exome sequencing combined with targeted ultra-deep targeted sequencing on biopsies from 5stage IV...

  10. Prognostic impact of HER-2 Subclonal Amplification in breast cancer.

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    Di Oto, Enrico; Brandes, Alba A; Cucchi, Maria C; Foschini, Maria P

    2017-06-02

    The presence of a limited number of cells with HER-2 amplification (Subclonal Amplification) in breast carcinomas is occasionally encountered, but its prognostic impact is poorly known. The purpose of this study is to evaluate the prognostic impact of HER-2 Subclonal Amplification in a retrospective series of breast cancers. Accordingly, 81 consecutive breast carcinomas showing HER-2 Subclonal Amplification were obtained from the histology files (case series). These cases were subdivided into two groups: (a) those cases in which the HER-2 Subclonal Amplification was consonant to the accepted criteria for amplification, showing clusters of amplified cells, and (b) those cases with rare HER-2 Subclonal Amplification that did not reflect the accepted criteria for amplification, showing scattered amplified cells only. The incidence of metastases and late recurrences of the case series was compared with a series composed of 109 consecutive cases, being HER-2 homogeneous (comprising 14 Amplified and 95 Non-Amplified cases), matched for grade and stage (control series). It appeared that cases showing Subclonal Amplification had an incidence of metastases intermediate between the cases Amplified and Non-Amplified. Specifically, Subclonal Amplification with clustered cells had a lower incidence of metastases than Amplified cases (12.9 versus 21.4%). On the contrary, Subclonal Amplification with scattered cells showed an incidence of metastases higher than Non-Amplified cases (14 versus 9.47%). In addition, patients Subclonal Amplification with clustered cells, who were treated with the specific monoclonal antibody, had a lower incidence of metastases than patients showing Subclonal Amplification with scattered cells, who did not receive target therapy. These data, together with those recently published, indicate that Subclonal Amplification has an impact on prognosis and should be taken into consideration to correctly plan the treatment of breast cancer patients.

  11. A comparative study of the FcepsilonRI molecule on human mast cell and basophil cell lines

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Dissing, S; Skov, P S

    2005-01-01

    Mast cells and basophils express the high-affinity IgE receptor FcepsilonRI. We have analysed the human mast cell line LAD2 and four subclones of the basophil cell line KU812 in order to reveal possible differences concerning the FcepsilonRI surface regulation, anti-IgE-triggered activation, Fcep...

  12. Oncogenic LINE-1 Retroelements Sustain Prostate Tumor Cells and Promote Metastatic Progression

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    2015-10-01

    The goal of this hypothesis development project is ( 1 ) to determine if ectopic expression of LINE- 1 elements in prostate cancer contribute to its...Furthermore, we have subcloned a LINE- 1 Open reading frame sequence and will determine the effect of its expression in non-tumorigenic prostate cells...Finally, we have cloned the PIWIL- 1 gene, known as a repressor of LINE- 1 retroelement sequences in the testis, and have it under the control of a

  13. A comparative study of the FcepsilonRI molecule on human mast cell and basophil cell lines

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Dissing, S; Skov, P S

    2005-01-01

    Mast cells and basophils express the high-affinity IgE receptor FcepsilonRI. We have analysed the human mast cell line LAD2 and four subclones of the basophil cell line KU812 in order to reveal possible differences concerning the FcepsilonRI surface regulation, anti-IgE-triggered activation, Fcep......, FcepsilonRIalpha protein stability and the mRNA level of FcepsilonRIalpha-, beta- and the truncated beta-chain (beta(T)), and thereby determine the utility of these cell lines in investigations of the FcepsilonRI biology....

  14. Resistance mechanisms to erlotinib in the non-small cell lung cancer cell line, HCC827 examined by RNA-seq

    DEFF Research Database (Denmark)

    Jacobsen, Kirstine; Alcaraz, Nicolas; Ditzel, Henrik

    Background: Erlotinib, an EGFR selective reversible inhibitor, has dramatically changed the treatment of non-small cell lung cancer (NSCLC) as approximately 70% of patients show significant tumor regression upon treatment. However, all patients eventually relapse due to development of acquired...... - in erlotinib-resistant subclones of the NSCLC cell line HCC827. Materials & Methods: We established 3 erlotinib-resistant subclones (resistant to 10, 20, 30 µM erlotinib, respectively), and prepared cDNA libraries of purified RNA from biological duplicates using TruSeq® Stranded Total RNA Ribo-Zero™ Gold...... (Illumina) prior to sequencing on an Illumina HiSeq platform (100bp paired end). The resistant subclones were examined both in presence and absence of erlotinib. The data was analyzed by an in-house developed pipeline including quality control by Trim Galore v0.3.3, mapping of reads to HG19 by TopHat2 v.2...

  15. Phenotypic identification of subclones in multiple myeloma with different chemoresistant, cytogenetic and clonogenic potential.

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    Paíno, T; Paiva, B; Sayagués, J M; Mota, I; Carvalheiro, T; Corchete, L A; Aires-Mejía, I; Pérez, J J; Sanchez, M L; Barcena, P; Ocio, E M; San-Segundo, L; Sarasquete, M E; García-Sanz, R; Vidriales, M-B; Oriol, A; Hernández, M-T; Echeveste, M-A; Paiva, A; Blade, J; Lahuerta, J-J; Orfao, A; Mateos, M-V; Gutiérrez, N C; San-Miguel, J F

    2015-05-01

    Knowledge about clonal diversity and selection is critical to understand multiple myeloma (MM) pathogenesis, chemoresistance and progression. If targeted therapy becomes reality, identification and monitoring of intraclonal plasma cell (PC) heterogeneity would become increasingly demanded. Here we investigated the kinetics of intraclonal heterogeneity among 116 MM patients using 23-marker multidimensional flow cytometry (MFC) and principal component analysis, at diagnosis and during minimal residual disease (MRD) monitoring. Distinct phenotypic subclones were observed in 35/116 (30%) newly diagnosed MM patients. In 10/35 patients, persistent MRD was detected after 9 induction cycles, and longitudinal comparison of patient-paired diagnostic vs MRD samples unraveled phenotypic clonal tiding after therapy in half (5/10) of the patients. After demonstrating selection of distinct phenotypic subsets by therapeutic pressure, we investigated whether distinct fluorescence-activated cell-sorted PC subclones had different clonogenic and cytogenetic profiles. In half (5/10) of the patients analyzed, distinct phenotypic subclones showed different clonogenic potential when co-cultured with stromal cells, and in 6/11 cases distinct phenotypic subclones displayed unique cytogenetic profiles by interphase fluorescence in situ hybridization, including selective del(17p13). Collectively, we unravel potential therapeutic selection of preexisting diagnostic phenotypic subclones during MRD monitoring; because phenotypically distinct PCs may show different clonogenic and cytogenetic profiles, identification and follow-up of unique phenotypic-genetic myeloma PC subclones may become relevant for tailored therapy.

  16. Quantifying Clonal and Subclonal Passenger Mutations in Cancer Evolution

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    Bozic, Ivana; Gerold, Jeffrey M.; Nowak, Martin A.

    2016-01-01

    The vast majority of mutations in the exome of cancer cells are passengers, which do not affect the reproductive rate of the cell. Passengers can provide important information about the evolutionary history of an individual cancer, and serve as a molecular clock. Passengers can also become targets for immunotherapy or confer resistance to treatment. We study the stochastic expansion of a population of cancer cells describing the growth of primary tumors or metastatic lesions. We first analyze the process by looking forward in time and calculate the fixation probabilities and frequencies of successive passenger mutations ordered by their time of appearance. We compute the likelihood of specific evolutionary trees, thereby informing the phylogenetic reconstruction of cancer evolution in individual patients. Next, we derive results looking backward in time: for a given subclonal mutation we estimate the number of cancer cells that were present at the time when that mutation arose. We derive exact formulas for the expected numbers of subclonal mutations of any frequency. Fitting this formula to cancer sequencing data leads to an estimate for the ratio of birth and death rates of cancer cells during the early stages of clonal expansion. PMID:26828429

  17. Label-free live cell imaging by Confocal Raman Microscopy identifies CHO host and producer cell lines.

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    Prats Mateu, Batirtze; Harreither, Eva; Schosserer, Markus; Puxbaum, Verena; Gludovacz, Elisabeth; Borth, Nicole; Gierlinger, Notburga; Grillari, Johannes

    2017-01-01

    As a possible viable and non-invasive method to identify high producing cells, Confocal Raman Microscopy was shown to be able to differentiate CHO host cell lines and derivative production clones. Cluster analysis of spectra and their derivatives was able to differentiate between different producer cell lines and a host, and also distinguished between an intracellular region of high lipid and protein content that in structure resembles the Endoplasmic Reticulum. This ability to identify the ER may be a major contributor to the identification of high producers. PCA enabled the discrimination even of host cell lines and their subclones with inherently higher production capacity. The method is thus a promising option that may contribute to early, non-invasive identification of high potential candidates during cell line development and possibly could also be used for proof of identity of established production clones. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Differential expression of the ufo/axl oncogene in human leukemia-lymphoma cell lines.

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    Challier, C; Uphoff, C C; Janssen, J W; Drexler, H G

    1996-05-01

    The ufo protein (also termed axl) is a member of a new family of receptor tyrosine kinases and is encoded by a transforming gene that was initially isolated from primary human myeloid leukemia cells by DNA-mediated transformation of NIH/3T3 cells. The ligand, Gas6, a protein S-related molecule lacking any known function yet, has recently been identified. We report the expression pattern of ufo mRNA in a panel of 76 human continuous leukemia-lymphoma cell lines. The gene was not expressed in cell lines derived from lymphoid malignancies (n=28), but transcription was seen in 3/11 myeloid, 0/6 monocytic, 9/13 erythroid and 11/18 megakaryocytic cell lines. Several cell lines were treated with phorbol ester leading to significant upregulation of the ufo message in constitutively positive cells. An apparent ufo mRNA overexpression was not found in any of the positive leukemia cell lines, but was identified in the drug-resistant subclones of the cervix carcinoma cell line HeLa. Southern blot analysis of restriction enzyme-digested genomic DNA did not provide evidence for gene amplification, but the HeLa subclones showed banding patterns suggestive of gene rearrangement. Two main ufo mRNA bands of 3.2 and 5.0 kb were identified; no differences in the half-lives (t1/2 = 2.5 h) of these two mRNA species could be identified. In summary, ufo, representing a novel type of receptor tyrosine kinase, is expressed solely in myeloid and erythro-megakaryocytic leukemias but not in lymphoid malignancies. These and previous data suggest an involvement of the ufo receptor tyrosine kinase in normal and malignant myelopoiesis; however, its exact role, if any, and mode of operation in leukemogenesis remains to be determined.

  19. Subclonal diversification of primary breast cancer revealed by multiregion sequencing

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    Yates, Lucy R; Gerstung, Moritz; Knappskog, Stian; Desmedt, Christine; Gundem, Gunes; Loo, Peter Van; Aas, Turid; Alexandrov, Ludmil B; Larsimont, Denis; Davies, Helen; Li, Yilong; Ju, Young Seok; Ramakrishna, Manasa; Haugland, Hans Kristian; Lilleng, Peer Kaare; Nik-Zainal, Serena; McLaren, Stuart; Butler, Adam; Martin, Sancha; Glodzik, Dominic; Menzies, Andrew; Raine, Keiran; Hinton, Jonathan; Jones, David; Mudie, Laura J; Jiang, Bing; Vincent, Delphine; Greene-Colozzi, April; Adnet, Pierre-Yves; Fatima, Aquila; Maetens, Marion; Ignatiadis, Michail; Stratton, Michael R; Sotiriou, Christos; Richardson, Andrea L; Lønning, Per Eystein; Wedge, David C; Campbell, Peter J

    2015-01-01

    Sequencing cancer genomes may enable tailoring of therapeutics to the underlying biological abnormalities driving a particular patient’s tumor. However, sequencing-based strategies rely heavily on representative sampling of tumors. To understand the subclonal structure of primary breast cancer, we applied whole genome and targeted sequencing to multiple samples from each of 50 patients’ tumors (total 303). The extent of subclonal diversification varied among cases and followed spatial patterns. No strict temporal order was evident, with point mutations and rearrangements affecting the most common breast cancer genes, including PIK3CA, TP53, PTEN, BRCA2 and MYC, occurring early in some tumors and late in others. In 13/50 cancers, potentially targetable mutations were subclonal. Landmarks of disease progression, such as resisting chemotherapy and acquiring invasive or metastatic potential, arose within detectable subclones of antecedent lesions. These findings highlight the importance of including analyses of subclonal structure and tumor evolution in clinical trials of primary breast cancer. PMID:26099045

  20. Identification of resistance mechanisms in erlotinib-resistant subclones of the non-small cell lung cancer cell line HCC827 by exome sequencing

    DEFF Research Database (Denmark)

    Jacobsen, Kirstine; Alcaraz, Nicolas; Lund, Rikke Raaen

    the SeqCap EZ Human Exome Library v3.0 kit and whole-exome sequencing of these (100 bp paired-end) were performed on an Illumina HiSeq 2000 platform. Using a recently developed in-house analysis pipeline the sequencing data were analyzed. The analysis pipeline includes quality control using Trim-Galore...

  1. Functional analysis of fatty acid binding protein 7 and its effect on fatty acid of renal cell carcinoma cell lines.

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    Takaoka, Naohisa; Takayama, Tatsuya; Ozono, Seiichiro

    2017-03-14

    Renal cell carcinomas (RCCs) overexpress fatty acid binding protein 7 (FABP7). We chose to study the TUHR14TKB cell line, because it expresses higher levels of FABP7 than other cell lines derived from renal carcinomas (OS-RC-2, 786-O, 769-P, Caki-1, and ACHN). FABP7 expression was detected using western blotting and real-time PCR. Cell proliferation was determined using an MTS assay and by directly by counting cells. The cell cycle was assayed using flow cytometry. Cell migration was assayed using wound-healing assays. An FABP7 expression vector was used to transfect RCC cell lines. The levels of FABP7 expressed by TUHR14TKB cells and their doubling times decreased during passage. High-passage TUHR14TKB cells comprised fewer G0/G1-phase and more S-phase cells than low-passage cells. Cell proliferation differed among subclones isolated from cultures of low-passage TUHR14TKB cells. The proliferation of TUHR14TKB cells decreased when FABP7 was overexpressed, and the cell migration property of TUHR14TKB cells were decreased when FABP7 was overexpressed. High concentrations of docosatetraenoic acid and eicosapentaenoic acid accumulated in TUHR14TKB cells that overexpressed FABP7, and docosatetraenoic acid enhanced cell proliferation. The TUHR14TKB cell line represents a heterogeneous population that does not express FABP7 when it rapidly proliferates. The differences in FABP7 function between RCC cell lines suggests that FABP7 affects cell proliferation depending on cell phenotype.

  2. Establishment and Characterization of a Novel Caco-2 Subclone with a Similar Low Expression Level of Human Carboxylesterase 1 to Human Small Intestine.

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    Ohura, Kayoko; Nishiyama, Hikaru; Saco, Saori; Kurokawa, Keisuke; Imai, Teruko

    2016-12-01

    Caco-2 cells predominantly express human carboxylesterase 1 (hCE1), unlike the human intestine that predominantly expresses human carboxylesterase 2 (hCE2). Transport experiments using Caco-2 cell monolayers often lead to misestimation of the intestinal absorption of prodrugs because of this difference, as prodrugs designed to increase the bioavailability of parent drugs are made to be resistant to hCE2 in the intestine, so that they can be hydrolyzed by hCE1 in the liver. In the present study, we tried to establish a new Caco-2 subclone, with a similar pattern of carboxylase expression to human intestine, to enable a more accurate estimation of the intestinal absorption of prodrugs. Although no subclone could be identified with high expression levels of only hCE2, two subclones, #45 and #78, with extremely low expression levels of hCE1 were subcloned from parental Caco-2 cells by the limiting dilution technique. Unfortunately, subclone #45 did not form enterocyte-like cell monolayers due to low expression of claudins and β-actin. However, subclone #78 formed polarized cell monolayers over 4 weeks and showed similar paracellular and transcellular transport properties to parental Caco-2 cell monolayers. In addition, the intestinal transport of oseltamivir, a hCE1 substrate, could be evaluated in subclone #78 cell monolayers, including P-glycoprotein-mediated efflux under nonhydrolysis conditions, unlike parental Caco-2 cells. Consequently, it is proposed that subclone #78 may provide a more effective system in which to evaluate the intestinal absorption of prodrugs that are intended to be hydrolyzed by hCE1. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  3. Genetic characteristics of the human hepatic stellate cell line LX-2.

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    Ralf Weiskirchen

    Full Text Available The human hepatic cell line LX-2 has been described as tool to study mechanisms of hepatic fibrogenesis and the testing of antifibrotic compounds. It was originally generated by immortalisation with the Simian Vacuolating Virus 40 (SV40 transforming (T antigen and subsequent propagation in low serum conditions. Although this immortalized line is used in an increasing number of studies, detailed genetic characterisation has been lacking. We here have performed genetic characterisation of the LX-2 cell line and established a single-locus short tandem repeat (STR profile for the cell line and characterized the LX-2 karyotype by several cytogenetic and molecular cytogenetic techniques. Spectral karyotyping (SKY revealed a complex karyotype with a set of aberrations consistently present in the metaphases analyses which might serve as cytogenetic markers. In addition, various subclonal and single cell aberrations were detected. Our study provides criteria for genetic authentication of LX-2 and offers insights into the genotype changes which might underlie part of its phenotypic features.

  4. Growth and Maintenance of Vero Cell Lines

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    Ammerman, Nicole C.; Beier-Sexton, Magda; Azad, Abdu F.

    2008-01-01

    Vero cells are derived from the kidney of an African green monkey, and are one of the more commonly used mammalian continuous cell lines in microbiology, and molecular and cell biology research. This unit includes protocols for the growth and maintenance of Vero cell lines in a research laboratory setting.

  5. LINE-1 Cultured Cell Retrotransposition Assay

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

    2016-01-01

    Summary The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells. PMID:26895052

  6. Long-term persistence of acquired resistance to 5-fluorouracil in the colon cancer cell line SW620

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    Tentes, I.K., E-mail: itentes@med.duth.gr [Department of Biochemistry, Medical School, Democritus University of Thrace, 6th km Alexandroupolis-Komotini (Dragana), 68100 Alexandroupolis (Greece); Schmidt, W.M. [Center for Anatomy and Cell Biology, Waehringer Strasse 13, 1090 Vienna (Austria); Krupitza, G. [Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna (Austria); Steger, G.G.; Mikulits, W. [Department of Medicine I, Medical University of Vienna, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna (Austria); Kortsaris, A. [Department of Biochemistry, Medical School, Democritus University of Thrace, 6th km Alexandroupolis-Komotini (Dragana), 68100 Alexandroupolis (Greece); Mader, R.M. [Department of Medicine I, Medical University of Vienna, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna (Austria)

    2010-11-15

    Treatment resistance to antineoplastic drugs represents a major clinical problem. Here, we investigated the long-term stability of acquired resistance to 5-fluorouracil (FU) in an in vitro colon cancer model, using four sub-clones characterised by increasing FU-resistance derived from the cell line SW620. The resistance phenotype was preserved after FU withdrawal for 15 weeks ({approx} 100 cell divisions) independent of the established level of drug resistance and of epigenetic silencing. Remarkably, resistant clones tolerated serum deprivation, adopted a CD133{sup +} CD44{sup -} phenotype, and further exhibited loss of membrane-bound E-cadherin together with predominant nuclear {beta}-catenin localisation. Thus, we provide evidence for a long-term memory of acquired drug resistance, driven by multiple cellular strategies (epithelial-mesenchymal transition and selective propagation of CD133{sup +} cells). These resistance phenomena, in turn, accentuate the malignant phenotype.

  7. A Quantitative Analysis of Subclonal and Clonal Gene Mutations before and after Therapy in Chronic Lymphocytic Leukemia.

    Science.gov (United States)

    Amin, Nisar A; Seymour, Erlene; Saiya-Cork, Kamlai; Parkin, Brian; Shedden, Kerby; Malek, Sami N

    2016-09-01

    Chronic lymphocytic leukemia (CLL)-associated gene mutations that influence CLL cell fitness and chemotherapy resistance should increase in clonal representation when measured before therapy and at relapse. To uncover mutations associated with CLL relapse, we have performed whole-exome sequencing in a discovery cohort of 61 relapsed CLL patients identifying 86 recurrently mutated genes. The variant allele fractions (VAF) of 19 genes with mutations in ≥3 of 61 cases were measured in 53 paired pre- and posttreatment CLL samples sorted to purity using panel-based deep resequencing or by droplet digital PCR. We identify mutations in TP53 as the dominant subclonal gene driver of relapsed CLL often demonstrating substantial increases in VAFs. Subclonal mutations in SAMHD1 also recurrently demonstrated increased VAFs at relapse. Mutations in ATP10A, FAT3, FAM50A, and MGA, although infrequent, demonstrated enrichment in ≥2 cases each. In contrast, mutations in NOTCH1, SF3B1, POT1, FBXW7, MYD88, NXF1, XPO1, ZMYM3, or CHD2 were predominantly already clonal prior to therapy indicative of a pretreatment pathogenetic driver role in CLL. Quantitative analyses of clonal dynamics uncover rising, stable, and falling clones and subclones without clear evidence that gene mutations other than in TP53 and possibly SAMHD1 are frequently selected for at CLL relapse. Data in aggregate support a provisional categorization of CLL-associated recurrently mutated genes into three classes (i) often subclonal before therapy and strongly enriched after therapy, or, (ii) mostly clonal before therapy or without further enrichments at relapse, or, (iii) subclonal before and after therapy and enriching only in sporadic cases. Clin Cancer Res; 22(17); 4525-35. ©2016 AACR. ©2016 American Association for Cancer Research.

  8. Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

    Directory of Open Access Journals (Sweden)

    Juliana Branco Novo

    2012-01-01

    Full Text Available Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher’s patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr− cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa and secreted (63–69 kDa form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources.

  9. Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

    Science.gov (United States)

    Novo, Juliana Branco; Morganti, Ligia; Moro, Ana Maria; Paes Leme, Adriana Franco; Serrano, Solange Maria de Toledo; Raw, Isaias; Ho, Paulo Lee

    2012-01-01

    Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr−) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa) and secreted (63–69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources. PMID:23091360

  10. 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.

  11. Improvement of the energy metabolism of recombinant CHO cells by cell sorting for reduced mitochondrial membrane potential.

    Science.gov (United States)

    Hinterkörner, Georg; Brugger, Gudrun; Müller, Dethardt; Hesse, Friedemann; Kunert, Renate; Katinger, Hermann; Borth, Nicole

    2007-05-10

    One of the major problems in process performance of mammalian cell cultures is the production of lactic acid. Cell specific glucose uptake rates usually correlate to glucose concentration and approximately 80% of the metabolised glucose is converted into lactic acid. As the mitochondrial membrane potential was shown to correlate to cell specific glucose uptake rates, we used Rhodamine 123, a lipophilic cationic dye for cell sorting to improve the energy metabolism of existing production cell lines. Two recombinant CHO cell lines with known differences in lactic acid production rate were used to evaluate Rhodamine 123 staining as a descriptor for glucose uptake rates and to determine whether it is possible to isolate subclones with altered metabolic properties. Such subclones would exhibit an improved process performance, and in addition could be used as models for genomic and metabolic studies. From the cell line with high lactate production, a subclone sorted for reduced mitochondrial membrane potential was found to have a lower specific lactate formation rate compared to the parental cell line in batch cultures. In addition, the glucose consumption rate was also reduced, while both the growth rate and the final cell concentration were increased. A subclone sorted for high mitochondrial membrane potential, on the other hand, had a higher glucose consumption rate, a higher lactate production rate and reduced growth. The potential of using flow cytometric cell sorting methods based on physiological activity for cell line optimisation is discussed.

  12. Susceptibility of cell lines to avian viruses

    Directory of Open Access Journals (Sweden)

    Simoni Isabela Cristina

    1999-01-01

    Full Text Available The susceptibility of the five cell lines - IB-RS-2, RK-13, Vero, BHK-21, CER - to reovirus S1133 and infectious bursal disease virus (IBDV vaccine GBV-8 strain was studied to better define satisfactory and sensitive cell culture systems. Cultures were compared for presence of CPE, virus titers and detection of viral RNA. CPE and viral RNA were detected in CER and BHK-21 cells after reovirus inoculation and in RK-13 cell line after IBDV inoculation and with high virus titers. Virus replication by production of low virus titers occurred in IB-RS-2 and Vero cells with reovirus and in BHK-21 cell line with IBDV.

  13. A cystic fibrosis pancreatic adenocarcinoma cell line.

    Science.gov (United States)

    Schoumacher, R A; Ram, J; Iannuzzi, M C; Bradbury, N A; Wallace, R W; Hon, C T; Kelly, D R; Schmid, S M; Gelder, F B; Rado, T A

    1990-05-01

    We established a pancreatic adenocarcinoma cell line (CFPAC-1) from a patient with cystic fibrosis (CF) and assessed some of its properties. The cells show epithelial morphology and express cytokeratin and oncofetal antigens characteristic of pancreatic duct cells. Basal and stimulated levels of cAMP and cAMP-dependent protein kinase and the biophysical properties of single Cl- channels in CFPAC-1 are similar to those of airway and sweat gland primary cultures and Cl(-)-secreting epithelial cell lines. Anion transport and single Cl- channel activity was stimulated by Ca2+ ionophores but not by forskolin, cAMP analogs, or phosphodiesterase inhibitors. The cells express the CF gene and manifest the most common CF mutation, deletion of three nucleotides resulting in a phenylalanine-508 deletion. These properties have been stable through greater than 80 passages (24 months), suggesting that CFPAC-1 can serve as a continuous cell line that displays the CF defect.

  14. 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

    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane...... repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique......, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested...

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

    Science.gov (United States)

    Talbot, N C; Caperna, T J; Garrett, W M

    2013-01-01

    Totipotent embryonic stem cell lines have not been established from ungulates; however, we have developed a somatic stem cell line from the in vitro culture of pig epiblast cells. The cell line, ARS-PICM-19, was isolated via colony cloning and was found to spontaneously differentiate into hepatic parenchymal epithelial cell types, namely hepatocytes and bile duct cells. Hepatocytes form as monolayers and bile duct cells as 3-dimensional bile ductules. Transmission electron microscopy revealed that the ductules were composed of radially arranged, monociliated cells with their cilia projecting into the lumen of the ductule whereas hepatocytes were arranged in monolayers with lateral canalicular structures containing numerous microvilli and connected by tight junctions and desmosomes. Extensive Golgi and rough endoplasmic reticulum networks were also present, indicative of active protein synthesis. Analysis of conditioned medium by 2-dimensional electrophoresis and mass spectrometry indicated a spectrum of serum-protein secretion by the hepatocytes. The PICM-19 cell line maintains a range of inducible cytochrome P450 activities and, most notably, is the only nontransformed cell line that synthesizes urea in response to ammonia challenge. The PICM-19 cell line has been used for several biomedical- and agricultural-related purposes, such as the in vitro replication of hepatitis E virus, a zoonotic virus of pigs, and a spaceflight experiment to evaluate somatic stem cell differentiation and liver cell function in microgravity. The cell line was also evaluated as a platform for toxicity testing and has been used in a commercial artificial liver rescue device bioreactor. A PICM-19 subclone, PICM-19H, which only differentiates into hepatocytes, was isolated and methods are currently under development to grow PICM-19 cells without feeder cells. Feeder-cell-independent growth will facilitate the study of mesenchymal-parenchymal interactions that influence the divergent

  16. Targeting SPARC by lentivirus-mediated RNA interference inhibits cervical cancer cell growth and metastasis

    Directory of Open Access Journals (Sweden)

    Chen Jie

    2012-10-01

    Full Text Available Abstract Background Secreted protein acidic and rich in cysteine (SPARC, a calcium-binding matricellular glycoprotein, is implicated in the progressions of some cancers. However, no information has been available to date regarding the function of SPARC in cervical cancer cell growth and metastasis. Methods In this study, we isolated and established high invasive subclones and low invasive subclones from human cervical cancer cell lines HeLa and SiHa by the limited dilution method. Real-time q-RT-PCR, Western Blot and ICC were performed to investigate SPARC mRNA and protein expressions in high invasive subclones and low invasive subclones. Then lentivirus vector with SPARC shRNA was constructed and infected the highly invasive subclones. Real-time q-RT-PCR, Western Blot and ICC were also performed to investigate the changes of SPARC expression after viral infection. In functional assays, effects of SPARC knockdown on the biological behaviors of cervical cancer cells were investigated. The mechanisms of SPARC in cervical cancer proliferation, apoptosis and invasion were also researched. Results SPARC was over-expressed in the highly invasive subclones compared with the low invasive subclones. Knockdown of SPARC significantly suppressed cervical cancer cell proliferation, and induced cell cycle arrest at the G1/G0 phase through the p53/p21 pathway, also caused cell apoptosis accompanied by the decreased ratio of Bcl-2/Bax, and inhibited cell invasion and metastasis accompanied by down-regulated MMP2 and MMP9 expressions and up-regulated E-cadherin expression. Conclusion SPARC is related to the invasive phenotype of cervical cancer cells. Knockdown of SPARC significantly suppresses cervical cancer cell proliferation, induces cell apoptosis and inhibits cell invasion and metastasis. SPARC as a promoter improves cervical cancer cell growth and metastasis.

  17. Comprehensive pharmacological profiling of neurofibromatosis cell lines.

    Science.gov (United States)

    Guo, Jianman; Grovola, Michael R; Xie, Hong; Coggins, Grace E; Duggan, Patrick; Hasan, Rukhsana; Huang, Jiale; Lin, Danny W; Song, Claire; Witek, Gabriela M; Berritt, Simon; Schultz, David C; Field, Jeffrey

    2017-01-01

    Patients with Neurofibromatosis type 1 (NF1) and Neurofibromatosis type 2 (NF2) are predisposed to tumors of the nervous system. NF1 patients predominantly develop neurofibromas, and Malignant Peripheral Nerve Sheath Tumors (MPNST) while NF2 patients develop schwannomas and meningiomas. Here we quantified the drug sensitivities of NF1 and NF2 tumor cell lines in a high throughput platform. The platform contained a comprehensive collection of inhibitors of MEK, RAF, RAS, farnesyl transferase, PAK and ERK, representative drugs against many other cancer pathways including Wnt, Hedgehog, p53, EGF, HDAC, as well as classical cytotoxic agents recommended for treating MPNST, such as doxorubicin and etoposide. We profiled seven NF1-associated MPNST cell lines (ST88-14, ST88-3, 90-8, sNF02.2, T265, S462TY, SNF96.2), one sporadic MPNST cell line (STS26), one schwannoma from a NF2 patient (HEI193), one NF2-deficient malignant meningioma (KT21-MG-Luc5D), one mouse NF2 schwannoma (SC4) and one sporadic rat schwannoma (RT4-67 or RT4). NF1 cells were primarily distinguished from NF2 cells and the sporadic MPNST cell line by their sensitivity to MEK and ERK inhibitors, and to a smaller extent their sensitivity to BH3 mimetics and farnesyl transferase inhibitors. The platform was highly successful in predicting the effects of clinical trials for Neurofibromas.

  18. 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. PMID:18927105

  19. Enhancement of effects of irradiation by gemcitabine in a glioblastoma cell line and cell line spheroids

    NARCIS (Netherlands)

    Genç, Mine; Castro Kreder, Natasja; Barten-van Rijbroek, Angelique; Stalpers, Lukas J. A.; Haveman, Jaap

    2004-01-01

    Background and purpose. To determine the cytotoxicity of, and radioenhancement by, gemcitabine on a glioma cell line grown as a monolayer and as spheroid cultures. Material and methods. We used a human glioma cell line, Gli-6, which originated from a biopsy specimen of a patient with a glioblastoma

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    Kashkolinejad-Koohi Tahere

    2015-03-01

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

  2. 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 liquid-.

  3. Transitional cell cancer: establishment and characterization of cell lines.

    Science.gov (United States)

    Elliott, A Y; Bronson, D L; Fraley, E E

    1978-12-01

    Eleven long-term (in culture more than 1 yr) cell lines were established from surgical specimens of human TCC. Characterization studies performed on the individual cell lines showed that each 1) demonstrated an abnormal human karyotype, 2) grew in soft agar, 3) exhibited rapid growth and multilayering 4) was free from microbial and HeLa cell contamination, 5) produced tumors in cheek pouches of immunosuppressed Syrian golden hamsters, 6) contained ultrastructural features consistently found in epithelial cells in culture, and 7) could be grown to high cell densities in roller-bottle cultures.

  4. Antiproliferative efficacy of Tabernaemontana divaricata against HEP2 cell line and Vero cell line.

    Science.gov (United States)

    Kumar, Arvind; Selvakumar, S

    2015-05-01

    Laryngeal cancer may also be called cancer of the larynx or laryngeal carcinoma. Conventional plants are a precious source of novel anticancer agents and are still in performance better role in health concern. The study was intended to estimation of the anticancer activity of the chloroformic extract of Tabernaemontana divaricata on the human epidermoid larynx carcinoma cell line (Hep 2). The aerial parts (leaves, stem, and flowers) of T. divaricata were tested for its inhibitory effect in 96 microplate formats against Hep 2 cell line. The anticancer activity of samples on Hep 2 and Vero was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and various enzymatic parameters like catalase, reduced glutathione (GSH), GSH peroxidase, and superoxide anion scavenging activity. Viable cells were determined by the absorbance at 540 nm. Measurements were performed, and the concentration required for a 50% inhibition of viability (IC50) was determined graphically. The effect of the samples on the proliferation of Hep 2 and Vero cells was expressed as the % cell viability. The extract on Hep 2 cell line up to 7.8 μg/ml and that IC50 value on Hep 2 cell line was 112 μg whereas 94 μg for Vero cell line. Hence, T. divaricata has lesser significant action on Vero cell line. Medicinal plant drug discovery continues to provide new and important leads against various pharmacological targets including cancer. Our results clearly indicate the anticancer property of the medicinal plant T. divaricata against the human laryngeal carcinoma cell lines (Hep 2 cell line).

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

    Energy Technology Data Exchange (ETDEWEB)

    Felthaus, O. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Ettl, T.; Gosau, M.; Driemel, O. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Brockhoff, G. [Department of Gynecology and Obstetrics, University of Regensburg (Germany); Reck, A. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Zeitler, K. [Institute of Pathology, University of Regensburg (Germany); Hautmann, M. [Department of Radiotherapy, University of Regensburg (Germany); Reichert, T.E. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Schmalz, G. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Morsczeck, C., E-mail: christian.morsczeck@klinik.uni-regensburg.de [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany)

    2011-04-01

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

  6. Loss of drug-induced activation of the CD95 apoptotic pathway in a cisplatin-resistant testicular germ cell tumor cell line

    NARCIS (Netherlands)

    Spierings, DCJ; de Vries, EGE; Vellenga, E; de Jong, S

    Testicular germ cell tumors (TGCTs) are unusually sensitive to cisplatin. In the present study the role of the CD95 death pathway in cisplatin sensitivity of TGCT cells was studied in Tera and its in vitro acquired cisplatin-resistant subclone Tera-CP. Cisplatin induced an increase in CD95 membrane

  7. A universal mammalian vaccine cell line substrate.

    Directory of Open Access Journals (Sweden)

    Jackelyn Murray

    Full Text Available Using genome-wide small interfering RNA (siRNA screens for poliovirus, influenza A virus and rotavirus, we validated the top 6 gene hits PV, RV or IAV to search for host genes that when knocked-down (KD enhanced virus permissiveness and replication over wild type Vero cells or HEp-2 cells. The enhanced virus replication was tested for 12 viruses and ranged from 2-fold to >1000-fold. There were variations in virus-specific replication (strain differences across the cell lines examined. Some host genes (CNTD2, COQ9, GCGR, NDUFA9, NEU2, PYCR1, SEC16G, SVOPL, ZFYVE9, and ZNF205 showed that KD resulted in enhanced virus replication. These findings advance platform-enabling vaccine technology, the creation of diagnostic cells substrates, and are informative about the host mechanisms that affect virus replication in mammalian cells.

  8. 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...

  9. 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...

  10. 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.

  11. [Cloning of ID4 gene expression regulation promoter and subcloning of recombinant ID4 promoter luciferase reporter].

    Science.gov (United States)

    Li, Wei; Huang, Yu; Yang, Bo; Chi, Xiao-Hua; Liu, Li-Hong; Zhang, Feng; Yan, Jiang-Wei; Lu, Xue-Chun

    2010-04-01

    vector, and the ligation was completed by T4 DNA ligase. After transformation to TOP10 competent E. coli. and screening for positive colonies, 5 subcloned recombinants of ID4 gene promoter and pGL3 Basic vector cells were constructed. In conclusion, 2.5 kb ID4 gene promoter with upstream expression regulatory sequence was successfully cloned and a series of ID4 promoter subclone-pGL3-Basic recombinant were constructed for further researches on activity, expression regulation and function of ID4 promoter.

  12. Regulatory networks define phenotypic classes of human stem cell lines

    OpenAIRE

    Müller, Franz-Josef; Laurent, Louise C.; Kostka, Dennis; Ulitsky, Igor; Williams, Roy; Lu, Christina; Park, In-Hyun; Rao, Mahendra?S.; Shamir, Ron; Schwartz, Philip H.; Schmidt, Nils O.; Loring, Jeanne F.

    2008-01-01

    Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal, and adult sources have been called stem cells, even though they range from pluripotent cells, typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation, to adult stem cell lines, which can generate a far more limited repertory of differentiated cell types. The...

  13. 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

  14. Cytotoxicity of Sambucus ebulus on cancer cell lines and protective ...

    African Journals Online (AJOL)

    Cytotoxicity of Sambucus ebulus on cancer cell lines and protective effects of vitamins C and E against its cytotoxicity on normal cell lines. ... Cytotoxicity of SEE on cancer (HepG2 and CT26) and normal (CHO and rat fibroblast) cell lines was evaluated by MTT assay. IC50 of SEE on ... African Journal of Biotechnology Vol.

  15. Cytotoxicity of Sambucus ebulus on cancer cell lines and protective ...

    African Journals Online (AJOL)

    DR. TONUKARI NYEROVWO

    2013-05-22

    May 22, 2013 ... Also, protective effects of vitamins C and E against SEE-induced cytotoxicity on normal cell lines were studied. Cytotoxicity of SEE on cancer (HepG2 and CT26) and normal (CHO and rat fibroblast) cell lines was evaluated by MTT assay. IC50 of SEE on the cell lines was assessed. Furthermore, IC50 of ...

  16. Maturation sensitive and resistant t(15;17) NB4 cell lines as tools for APL physiopathology: nomenclature of cells and repertory of their known genetic alterations and phenotypes.

    Science.gov (United States)

    Roussel, M J; Lanotte, M

    2001-10-29

    Chromosomal translocations, leading to gene rearrangements that generate chimerical proteins, represent one of the initiating events of leukemia. Preleukemia cells eventually develop into overt leukemia by occurrence of secondary genetic alterations (tumor progression). The physiopathology of leukemia has made considerable progress during the last two decades, due to molecular biology investigations on the role played by the altered genes, during neoplasic hemopoiesis. In vitro studies have been facilitated by the establishment of stable leukemia cell lines bearing these gene rearrangements and secondary gene mutations. Investigations on acute promyelocytic leukemia (APL) have benefited from maturation sensitive and resistant cell lines (NB4 and UF-1) derived from APL patient's leukemia cells and bearing the t(15;17). The information concerning the NB4 cell line (responsiveness to retinoid/rexinoid, cAMP, arsenic, mutations causing resistance) is spread in an abundant literature. In this paper, we briefly recapitulate the cellular and molecular features of this cell line and its subclones with the aim of facilitating investigators in their choice of the most appropriate tool for their studies. As redundancy of several names given to NB4 sublines has sometimes created difficulties, we propose a nomenclature for the various NB4 sublines that most investigators certainly would be agreed with.

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

    Directory of Open Access Journals (Sweden)

    Mercedes Fernández-Moreno

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

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

    Science.gov (United States)

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

    2016-01-01

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

  19. The response effect of pheochromocytoma (PC12) cell lines to ...

    African Journals Online (AJOL)

    The toxicity of oxidized multi-walled carbon nanotubes (o-MWCNTs) are of utmost concern and in most in-vitro studies conducted so far are on dendritic cell (DC) lines with limited data on PC12 cell lines. Objectives: We focused on the effect of o-MWCNTs in PC12 cells in vitro: a common model cell for neurotoxicity.

  20. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

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

    1981-05-01

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

  1. MUCOUS CELLS IN THE EPITHELIAL LINING OF DENTIGEROUS CYST

    OpenAIRE

    Pratiksha; Sangeeta; Sumanta; Prashanti; Sathyajitraje; Rajkumar

    2014-01-01

    The epithelial lining of both the developmental and inflammatory cysts of odontogenic origin are primarily composed of squamous epithelium. Various forms of metaplasia and degenerations are observed in these epithelial linings e.g. mucous cells, ciliated cells, para and/or orthokeratinization and formation of hyaline bodies. The present study was designed to investigate the incidences of mucous cells in the epithelial lining of dentigerous cyst. Mucous cells were observed ...

  2. 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.

  3. Molecular basis for vulnerability to mitochondrial and oxidative stress in a neuroendocrine CRI-G1 cell line.

    Directory of Open Access Journals (Sweden)

    Natasha Chandiramani

    2011-01-01

    Full Text Available Many age-associated disorders (including diabetes, cancer, and neurodegenerative diseases are linked to mitochondrial dysfunction, which leads to impaired cellular bioenergetics and increased oxidative stress. However, it is not known what genetic and molecular pathways underlie differential vulnerability to mitochondrial dysfunction observed among different cell types.Starting with an insulinoma cell line as a model for a neuronal/endocrine cell type, we isolated a novel subclonal line (named CRI-G1-RS that was more susceptible to cell death induced by mitochondrial respiratory chain inhibitors than the parental CRI-G1 line (renamed CRI-G1-RR for clarity. Compared to parental RR cells, RS cells were also more vulnerable to direct oxidative stress, but equally vulnerable to mitochondrial uncoupling and less vulnerable to protein kinase inhibition-induced apoptosis. Thus, differential vulnerability to mitochondrial toxins between these two cell types likely reflects differences in their ability to handle metabolically generated reactive oxygen species rather than differences in ATP production/utilization or in downstream apoptotic machinery. Genome-wide gene expression analysis and follow-up biochemical studies revealed that, in this experimental system, increased vulnerability to mitochondrial and oxidative stress was associated with (1 inhibition of ARE/Nrf2/Keap1 antioxidant pathway; (2 decreased expression of antioxidant and phase I/II conjugation enzymes, most of which are Nrf2 transcriptional targets; (3 increased expression of molecular chaperones, many of which are also considered Nrf2 transcriptional targets; (4 increased expression of β cell-specific genes and transcription factors that specify/maintain β cell fate; and (5 reconstitution of glucose-stimulated insulin secretion.The molecular profile presented here will enable identification of individual genes or gene clusters that shape vulnerability to mitochondrial dysfunction and

  4. Polyunsaturated fatty acid metabolism in enterocyte models: T84 cell line vs. Caco-2 cell line.

    Science.gov (United States)

    Beguin, Pauline; Schneider, Anne-Catherine; Mignolet, Eric; Schneider, Yves-Jacques; Larondelle, Yvan

    2014-02-01

    Human colon carcinoma cell lines such as Caco-2 cells, model of mature enterocytes and T84 cells, model of crypt cells are useful to study interactions between nutrient processing and metabolic functions at intestinal level. Our study aimed at comparing the ability of Caco-2 and T84 cells (1) to incorporate dietary polyunsaturated fatty acids (PUFA), (2) to process them and (3) to sort them into neutral lipids (NL), free fatty acids (FFA) and phospholipids (PL). Caco-2 and T84 cells were exposed to a 7-day long supplementation with PUFA. The amounts of fatty acids accumulated and incorporated into the NL, FFA or PL fractions were higher in Caco-2 than in T84 cells. Caco-2 cells were able to significantly elongate C18 PUFA and C20 PUFA of both n-3 and n-6 families. In contrast, T84 cells were unable to elongate the n-6 fatty acids whereas elongation of n-3 fatty acids was detectable but marginal. Similarly, a Δ6 desaturase activity was observed in Caco-2 but not in T84 cells. In T84 cells, each exogenous fatty acid was predominantly accumulated in the PL fraction. In Caco-2 cells, C20 fatty acids and C18:2n-6 was preferentially accumulated in the PL fraction, while C22 PUFA and C18:3n-3 was preferentially accumulated in the NL fraction. Overall, this study has shown that Caco-2 and T84 cells, as models of intestinal mucosal cells, present large differences in PUFA accumulation capacity, specific elongase and desaturase activities and distribution pattern of exogenous PUFA and of their metabolites in the lipid classes.

  5. [Screening of differentially expressed proteins associated with directional highly lymphatic metastasis in ovarian carcinoma cell lines using SELDI-TOF-MS technology].

    Science.gov (United States)

    Guan, Xiao; Li, Dan-Rong; Wang, Qi; Li, Li; Ruan, He-Yun; Zhang, Wei

    2008-11-01

    The ovarian serous papillary adenocarcinoma cell line SKOV3 and its subclones SKOV3-pm2 and SKOV3-pm3 are cell models to investigate the molecular mechanism of invasion and metastasis of ovarian cancers. This study was to screen differentially expressed proteins between ovarian carcinoma cell lines with directional (SKOV3-pm2 and SKOV3-pm3) and non-directional (SKOV3) highly lymphatic metastasis potentials using time-of-flight mass spectrometry technology and protein chips. The lymphatic metastasis rates of the three cell lines were detected in animal models. Proteins in endochylema and supernatants of the three cell lines were screened using surface-enhanced laser desorption and ionization-time of flight-mass spectrometry (SELDI-TOF-MS). Each sample was examined using weak cation exchange (CM-10) protein chip assay and immobilized metal affinity capture (IMAC-3) SELDI ProteinChip array. Detected protein peaks were filtrated and analyzed using Ciphergen proteinchip software 3.2.0 and Biomarker Wizard software. Differentially expressed proteins were defined as those whose absolute ratio values were greater than 0.5. Lymphatic metastasis rates in SKOV3, SKOV3-pm2 and SKOV3-pm3 cell xenografts in nude mice were 20%, 90% and 100%, respectively (P<0.05). Proteins in endochylema whose mass-to-charge ratio (m/z) were 6971, 7475, 9089, 9453, 10103, 11655, and the protein in supernatants whose m/z was 4746 were differentially expressed in SKOV3, SKOV3-pm2 and SKOV3-pm3 cells. Combined with weak cation exchange protein chip assay and immobilized metal affinity capture SELDI ProteinChip array, SELDI-TOF-MS technology can be used to screen and identify differentially expressed proteins associated with directional highly lymphatic metastasis in ovarian carcinoma cell lines.

  6. Characterization and properties of nine human ovarian adenocarcinoma cell lines.

    Science.gov (United States)

    Langdon, S P; Lawrie, S S; Hay, F G; Hawkes, M M; McDonald, A; Hayward, I P; Schol, D J; Hilgers, J; Leonard, R C; Smyth, J F

    1988-11-01

    Four series of cell lines have been derived from patients with ovarian adenocarcinoma. Nine cell lines have been established at one from a solid metastasis. Six lines were derived from the ascites or pleural effusion of patients with poorly differentiated adenocarcinoma: PEO1, PEO4, and PEO6 from one patient, PEA1 and PEA2 from a second, and PEO16 from a third. Three lines (PEO14 and PEO23 from ascites and TO14 from a solid metastasis) were derived from a patient with a well-differentiated serous adenocarcinoma. Each set of cell lines was morphologically distinct. The five cell lines PEO1, PEO4, PEO6, PEA1, and PEA2 had cloning efficiencies on plastic of 1-2% and only a few cells in these lines expressed alkaline phosphatase or vimentin. Only a low percentage of these cells reacted with the monoclonal antibodies 123C3 and 123A8 but most reacted with OC125. Conversely the cell lines PEO14, TO14, PEO23, and PEO16 were characterized by low cloning efficiency values (less than 0.05%), marked expression of alkaline phosphatase and vimentin, and good reaction with 123C3 and 123A8 but not OC125. These four cell lines also exhibited dome formation. Four of the cell lines, PEO1, PEO4, PEO6, and PEO16, have been xenografted into immune-deprived mice and found to be tumorigenic.

  7. Comparative proteomic analysis of fibrosarcoma and skin fibroblast cell lines.

    Science.gov (United States)

    Meral, Ogunc; Uysal, Hamdi

    2015-02-01

    Comparative proteomic analysis of normal and cancer cell lines provides for a better understanding of the molecular mechanism of cancer development and is essential for developing more effective strategies for new biomarker or drug target discovery. The purpose of this study is to compare protein expression levels between fibrosarcoma and fibroblast cell lines. In our study, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) techniques were carried out to compare the protein profile between fibrosarcoma and fibroblast cell lines. We prepared cell lysate samples to analyze intracellular proteins and secretome samples to analyze extracellular proteins in both cell lines. Our results revealed 13 upregulated proteins and 1 downregulated protein of which all of them identified in fibrosarcoma cell line after the comparison with fibroblast cell line cell lysates. When comparing secretome profiles of both cell lines, we found and identified 13 proteins only expressed in fibrosarcoma cell line. These identified proteins have common functions such as cell proliferation, cell differentiation, invasion, metastasis, and apoptosis in cancer. The data obtained from this study indicates that these proteins have importance on understanding the molecular mechanism of fibrosarcoma. These proteins may serve as candidate biomarkers and drug targets for future clinical studies.

  8. Antiproliferative action of metformin in human lung cancer cell lines.

    Science.gov (United States)

    Ashinuma, Hironori; Takiguchi, Yuichi; Kitazono, Satoru; Kitazono-Saitoh, Miyako; Kitamura, Atsushi; Chiba, Tetsuhiro; Tada, Yuji; Kurosu, Katsushi; Sakaida, Emiko; Sekine, Ikuo; Tanabe, Nobuhiro; Iwama, Atsushi; Yokosuka, Osamu; Tatsumi, Koichiro

    2012-07-01

    The oral antidiabetic agent metformin has anticancer properties, probably via adenosine monophosphate-activated protein kinase activation. In the present study, growth inhibition was assessed by a clonogenic and by a cell survival assay, apoptosis induction was assessed by Hoechst staining and caspase activities and cell cycle alteration after exposure to metformin, and the interaction of metformin with cisplatin in vitro were elucidated in four human lung cancer cell lines representing squamous, adeno-, large cell and small cell carcinoma. Clonogenicity and cell proliferation were inhibited by metformin in all the cell lines examined. This inhibitory effect was not specific to cancer cells because it was also observed in a non-transformed human mesothelial cell line and in mouse fibroblast cell lines. Inhibition of clonogenicity was observed only when the cells were exposed to metformin for a long period, (10 days) and the surviving fraction, obtained after inhibiting proliferation by increasing the dose, reached a plateau at approximately 0.1-0.3, indicating the cytostatic characteristics of metformin. Metformin induced significant apoptosis only in the small cell carcinoma cell line. A tendency of cell cycle accumulation at the G0/G1 phase was observed in all four cell lines. Cisplatin, in a dose-dependent manner, severely antagonized the growth inhibitory effect of metformin, and even reversed the effect in three cell lines but not in the adenocarcinoma cell line. The present data obtained using various histological types of human lung cancer cell lines in vitro illustrate the cytostatic nature of metformin and its cytoprotective properties against cisplatin.

  9. 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

  10. Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

    Directory of Open Access Journals (Sweden)

    Farzaneh Shafaghat

    2015-03-01

    Full Text Available Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs. We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems.

  11. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

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

    2011-01-01

    The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...... (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 mu M were...... incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size...

  12. Comparison of abnormal isoform of prion protein in prion-infected cell lines and primary-cultured neurons by PrPSc-specific immunostaining.

    Science.gov (United States)

    Tanaka, Misaki; Fujiwara, Ai; Suzuki, Akio; Yamasaki, Takeshi; Hasebe, Rie; Masujin, Kentaro; Horiuchi, Motohiro

    2016-08-01

    We established abnormal isoform of prion protein (PrPSc)-specific double immunostaining using mAb 132, which recognizes aa 119-127 of the PrP molecule, and novel PrPSc-specific mAb 8D5, which recognizes the N-terminal region of the PrP molecule. Using the PrPSc-specific double immunostaining, we analysed PrPSc in immortalized neuronal cell lines and primary cerebral-neuronal cultures infected with prions. The PrPSc-specific double immunostaining showed the existence of PrPSc positive for both mAbs 132 and 8D5, as well as those positive only for either mAb 132 or mAb 8D5. This indicated that double immunostaining detects a greater number of PrPSc species than single immunostaining. Double immunostaining revealed cell-type-dependent differences in PrPSc staining patterns. In the 22 L prion strain-infected Neuro2a (N2a)-3 cells, a subclone of N2a neuroblastoma cell line, or GT1-7, a subclone of the GT1 hypothalamic neuronal cell line, granular PrPSc stains were observed at the perinuclear regions and cytoplasm, whereas unique string-like PrPSc stains were predominantly observed on the surface of the 22 L strain-infected primary cerebral neurons. Only 14 % of PrPSc in the 22 L strain-infected N2a-3 cells were positive for mAb 8D5, indicating that most of the PrPSc in N2a-3 lack the N-terminal portion. In contrast, nearly half PrPSc detected in the 22 L strain-infected primary cerebral neurons were positive for mAb 8D5, suggesting the abundance of full-length PrPSc that possesses the N-terminal portion of PrP. Further analysis of prion-infected primary neurons using PrPSc-specific immunostaining will reveal the neuron-specific mechanism for prion propagation.

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

    Science.gov (United States)

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

  14. Human rhabdomyosarcoma cell lines for rhabdomyosarcoma research: Utility and pitfalls

    Directory of Open Access Journals (Sweden)

    Ashley R.P. Hinson

    2013-07-01

    Full Text Available Rhabdomyosarcoma (RMS is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis.

  15. Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes.

    Science.gov (United States)

    Ramirez-Gordillo, Daniel; Trujillo-Provencio, Casilda; Knight, V Bleu; Serrano, Elba E

    2011-10-01

    The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. As part of this effort, we aimed to optimize physical (electroporation), chemical (lipid-mediated; Lipofectamine™ 2000, Metafectene® Pro), and biological (viral-mediated; BacMam virus Cellular Lights™ Tubulin-RFP) gene delivery methods in amphibian (Xenopus; A6) cells and mammalian (Chinese hamster ovary (CHO)) cells. We successfully introduced the commercially available pEGFP-N3, pmCherry-N1, pEYFP-Tubulin, and Cellular Lights™ Tubulin-RFP fluorescent constructs to cells and evaluated their transfection or transduction efficiencies using the three gene delivery methods. In addition, we analyzed the transfection efficiency of a novel construct synthesized in our laboratory by cloning the Xenopus inner ear calcium-activated potassium channel β1 subunit, then subcloning the subunit into the pmCherry-N1 vector. Every gene delivery method was significantly more effective in CHO cells. Although results for the A6 cell line were not statistically significant, both cell lines illustrate a trend towards more efficient gene delivery using viral-mediated methods; however the cost of viral transduction is also much higher. Our findings demonstrate the need to improve gene delivery methods for amphibian cells and underscore the necessity for a greater understanding of amphibian cell biology.

  16. Molecular characterization of immortalized normal and dysplastic oral cell lines.

    Science.gov (United States)

    Dickman, Christopher T D; Towle, Rebecca; Saini, Rajan; Garnis, Cathie

    2015-05-01

    Cell lines have been developed for modeling cancer and cancer progression. The molecular background of these cell lines is often unknown to those using them to model disease behaviors. As molecular alterations are the ultimate drivers of cell phenotypes, having an understanding of the molecular make-up of these systems is critical for understanding the disease biology modeled. Six immortalized normal, one immortalized dysplasia, one self-immortalized dysplasia, and two primary normal cell lines derived from oral tissues were analyzed for DNA copy number changes and changes in both mRNA and miRNA expression using SMRT-v.2 genome-wide tiling comparative genomic hybridization arrays, Agilent Whole Genome 4x44k expression arrays, and Exiqon V2.M-RT-PCR microRNA Human panels. DNA copy number alterations were detected in both normal and dysplastic immortalized cell lines-as well as in the single non-immortalized dysplastic cell line. These lines were found to have changes in expression of genes related to cell cycle control as well as alterations in miRNAs that are deregulated in clinical oral squamous cell carcinoma tissues. Immortal lines-whether normal or dysplastic-had increased disruption in expression relative to primary lines. All data are available as a public resource. Molecular profiling experiments have identified DNA, mRNA, and miRNA alterations for a panel of normal and dysplastic oral tissue cell lines. These data are a valuable resource to those modeling diseases of the oral mucosa, and give insight into the selection of model cell lines and the interpretation of data from those lines. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Establishment and characterization of eight feline mammary adenocarcinoma cell lines.

    Science.gov (United States)

    Uyama, Rina; Hong, Sung-Hyeok; Nakagawa, Takayuki; Yazawa, Mitsuhiro; Kadosawa, Tsuyoshi; Mochizuki, Manabu; Tsujimoto, Hajime; Nishimura, Ryohei; Sasaki, Nobuo

    2005-12-01

    Eight new feline mammary adenocarcinoma cell lines derived from either primary or metastatic lesions were established. The morphology of all the cell lines was epithelioid and round to spindle in shape, with cell growth occurring in a monolayer fashion. On immunohistochemistry, these cells reacted with anti-keratin and anti-vimentin antisera. The doubling time of these cells was between 19 and 54 hr. Tumor masses were developed in nude mice by subcutaneous inoculation of the cells that were histologically identical to their original mammary tumor lesions. Telomerase activities measured using the telomeric repeat amplification protocol assay revealed high telemetric activity in all of the cells.

  18. Licochalcone A exerts antitumor activity in bladder cancer cell lines ...

    African Journals Online (AJOL)

    State and Federal laws, standards of the US. Department of Health and Human Services, and guidelines established by Tulane University. Animal Care and Use Committee, accredited by. Association for the Assessment and. Accreditation of Laboratory Animal Care [16]. Tumor cell line. The bladder cancer cell lines, ...

  19. Trichloroethylene toxicity in a human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-12-31

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

  20. Cancer and inflammation studies using zebrafish cell lines

    NARCIS (Netherlands)

    He, Shuning

    2010-01-01

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

  1. Beryllium-stimulated apoptosis in macrophage cell lines.

    Science.gov (United States)

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

    2000-08-21

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

  2. The ST131 Escherichia coli H22 subclone from human intestinal microbiota: Comparison of genomic and phenotypic traits with those of the globally successful H30 subclone.

    Science.gov (United States)

    Nicolas-Chanoine, Marie-Hélène; Petitjean, Marie; Mora, Azucena; Mayer, Noémie; Lavigne, Jean-Philippe; Boulet, Olivier; Leflon-Guibout, Véronique; Blanco, Jorge; Hocquet, Didier

    2017-03-27

    In 2006, we found healthy subjects carrying ST131 Escherichia coli in their intestinal microbiota consisting of two populations: a subdominant population of fluoroquinolone-resistant E. coli belonging to subclone H30 (H30-R or subclade C1), the current worldwide dominant ST131 subclone, and a dominant E. coli population composed of antibiotic-susceptible E. coli belonging to subclone H22 (clade B), the precursor of subclone H30. We sequenced the whole genome of fecal H22 strain S250, compared it to the genomes of ExPEC ST131 H30-Rx strain JJ1886 and commensal ST131 H41 strain SE15, sought the H22-H30 genomic differences in our fecal strains and assessed their phenotypic consequences. We detected 173 genes found in the Virulence Factor Database, of which 148 were shared by the three ST131 genomes, whereas some were genome-specific, notably those allowing determination of virotype (D for S250 and C for JJ1886). We found three sequences of the FimH site involved in adhesion: two in S250 and SE15 close and identical, respectively, to that previously reported to confer strong intestinal adhesion, and one in JJ1886, corresponding to that commonly present in uropathogenic E. coli. Among the genes involved in sugar metabolism, one encoding a gluconate kinase lacked in S250 and JJ1886. Although this gene was also absent in both our fecal H22 and H30-R strains, H22 strains showed a higher capacity to grow in minimal medium with gluconate. Among the genes involved in gluconate metabolism, only the ghrB gene differed between S250/H22 and JJ1886/H30-R strains, resulting in different gluconate reductases. Of the genes involved in biofilm formation, two were absent in the three genomes and one, fimB, in the JJ1886 genome. Our fecal H30-R strains lacking intact fimB displayed delayed biofilm formation relative to our fecal H22 strains. The H22 strains differed by subclade B type and plasmid content, whereas the H30-R strains were identical. Phenotypic analysis of our fecal strains

  3. Effect of lycopene on cell viability and cell cycle progression in human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Teodoro Anderson

    2012-08-01

    Full Text Available Abstract Background Lycopene, a major carotenoid component of tomato, has a potential anticancer activity in many types of cancer. Epidemiological and clinical trials rarely provide evidence for mechanisms of the compound’s action, and studies on its effect on cancer of different cell origins are now being done. The aim of the present study was to determine the effect of lycopene on cell cycle and cell viability in eight human cancer cell lines. Methods Human cell lines were treated with lycopene (1–5 μM for 48 and 96 h. Cell viability was monitored using the method of MTT. The cell cycle was analyzed by flow cytometry, and apoptotic cells were identified by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling (TUNEL and by DAPI. Results Our data showed a significant decrease in the number of viable cells in three cancer cells lines (HT-29, T84 and MCF-7 after 48 h treatment with lycopene, and changes in the fraction of cells retained in different cell cycle phases. Lycopene promoted also cell cycle arrest followed by decreased cell viability in majority of cell lines after 96 h, as compared to controls. Furthermore, an increase in apoptosis was observed in four cell lines (T-84, HT-29, MCF-7 and DU145 when cells were treated with lycopene. Conclusions Our findings show the capacity of lycopene to inhibit cell proliferation, arrest cell cycle in different phases and increase apoptosis, mainly in breast, colon and prostate lines after 96 h. These observations suggest that lycopene may alter cell cycle regulatory proteins depending on the type of cancer and the dose of lycopene administration. Taken together, these data indicated that the antiproliferative effect of lycopene was cellular type, time and dose-dependent.

  4. Comparison of steroid receptors from the androgen responsive DDT1 cell line and the nonresponsive HVP cell line.

    Science.gov (United States)

    Norris, J S; Kohler, P O

    1978-01-01

    Two hamster cell lines have been isolated from androgen target tissue. The DDT1 cells derived from ductus deferens tissue exhibit a growth response to androgens, while the HVP cells derived from ventral prostate are androgen unresponsive. Both cell lines contain androgen receptors, that are similar when compared by kinetic methods, sedimentation velocity, chromatographic procedures or nuclear translocation ability. The forms of the high salt extracted nuclear receptors are indistinguishable chromatographically. Therefore, we postulate that the lesion preventing androgen induced growth in the HVP cell line is subseqent to nuclear translocation of the steroid receptor complex.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Khordadmehr, M.,

    2014-05-01

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

  7. Induction of DNA-strand breaks after X- irradiation in murine bone cells of various differentiation capacities

    Science.gov (United States)

    Lau, P.; Hellweg, C. E.; Kirchner, S.; Arenz, A.; Baumstark-Khan, C.; Horneck, G.

    Bone loss resulting from long-duration space flight is a well known medical risk for space travellers, as a weakened skeleton is more susceptible to bone fractures. In addition to weightlessness the astronaut is also exposed to cosmic ionizing radiation. In order to elucidate changes in bone cell metabolism by ionizing radiation, a ground-based bone cell model has been developed. This model consists of a bunch of immortalized murine osteocyte, osteoblast and pre-osteoblast cell lines representing discrete stages of differentiation: The osteocyte cell line MLO-Y4 (obtained from L. Bonewald, Kansas City, USA), the osteoblast cell line OCT-1 (obtained from D. Chen, San Antonio, USA), and the subclones 4 and 24 of the osteoblast cell line MC3T3-E1 (obtained from ATCC, Manassas, Virginia, USA). Regarding their growth properties, MLO-Y4 cells show the highest growth velocity with a doubling time of 15.8 h. The osteoblast cell line OCT-1 has a doubling time of 27.3 h. The respective values for MC3T3-E1 subclone 24 and S4 are 90.5 h and 51.6 h. To investigate the stage of differentiation, the expression of alkaline phosphatase, of osteocalcin and of E11 was examined. Survival after X-ray exposure was determined using the colony forming ability test. The resulting dose-effect relationships revealed significant differences. The parameter D0 of the survival curves ranges between 1.8 Gy for OCT-1, 1.9 Gy for MLO-Y4, 2.0 Gy for subclone 24 and 2,3 Gy for subclone 4. The quantitative acquisition of DNA-strand breaks was performed by Fluorescent Analysis of DNA-Unwinding (FADU). The results can be correlated with the corresponding survival curve. In conclusion, the cell lines with higher differentiation levels are less sensitive to radiation when compared to the lower differentiated osteoblast cell lines.

  8. Radiation-induced adaptive response in fish cell lines.

    Science.gov (United States)

    Ryan, Lorna A; Seymour, Colin B; O'Neill-Mehlenbacher, Alicia; Mothersill, Carmel E

    2008-04-01

    There is considerable interest at present in low-dose radiation effects in non-human species. In this study gamma radiation-induced adaptive response, a low-dose radiation effect, was examined in three fish cell lines, (CHSE-214 (Chinook salmon), RTG-2 (rainbow trout) and ZEB-2J (zebrafish)). Cell survival after exposure to direct radiation with or without a 0.1 Gy priming dose, was determined using the colony forming assay for each cell line. Additionally, the occurrence of a bystander effect was examined by measuring the effect of irradiated cell culture medium from the fish cell lines on unexposed reporter cells. A non-linear dose response was observed for all cell lines. ZEB-2J cells were very sensitive to low doses and a hyper-radiosensitive (HRS) response was observed for doses fish cell lines tested. Rather, it was found that pre-exposure of these cells to 0.1 Gy radiation sensitized the cells to subsequent high doses. In CHSE-214 cells, increased sensitivity to subsequent high doses of radiation was observed when the priming and challenge doses were separated by 4 h; however, this sensitizing effect was no longer present when the interval between doses was greater than 8 h. Additionally, a "protective" bystander response was observed in these cell lines; exposure to irradiated medium from fish cells caused increased cloning efficiency in unirradiated reporter cells. The data confirm previous conclusions for mammalian cells that the adaptive response and bystander effect are inversely correlated and contrary to expectations probably have different underlying mechanisms.

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

    Science.gov (United States)

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

    2014-01-01

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

  10. Vaccine production: upstream processing with adherent or suspension cell lines.

    Science.gov (United States)

    Genzel, Yvonne; Rödig, Jana; Rapp, Erdmann; Reichl, Udo

    2014-01-01

    The production of viral vaccines in cell culture can be accomplished with primary, diploid, or continuous (transformed) cell lines. Each cell line, each virus type, and each vaccine preparation require the specific design of upstream and downstream processing. Media have to be selected as well as production vessels, cultivation conditions, and modes of operation. Many viruses only replicate to high titers in adherently growing cells, but similar to processes established for recombinant protein production, an increasing number of suspension cell lines is being evaluated for future use. Here, we describe key issues to be considered for the establishment of large-scale virus production in bioreactors. As an example upstream processing of cell culture-derived influenza virus production is described in more detail for adherently growing and for suspension cells. In particular, use of serum-containing, serum-free, and chemically defined media as well as choice of cultivation vessel are considered.

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

    Science.gov (United States)

    Machatková, M; Jurmanová, K; Snejdar, V

    1986-07-01

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

  12. Differential heat shock response of primary human cell cultures and established cell lines

    DEFF Research Database (Denmark)

    Richter, W W; Issinger, O G

    1986-01-01

    degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized...

  13. Transcription profiles of non-immortalized breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Holland James F

    2006-04-01

    Full Text Available Abstract Background Searches for differentially expressed genes in tumours have made extensive use of array technology. Most samples have been obtained from tumour biopsies or from established tumour-derived cell lines. Here we compare cultures of non-immortalized breast cancer cells, normal non-immortalized breast cells and immortalized normal and breast cancer cells to identify which elements of a defined set of well-known cancer-related genes are differentially expressed. Methods Cultures of cells from pleural effusions or ascitic fluids from breast cancer patients (MSSMs were used in addition to commercially-available normal breast epithelial cells (HMECs, established breast cancer cell lines (T-est and established normal breast cells (N-est. The Atlas Human Cancer 1.2 cDNA expression array was employed. The data obtained were analysed using widely-available statistical and clustering software and further validated through real-time PCR. Results According to Significance Analysis of Microarray (SAM and AtlasImage software, 48 genes differed at least 2-fold in adjusted intensities between HMECs and MSSMs (p Conclusion The expression profiles of 1176 genes were determined in finite life-span cultures of metastatic breast cancer cells and of normal breast cells. Significant differences were detected between the finite life-span breast cancer cell cultures and the established breast cancer cell lines. These data suggest caution in extrapolating information from established lines for application to clinical cancer research.

  14. UCI-VULV-1, a vulvar squamous carcinoma cell line.

    Science.gov (United States)

    Carpenter, P M; Gamboa-Vujicic, G; Mascarello, J T; Wilczynski, S; Bhaumik, M; Dorion, G; Manetta, A

    1995-05-01

    Squamous carcinoma of the vulva (SCV) is an uncommon neoplasm of uncertain etiology. There is evidence that there are two subgroups of SCV, one associated with human papilloma virus (HPV) and a second HPV-negative group. The UCI-VULV-1 cell line, obtained from a lymph node metastasis of an SCV, grows with a population doubling time of approximately 60 hr. The saturation density is 10(5) cells/cm2. The cell line does not exhibit anchorage independence and is weakly tumorigenic. The cells range in appearance from an abundant spindle cell to a less common larger, flat cell. All of the cells are immunoreactive for high-molecular-weight keratin, but only the flat cells, which form squamous pearls in vivo, are immunoreactive for low-molecular-weight keratin. The cell line expresses epidermal growth factor (EGF), transforming growth factor-alpha, the EGF receptor, and p53 protein. Polymerase chain reaction revealed no HPV DNA within the cells. Early passage cells exhibited karyotypic heterogeneity with few similarities to previous described SCV karyotypes. The cells display sensitivity to cis-platinum in concentrations toxic to many ovarian and cervical carcinoma lines. UCI-VULV-1 may be helpful for studying the properties of the HPV-negative form of SCV.

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

    Directory of Open Access Journals (Sweden)

    Julian Biau

    2016-08-01

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

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

    Science.gov (United States)

    Relan, Vandana; Morrison, Leanne; Parsonson, Kylie; Clarke, Belinda E; Duhig, Edwina E; Windsor, Morgan N; Matar, Kevin S; Naidoo, Rishendran; Passmore, Linda; McCaul, Elizabeth; Courtney, Deborah; Yang, Ian A; Fong, Kwun M; Bowman, Rayleen V

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Vandana Relan

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

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

    Science.gov (United States)

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

    2010-01-01

    Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide-ranging transfectant population. This identification process is on the critical path for first-in-human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One-hundred and seventy-five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines. © 2010 American Institute of Chemical Engineers

  19. Determinants of intrinsic radiosensitivity of mammalian cell lines

    Energy Technology Data Exchange (ETDEWEB)

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

    1998-12-31

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

  20. 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.

  1. Molecular cytogenetic characterization of two established ESFT cell lines.

    Science.gov (United States)

    Ishiguro, Masako; Yuki, Mutsumi; Fukushige, Tomoko; Mizoguchi, Mikio; Kaneko, Yasuhiko; Morishige, Takeshita; Iwasaki, Hiroshi

    2017-01-01

    Ewing's sarcoma/primitive neuroectodermal tumor/Askin's tumor (Ewing`s sarcoma family of tumors: ESFT) is the most common type of malignant tumor of bone and soft tissue in children and young adults, and morphologically is a member of a group of small round cell tumors. We report, here, on the establishment of two human ESFT cell lines, FU-PNET-3 and FU-PNET-4, from the iliac and the chest wall, respectively, the cells of both cell lines were tumorigenic in immunodeficient mice. Histologically, both original and xenograft tumors and cultured cells were composed of small round cells with positive immunoreactivity for CD99 and Nkx2.2. Molecular biological examination demonstrated chimeric transcripts of EWSR1 exon 7 to FLI1 exon 6 in FU-PNET-3 cells, and EWSR1 exon 10 to FLI1 exon 6 in FU-PNET-4 cells. Cytogenetic analysis revealed chromosome translocation t(11;22)(q24;q12) and some secondary changes in both cultured cells. These histological, molecular biological, and cytogenetical findings indicate ESFT in both cell lines. ESFT is well studied, but its recurrent fusion genes are heterogeneous and its biological behaviors are unclear. The FU-PNET-3 and FU-PNET-4 cell lines have been well examined and may become useful tools for studying the genetic and biological behavioral properties of ESFT.

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

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2006-11-01

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

  3. 77 FR 5489 - Identification of Human Cell Lines Project

    Science.gov (United States)

    2012-02-03

    ... at the National Center For Biotechnology Information (NCBI). DATES: On the first of each month... will be posted in a publically held database. The use of misidentified cell lines in cancer and other...

  4. Recent developments on human cell lines for the bioartificial liver

    NARCIS (Netherlands)

    Hoekstra, R.; Chamuleau, R. A. F. M.

    2002-01-01

    Most bioartificial liver (BAL) devices contain porcine primary hepatocytes as their biological component. However, alternatives are needed due to xenotransplantation associated risks. Human liver cell lines have excellent growth characteristics and are therefore candidates for application in BAL

  5. Surface charge characteristics of cells from malignant cell lines and normal cell lines of the human hematopoietic system.

    Science.gov (United States)

    Marikovsky, Y; Ben-Bassat, H; Leibovich, S J; Cividalli, L; Fischler, H; Danon, D

    1979-02-01

    Cells from malignant and normal lines of human hematopoietic origin were studied for their surface charge characteristics with the use of the following criteria: 1) the electron microscopic appearance of cell membranes after labeling with cationized ferritin (CF) either before or after glutaraldehyde fixation, 2) electrophoretic mobility, 3) total sialic acid content, and 4) agglutinability with poly-L-lysine (PLL). CF induced a time-dependent redistribution of surface receptors in unfixed malignant cells but not in unfixed normal cells. After 10 seconds of labeling with CF, both normal and malignant unfixed cells showed a uniform and even labeling pattern. After 5 minutes of labeling, malignant cells exhibited a highly pronounced pattern of clusters and patches, as distinct from a random and even pattern exhibited by normal cells. Both normal and malignant cells after fixation exhibited an equivalent random and even labeling pattern with CF, independent of the duration of labeling. The malignant cells studied possessed less sialic acid, had a lower electric mobility, and were agglutinated more readily with PLL than were the normal cells.

  6. Selection of hydroxyproline-resistant cell lines of Solanum melongena

    Energy Technology Data Exchange (ETDEWEB)

    Hogetu, Daisuke (Tokyo Metropolitan Isotope Research Center (Japan))

    1991-02-01

    Plating cell clusters of Solanum melongena the diameter of which were between 177 {mu}m and 64 {mu}m, gave high and stable plating efficiencies. The effect of various mutagen treatment on the appearance of hydroxyproline-resistant cell lines were compared using the plating technique. Gamma-ray, UV and MNNG, at intensities around LD 50, raised mutation rate eight-, thirteen- and six-times that of spontaneous mutation, respectively. Proline content of hydroxyproline-resistant cell lines obtained by gamma-ray irradiation were higher than that of hydroxyproline-sensitive cell. (author).

  7. RC-IAL cell line: sensitivity of rubella virus grow

    Directory of Open Access Journals (Sweden)

    Figueiredo Cristina A

    2000-01-01

    Full Text Available OBJECTIVE: The rapid growth of the rubella virus in RC-IAL² with development of cytopathic effect, in response to rubella virus infection, is described. For purposes of comparison, the rubella virus RA-27/3 strain was titered simultaneously in the RC-IAL, Vero, SIRC and RK13 cell lines. METHODS: Rubella virus RA-27/3 strain are inoculated in the RC-IAL cell line (rabbit Kidney, Institute Adolfo Lutz. Plates containing 1.5x10(5 cells/ml of RC-IAL line were inoculated with 0.1ml s RA-27/3 strain virus containing 1x 10(4TCID50/0.1ml. A 25% cytopathic effect was observed after 48 hours and 100% after 96 hours. The results obtained were compared to those observed with the SIRC, Vero and RK13 cell lines. Rubella virus was detected by immunohistochemistry. RESULTS: With the results, it was possible to conclude that the RC-IAL cell line is a very good substrate for culturing rubella virus. The cells inoculated with rubella virus were examined by phase contrast microscopy and showed the characteristic rounded, bipolar and multipolar cells. The CPE in RC-IAL was observed in the first 48 hours and the curve of the increased infectivity was practically the same as observed in other cell lines. CONCLUSIONS: These findings are important since this is one the few cell lines described in the literature with a cytopathic effect. So it can be used for antigen preparation and serological testing for the diagnosis of specific rubella antibodies.

  8. Bortezomib resistance in mantle cell lymphoma is associated with plasmacytic differentiation

    DEFF Research Database (Denmark)

    Pérez-Galán, Patricia; Mora-Jensen, Helena; Weniger, Marc A

    2011-01-01

    Bortezomib induces remissions in 30%-50% of patients with relapsed mantle cell lymphoma (MCL). Conversely, more than half of patients' tumors are intrinsically resistant to bortezomib. The molecular mechanism of resistance has not been defined. We generated a model of bortezomib-adapted subclones...... of the MCL cell lines JEKO and HBL2 that were 40- to 80-fold less sensitive to bortezomib than the parental cells. Acquisition of bortezomib resistance was gradual and reversible. Bortezomib-adapted subclones showed increased proteasome activity and tolerated lower proteasome capacity than the parental lines....... Using gene expression profiling, we discovered that bortezomib resistance was associated with plasmacytic differentiation, including up-regulation of IRF4 and CD38 and expression of CD138. In contrast to plasma cells, plasmacytic MCL cells did not increase immunoglobulin secretion. Intrinsically...

  9. A Focused Microarray for Screening Rat Embryonic Stem Cell Lines

    OpenAIRE

    Hong, James; He, Hong; Bui, Phuoc; Ryba-White, Ben; Rumi, Mohammad A.K.; Soares, Michael J.; Dutta, Debasree; Paul, Soumen; Kawamata, Masaki; Ochiya, Takahiro; Ying, Qi-Long; Rajanahalli, Pavan; Mark L. Weiss

    2012-01-01

    Here, we describe a focused microarray for screening rat embryonic stem cells (ESCs) and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem (TS) cells, rat extraembryonic endoderm cells, mouse embryonic fibroblast feeder cells, and differentiated rat ESCs. Using this tool, genuine rat ESC lines, which have been expanded in a conventional rat ESC medium containing two inhibitors (2i), for example, glycogen synthase kinase 3 (GSK3) and mi...

  10. Antitumor Activity of Propolis on Differantiated Cancer Cell Lines

    OpenAIRE

    Neşe Ersöz Gülçelik

    2012-01-01

    Propolis is a natural bee product with several pharmacological activities. Nowadays, it is also investigated for its antitumor properties. There are contraversies on the antitumor activity of propolis, not all tumour cells seem to respond to propolis treatment. The aim of our study is to evaluate the activity of propolis on differantiated thyroid cancer cell lines. Tyripan blue test and MTT assay were performed to evaluate the cell viability of B-CPAP cells after propolis treatment and compar...

  11. Antitumor Activity of Propolis on Differantiated Cancer Cell Lines

    OpenAIRE

    Neşe Ersöz Gülçelik; Zeybek, Dilara; Kaymaz, Figen; Gencay, Ömür; Salih, Bekir; Asan, Esin; Sorkun, Kadriye; Usman, Aydan

    2014-01-01

    Propolis is a natural bee product with several pharmacological activities. Nowadays, it is also investigated for its antitumor properties. There are contraversies on the antitumor activity of propolis, not all tumour cells seem to respond to propolis treatment. The aim of our study is to evaluate the activity of propolis on differantiated thyroid cancer cell lines. Tyripan blue test and MTT assay were performed to evaluate the cell viability of B-CPAP cells after propolis treatment and compar...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-01-01

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

  13. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

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

    1981-05-01

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

  14. Cancer stem cell-like side population cells in clear cell renal cell carcinoma cell line 769P.

    Science.gov (United States)

    Huang, Bin; Huang, Yi Jun; Yao, Zhi Jun; Chen, Xu; Guo, Sheng Jie; Mao, Xiao Peng; Wang, Dao Hu; Chen, Jun Xing; Qiu, Shao Peng

    2013-01-01

    Although cancers are widely considered to be maintained by stem cells, the existence of stem cells in renal cell carcinoma (RCC) has seldom been reported, in part due to the lack of unique surface markers. We here identified cancer stem cell-like cells with side population (SP) phenotype in five human RCC cell lines. Flow cytometry analysis revealed that 769P, a human clear cell RCC cell line, contained the largest amount of SP cells as compared with other four cell lines. These 769P SP cells possessed characteristics of proliferation, self-renewal, and differentiation, as well as strong resistance to chemotherapy and radiotherapy that were possibly related to the ABCB1 transporter. In vivo experiments with serial tumor transplantation in mice also showed that 769P SP cells formed tumors in NOD/SCID mice. Taken together, these results indicate that 769P SP cells have the properties of cancer stem cells, which may play important roles in tumorigenesis and therapy-resistance of RCC.

  15. Cloning of a Recombinant Plasmid Encoding Thiol-Specific Antioxidant Antigen (TSA) Gene of Leishmania majorand Expression in the Chinese Hamster Ovary Cell Line.

    Science.gov (United States)

    Fatemeh, Ghaffarifar; Fatemeh, Tabatabaie; Zohreh, Sharifi; Abdolhosein, Dalimiasl; Mohammad Zahir, Hassan; Mehdi, Mahdavi

    2012-01-01

    TSA (thiol-specific antioxidant antigen) is the immune-dominant antigen of Leishmania major and is considered to be the most promising candidate molecule for a recombinant or DNA vaccine against leishmaniasis. The aim of the present work was to express a plasmid containing the TSA gene in eukaryotic cells. Genomic DNA was extracted, and the TSA gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pTZ57R/T vector, followed by subcloning into the eukaryotic expression vector pcDNA3 (EcoRI and HindIII sites). The recombinant plasmid was characterised by restriction digest and PCR. Eukaryotic Chinese hamster ovary cells were transfected with the plasmid containing the TSA gene. Expression of the L. major TSA gene was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting. The plasmid containing the TSA gene was successfully expressed, as demonstrated by a band of 22.1 kDa on Western blots. The plasmid containing the TSA gene can be expressed in a eukaryotic cell line. Thus, the recombinant plasmid may potentially be used as a DNA vaccine in animal models.

  16. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

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

    2011-01-01

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

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

    DEFF Research Database (Denmark)

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

    1985-01-01

    Specimens from a human small cell carcinoma of the lung were established as a cell line in vitro. Flow cytometric DNA analysis demonstrated only one tumor cell population in the parent tumor as well as in the early passages in vitro. After six passages in vitro, two new subpopulations...... content was examined regularly by flow cytometric DNA analysis and instability was found in one of the cloned cell lines. Chromosome analysis showed that the cloned cell lines consisted of more than one population after 17 in vitro passages. Both cloned cell lines produced tumors in nude mice. Genetic...... with different DNA content appeared. By cloning, permanent cell lines were established from the new subpopulations, whereas the original population stopped growing. The cloned cell lines were characterized by morphology, chromosomes analysis, electron microscopy and plating efficiency; the stability of the DNA...

  18. SubClonal Hierarchy Inference from Somatic Mutations: Automatic Reconstruction of Cancer Evolutionary Trees from Multi-region Next Generation Sequencing.

    Directory of Open Access Journals (Sweden)

    Noushin Niknafs

    2015-10-01

    Full Text Available Recent improvements in next-generation sequencing of tumor samples and the ability to identify somatic mutations at low allelic fractions have opened the way for new approaches to model the evolution of individual cancers. The power and utility of these models is increased when tumor samples from multiple sites are sequenced. Temporal ordering of the samples may provide insight into the etiology of both primary and metastatic lesions and rationalizations for tumor recurrence and therapeutic failures. Additional insights may be provided by temporal ordering of evolving subclones--cellular subpopulations with unique mutational profiles. Current methods for subclone hierarchy inference tightly couple the problem of temporal ordering with that of estimating the fraction of cancer cells harboring each mutation. We present a new framework that includes a rigorous statistical hypothesis test and a collection of tools that make it possible to decouple these problems, which we believe will enable substantial progress in the field of subclone hierarchy inference. The methods presented here can be flexibly combined with methods developed by others addressing either of these problems. We provide tools to interpret hypothesis test results, which inform phylogenetic tree construction, and we introduce the first genetic algorithm designed for this purpose. The utility of our framework is systematically demonstrated in simulations. For most tested combinations of tumor purity, sequencing coverage, and tree complexity, good power (≥ 0.8 can be achieved and Type 1 error is well controlled when at least three tumor samples are available from a patient. Using data from three published multi-region tumor sequencing studies of (murine small cell lung cancer, acute myeloid leukemia, and chronic lymphocytic leukemia, in which the authors reconstructed subclonal phylogenetic trees by manual expert curation, we show how different configurations of our tools can

  19. Novel human multiple myeloma cell line UHKT-893

    Czech Academy of Sciences Publication Activity Database

    Uherková, L.; Vančurová, I.; Vyhlídalová, I.; Pleschnerová, M.; Špička, I.; Mihalová, R.; Březinová, J.; Hodný, Zdeněk; Čermáková, K.; Polanská, V.; Marinov, I.; Jedelský, P.L.; Kuželová, K.; Stöckbauer, P.

    2013-01-01

    Roč. 37, č. 3 (2013), s. 320-326 ISSN 0145-2126 Institutional support: RVO:68378050 Keywords : human myeloma cell line * human multiple myeloma * plasma cell * IL-6 dependence * immunoglobulin * free light chain Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.692, year: 2013

  20. The secretome signature of malignant mesothelioma cell lines.

    Science.gov (United States)

    Manfredi, Marcello; Martinotti, Simona; Gosetti, Fabio; Ranzato, Elia; Marengo, Emilio

    2016-08-11

    The secretome is the complex set of molecules secreted by cells; these molecules play a key role in cell signaling, communication and migration. Secretomics has been already used to discover new potential diagnostic biomarkers and therapeutic agents and to elucidate key autocrine pathways. Malignant mesothelioma (MMe), an extremely aggressive tumor, is characterized by a long latency period (20-30years), a poor prognosis, and limited effective therapies. MMe has a highly secretory cell type, and the factors released by cells may act in an autocrine or paracrine fashion on tumor and stroma, where they may modulate the extracellular environment. The aim of this work is to characterize the secretome of two MMe cell lines, MM98 and REN, in comparison with a mesothelial cell line Met5A, in order to evaluate differences and similarities of these two different MMe cancer model systems, and to identify potential biomarkers. We performed quantitative shotgun proteomics using SWATH-MS technology and we identified a total of 421 proteins, 112 expressed in the secretome of REN cells, 208 expressed in the secretome of MM98 cells and 189 secreted by mesothelial cells; 25 proteins are shared by the two mesothelioma cell lines. This study characterizes the secretome signature of the REN and MM98 cell lines, confirming the availability of a cell-culture based model in order to describe the cell-specific properties, and to provide a list of putative cancer biomarkers. This work constitutes the first qualitative and quantitative proteomic approach performed on MMe secretome. Moreover, since the data were acquired in SWATH-MS acquisition mode, they can be successively re-mined without performing a new analysis of the sample, which is extremely useful for retrospective analyses. The overall aim was to identify novel tumor-derived protein biomarkers with the potential to be applied for early diagnosis, prognosis, therapy prediction and/or disease monitoring of MMe. Copyright © 2016

  1. Isolation of Oct4-expressing extraembryonic endoderm precursor cell lines.

    Directory of Open Access Journals (Sweden)

    Bisrat G Debeb

    Full Text Available BACKGROUND: The extraembryonic endoderm (ExEn defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines ("XEN-P cell lines", which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo. CONCLUSIONS/SIGNIFICANCE: Our findings (i suggest strongly that the ExEn precursor is a self-renewable entity, (ii indicate that active Oct4 gene expression (transcription plus translation is part of its molecular identity, and (iii provide an in vitro model of early ExEn differentiation.

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

    Directory of Open Access Journals (Sweden)

    Gary Crameri

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

  3. Non-targeted radiation effects in vertebrate cell lines

    Science.gov (United States)

    Ryan, Lorna

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

  4. Characterization of a human ovarian carcinoma cell line: UCI 101.

    Science.gov (United States)

    Fuchtner, C; Emma, D A; Manetta, A; Gamboa, G; Bernstein, R; Liao, S Y

    1993-02-01

    A new epithelial ovarian carcinoma cell line (UCI 101) has been established from the ascitic fluids and solid tumor of a patient with progressive papillary adenocarcinoma of the ovary shown previously to be refractory to combination chemotherapy consisting of cyclophosphamide, Adriamycin, and cisplatin as well as single-agent chemotherapy of taxol and high-dose cisplatin. The UCI 101 cell line grows well with an in vitro doubling time of 24 hr. The cell line expresses the B 72.3 (Tag 72), CA125, MH99 (ESA), and E29 (EMA) cell surface antigens and AE1/AE3 cytokeratins. This cell line overexpresses (as determined by immunocytochemistry) both p-glycoprotein and the epidermal growth factor receptor. The in vitro drug response to single agents including Adriamycin, cisplatin, dequalinium chloride, etoposide, 5-fluorouracil, taxol, and tumor necrosis factor was examined. Intraperitoneal transplantation of the cells into athymic mice resulted in foci of tumor on all peritoneal surfaces including the viscera and diaphragm ultimately leading to solid bulky disease with massive production of ascites. High levels of CA125 (> 500 units/ml) were detected in the serum of tumor-bearing mice. Cytogenetic analysis of cultured cells shows several marker chromosomes containing deletions, duplications, and translocations. Cytologic and histologic evaluation of the xenograft revealed morphological characteristics identical to those of the original tumor.

  5. Sensory hair cell regeneration in the zebrafish lateral line.

    Science.gov (United States)

    Lush, Mark E; Piotrowski, Tatjana

    2014-10-01

    Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling, and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. Copyright © 2014 Wiley Periodicals, Inc.

  6. Phosphonium Salt Displays Cytotoxic Effects Against Human Cancer Cell Lines.

    Science.gov (United States)

    Dhanya, Dhanyalayam; Palma, Giuseppe; Cappello, AnnaRita; Mariconda, Annaluisa; Sinicropi, Maria Stefania; Giordano, Francesca; Vecchio, Vitale Del; Ramunno, Anna; Arra, Claudio; Longo, Pasquale; Saturnino, Carmela

    2017-07-19

    Aims/ Objective: Phosphonium salts are compounds whose structural characteristics enable them to cross the plasma and mitochondrial membrane with ease. Cancer cells have higher plasma membrane potentials than normal cells, phosphonium salts selectively accumulate in the mitochondria of neoplastic cells and inhibit mitochondrial function. In the presente work, we investigate the cytotoxic activity of lipophilic phosphonium salt (11-methoxy11-oxo-undecyl) triphenylphosphonium bromide (MUTP) as well as of two new phosphine oxide salts, 3,3'-(methylphosphoryl) dibenzenaminium chloride (SBAMPO) and 3,3' (phenylphosphoryl) dibenzenaminium chloride (SBAPPO) on the proliferation of breast cancer cell line (MCF-7) and human uterin cervix adenocarcinoma cells (HeLa). We show that only MUTP exhibits antiproliferative effects on both cell lines, without affecting normal breast epithelial cell proliferation. More specifically, we demonstrate that MUTP treatment of breast cancer cells is associated with impaired cell-cycle progression and metabolically induces mitochondrial damage and triggers apoptotic cell death in MCF-7 and HeLa cells. Taken together, these findings suggest that MUTP may be capable of selectively targeting neoplastic cell growth and therefore has potential applications as anticancer agent. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

    DEFF Research Database (Denmark)

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

    1992-01-01

    Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data demonstr...

  8. Identification of cancer stem-like side population cells in ovarian cancer cell line OVCAR-3.

    Science.gov (United States)

    Gao, Quanli; Geng, Li; Kvalheim, Gunnar; Gaudernack, Gustav; Suo, Zhenhe

    2009-01-01

    Side population (SP) cells may enrich stem-like cells in many normal and malignant tissues. However, SP method application has drawn special attention to the field of stem cell research, and the existence of SP cells in cell culture is being debated, most probably because different cell lines require different technical modifications, especially when cell staining is considered. In this study, the authors aimed to disclose whether the hoechst33342 staining required extensive optimization for identifying SP cells in the human ovarian cancer cell line OVCAR-3. After systematic evaluations, it was found that only 2.5 microg/mL hoechst33342 staining of the cells for 60 min could get an ideal SP population, which accounted for 0.9% of the whole cell population. The sorted SP cells showed significantly higher colony formation efficiency than the non-side population (NSP) cells, and only the SP cells could form holoclones. Real-time PCR disclosed that SP cells expressed higher levels of "stemness" gene Oct3/4 than the NSP cells did, indicating that the SP cells might harbor cancer stem cells in this cell line. The results highlight the necessity of SP method optimization in cell studies, and the SP cells in this cell line merit further studies when cancer stem cell identification and isolation are considered.

  9. Derivation and Characterization of a Pig Embryonic-Stem-Cell-Derived Exocrine Pancreatic Cell Line.

    Science.gov (United States)

    Talbot, Neil C; Shannon, Amy E; Phillips, Caitlin E; Garrett, Wesley M

    2017-07-01

    The aim of this study was to identify an epithelial cell line isolated from the spontaneous differentiation of totipotent pig epiblast cells. PICM-31 and its colony-cloned derivative cell line, PICM-31A, were established from the culture and differentiation of an epiblast mass isolated from an 8-day-old pig blastocyst. The cell lines were analyzed by transmission electron microscopy, marker gene expression, and mass spectroscopy-based proteomics. The PICM-31 cell lines were continuously cultured and could be successively colony cloned. They spontaneously self-organized into acinarlike structures. Transmission electron microscopy indicated that the cell lines' cells were epithelial and filled with secretory granules. Candidate gene expression analysis of the cells showed an exocrine pancreatic profile that included digestive enzyme expression, for example, carboxypeptidase A1, and expression of the fetal marker, α-fetoprotein. Pancreatic progenitor marker expression included pancreatic and duodenal homeobox 1, NK6 homeobox 1, and pancreas-specific transcription factor 1a, but not neurogenin 3. Proteomic analysis of cellular proteins confirmed the cells' production of digestive enzymes and showed that the cells expressed cytokeratins 8 and 18. The PICM-31 cell lines provide in vitro models of fetal pig pancreatic exocrine cells. They are the first demonstration of continuous cultures, that is, cell lines, of nontransformed pig pancreas cells.

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

    Directory of Open Access Journals (Sweden)

    Emanuela Mari

    2016-11-01

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

  11. Dipeptidyl peptidase IV in two human glioma cell lines

    Directory of Open Access Journals (Sweden)

    A Sedo

    2009-12-01

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

  12. 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

  13. Differential ectonucleotidase expression in human bladder cancer cell lines.

    Science.gov (United States)

    Stella, Joséli; Bavaresco, Luci; Braganhol, Elizandra; Rockenbach, Liliana; Farias, Patrícia Fernandes; Wink, Márcia R; Azambuja, Alan A; Barrios, Carlos Henrique; Morrone, Fernanda Bueno; Oliveira Battastini, Ana Maria

    2010-01-01

    Bladder cancer is the most prevalent tumor in the genitourinary tract. Nucleotides are important molecules that regulate many pathophysiological functions in the extracellular space. Studies have revealed evidence of a relationship between purinergic signaling and urothelial malignancies. Nucleotide-mediated signaling is controlled by a highly efficient enzymatic cascade, which includes the members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDases), ectonucleotide pyrophosphatase/phosphodiesterase (E-NPPs), ecto-alkaline phosphatases, and ecto-5'-nucleotidase/CD73. In an attempt to identify possible differential expression of ectonucleotidases during bladder cancer progression, a comparative analysis between RT4 (grade 1) and T24 (grade 3) bladder cancer cell lines was performed. In RT4 cells, the hydrolysis of tri- and diphosphate nucleosides was higher than monophosphonucleosides. T24 cells, however, presented the opposite profile, a low level of hydrolysis of tri- and diphosphate nucleosides and a high level of hydrolysis of monophosphates. Phosphodiesterase activity was negligible in both cell lines at physiological pH, indicating that these enzymes are not active under our assay conditions, although they are expressed in both cell lines. The T24 cells expressed NTPDase5 mRNA, while the RT4 cells expressed NTPDase3 and NTPDase5 mRNA. Both cell lines expressed ecto-5'-nucleotidase/CD73 mRNA. The present work describes, for the first time, the differential pattern of ectonucleotidases in the more malignant bladder cancer cells compared with cells derived from an early stage of bladder cancer. Our results open new avenues for research into the physiological roles of this family of enzymes and their possible therapeutic potential in bladder cancer. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  14. Primed Pluripotent Cell Lines Derived from Various Embryonic Origins and Somatic Cells in Pig

    OpenAIRE

    Jin-Kyu Park; Hye-Sun Kim; Kyung-Jun Uh; Kwang-Hwan Choi; Hyeong-Min Kim; Taeheon Lee; Byung-Chul Yang; Hyun-Jong Kim; Hak-Hyun Ka; Heebal Kim; Chang-Kyu Lee

    2013-01-01

    Since pluripotent embryonic stem cell (ESC) lines were first derived from the mouse, tremendous efforts have been made to establish ESC lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to maintain an ESC-like state in pluripotent porcine cell lines due to the frequent occurrence of spontaneous differentiation into an epiblast stem cell (EpiSC)-like state during culture. We have been able to derive EpiSC...

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

    Science.gov (United States)

    Ghashm, Abdulmlik A; Othman, Nor H; Khattak, Mohammed N; Ismail, Noorliza M; Saini, Rajan

    2010-09-14

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

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

    Directory of Open Access Journals (Sweden)

    Ismail Noorliza M

    2010-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-12-01

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

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

    Science.gov (United States)

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

    2013-10-01

    In vitro culture of parasitic helminths provides an important tool to study cell regeneration and physiology, as well as for molecular biology and genetic engineering studies. In the present study, we established in vitro propagation of cells from Echinococcus granulosus germinal cyst layer. E. granulosus germinal cells grew beyond 100 passages and showed no signs of reduced proliferation capacity. Microscopic analysis revealed that cells grew both attached to the substrate and in suspension, forming three-dimensional structures like mammalian stem cell aggregates. Examination of the chromosome number of attached germinal cells showed a high degree of heteroploidy, suggesting the occurrence of transformation during culture. Monolayer cells survived cryopreservation and were able to proliferate after thawing. Based on the characteristics displayed by E. granulosus germinal cells, we establish a cell line from the E. granulosus germinal layer. Furthermore, we propose that this cell line could be useful for drug screening and for obtaining parasite material. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Investigation of factors responsible for cell line cytoplasmic expression differences

    Directory of Open Access Journals (Sweden)

    Finn Jonathan D

    2005-05-01

    Full Text Available Abstract Background Previous work has described a novel cytoplasmic expression system that results in a 20-fold increase in the levels of gene expression over a standard CMV-based nuclear expression system, as compared with a 2–3 fold increase seen with previous similar systems. While this increase was seen with BHK and Neuro-2a cells, further studies revealed that some cell lines, such as COS-7, demonstrated relatively poor levels of cytoplasmic expression. The objective of this study was to determine what factors were responsible for the different expression levels between BHK (a high expressing cell line and COS-7 (a low expressing cell line. Results The main findings of this work are that the individual elements of the cytoplasmic expression system (such as the T7 RNAP gene and Internal Ribosome Entry Sequence are functioning similarly in both cell types. Both cell types were found to have the same amount of cytosolic nuclease activity, and that the cells appeared to have differences in the intra-cellular processing of DNA -cationic lipid complexes. Conclusion After exploring many factors, it was found that differences in the intra-cellular processing of the DNA-cationic lipid complex was the most probable factor responsible for the difference in cytoplasmic gene expression.

  20. SubClones (Alan Parsons) visit CERN – and the subatomic world

    CERN Multimedia

    Jordan Juras

    2011-01-01

    While the LHC has been creating subatomic particles, Alan Parsons has re-entered the studio with his new project, SubClones. The three-piece electronic rock group has joined the legendary Alan Parsons Live Project on tour, and their common friend Patrick Geeraert gave them the chance to drop into CERN for a visit.   Alan Parsons during his visit to CERN. The Alan Parsons Live Project is back on tour, with dates scheduled for the end of the summer across Europe and the Americas. A stop at CERN recently complemented a day off between two cities and allowed the band to move from the frontier of the music industry to the frontier of science. “We saw everything there was to see at CERN,” explains Alan Parsons. “It was all fascinating stuff but unfortunately we couldn’t see what was underground, though, so I think we will have to come back.” Parsons’ new project, SubClones, features three members whose identities remain a secret. Althoug...

  1. Maslinic acid inhibits proliferation of renal cell carcinoma cell lines and suppresses angiogenesis of endothelial cells

    Directory of Open Access Journals (Sweden)

    Parth Thakor

    2017-03-01

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

  2. [Isolation and identification of side population cells in human lung adenocarcinoma cell line A549].

    Science.gov (United States)

    Xie, Tong; Li, Li; Li, Dan-rong; Mao, Nai-quan; Liu, De-seng; Zuo, Chuan-tian; Zhang, Wei; Huang, Ding-ming

    2011-02-01

    To isolate and characterize the side population cells (SP cells) in the lung adenocarcinomas cell line A549. The protein expression of ABCG2 in human lung adenocarcinoma cell line A549 was detected by immunohistochemistry. SP and NSP cells in the cell line A549 were isolated by FACS, and their differentiation was analysed. ABCG2 expression in the two cell subsets was detected by RT-PCR. The cell growth curves, cell division indexes, cell cycles, plate clone formation tests, migration and invasion assays, chemotherapeutic susceptibility tests, tests of the intracellular drug levels, and the tumor cell implantation experiments on nude mice were applied to study the biological properties of the two cell subsets. The expression of ABCG2 in the transplanted tumor in nude mice was detected by immunohistochemistry and RT-PCR. The positive rate of ABCG2 expression in the A549 cells by immunohistochemistry was 2.13%. SP and NSP cells were isolated by FACS. The SP cells could produce both SP and NSP cells, while NSP cells only produced NSP cells. SP cells expressed ABCG2, but NSP cells did not. The proliferation and migration abilities of the two cell subsets were similar, but the invasion and tumorigenic ability of SP cells was significantly higher than that of NSP cells. The susceptibilities to DDP and its intracellular levels of the two cell subsets were similar, but the susceptibilities to 5-FU, VP16, NVB and GEM and their intracellular levels of NSP cells were significantly higher than those of the SP cells. SP cells in the human lung adenocarcinomas cell line A549 is enriched with tumor stem cells. An effective way to get lung adenocarcinomas stem cells is to isolate SP cells by FACS.

  3. Characterization of stem-like cells in a new astroblastoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Coban, Esra Aydemir; Kasikci, Ezgi [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Karatas, Omer Faruk [Molecular Biology and Genetics Department, Erzurum Technical University, Erzurum (Turkey); Suakar, Oznur; Kuskucu, Aysegul [Department of Medical Genetics, Yeditepe University Medical School and Yeditepe University Hospital, Istanbul (Turkey); Altunbek, Mine [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Türe, Uğur [Department of Neurosurgery, Yeditepe University School of Medicine, Istanbul (Turkey); Sahin, Fikrettin [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Bayrak, Omer Faruk, E-mail: ofbayrak@yeditepe.edu.tr [Department of Medical Genetics, Yeditepe University Medical School and Yeditepe University Hospital, Istanbul (Turkey)

    2017-03-15

    Cell lines established from tumors are the most commonly used models in cancer research, and their use in recent years has enabled a greater understanding of the biology of cancer and the means to develop effective treatment strategies. Astroblastomas are uncommon neuroepithelial tumors of glial origin, predominantly affecting young people, mainly teenagers and children, predominantly females. To date, only a single study has reported that astroblastomas contain a large number of neural stem-like cells, which had only a partial proliferation capacity and differentiation. Our objective was to establish an astroblastoma cell line to investigate the presence of astroblastic cells and cancer stem-like cells. The migratory and invasion abilities of the cells were quantified with invasion and migration assays and compared to a glioblastoma cell line. The presence of stem cells was detected with surface-marker analysis by using flow cytometry, and measuring the differentiation ability with a differentiation assay and the self-renewal capacity with a sphere-forming assay. These characteristics may determine whether this novel cell line is a model for astroblastomas that may have stem-cell characteristics. With this novel cell line, scientists can investigate the molecular pathways underlying astroblastomas and develop new therapeutic strategies for patients with these tumors. - Highlights: • An establishment of a novel astroblastoma cell line was proposed. • The presence of astroblastic cells and cancer stem-like cells was investigated. • The molecular pathways underlying astroblastomas may be investigated. • New therapeutic strategies for patients with astroblastoma may be developed.

  4. 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.

  5. Interaction of Leishmania (L. chagasi with the Vero cell line

    Directory of Open Access Journals (Sweden)

    Pessotti J.H.

    2004-03-01

    Full Text Available The Vero cell line, a non-phagocytic cell, has supported the intracellular mechanism of Leishmania (L. chagasi. This strain (MHOM/BR/501/MS00 was isolated from a human case of visceral leishmaniasis in Mato Grosso do Sul, Brazil and cultivated in Schneider's Drosophila medium with 20 % of heat inactivated fetal calf serum. It was allowed to infect the Vero cells at a ratio of 10 to 20 promastigotes per cell. Within six hours of incubation, promastigote forms were found attached to Vero cells without any particular orientation. The number of amastigotes per cell increased during the incubation period. Results showed that promastigotes of L. (L.. chagasi could interact, transform to amastigote forms and multiply in non-phagocytic cells, demonstrating a new model to study the intracellular cycle of this protozoan.

  6. Interaction of Leishmania (L.) chagasi with the Vero cell line.

    Science.gov (United States)

    Pessotti, J H; Zaverucha Do Valle, T; Corte-Real, S; Gonçalves Da Costa, S C

    2004-03-01

    The Vero cell line, a non-phagocytic cell, has supported the intracellular mechanism of Leishmania (L.) chagasi. This strain (MHOM/BR/501/MS00) was isolated from a human case of visceral leishmaniasis in Mato Grosso do Sul, Brazil and cultivated in Schneider's Drosophila medium with 20% of heat inactivated fetal calf serum. It was allowed to infect the Vero cells at a ratio of 10 to 20 promastigotes per cell. Within six hours of incubation, promastigote forms were found attached to Vero cells without any particular orientation. The number of amastigotes per cell increased during the incubation period. Results showed that promastigotes of L. (L.) chagasi could interact, transform to amastigote forms and multiply in non-phagocytic cells, demonstrating a new model to study the intracellular cycle of this protozoan.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-06-01

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

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

    Science.gov (United States)

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

    2013-10-01

    Cellular immortality happens upon impairment of cell-cycle checkpoint pathways (p53/p16/pRb), reactivation or up-regulation of telomerase enzyme, or upregulation of some oncogenes or oncoproteins leading to a higher rate of cell division.There are also some other factors and mechanisms involved in immortalisation, which need to be discovered. Immortalisation of cells derived from different sources and establishment of immortal cell lines has proven useful in understanding the molecular pathways governing cell developmental cascades in eukaryotic, especially human, cells. After the breakthrough of achieving the immortal cells and understanding their critical importance in the field of molecular biology, intense efforts have been dedicated to establish cell lines useful for elucidating the functions of telomerase, developmental lineage of progenitors, self-renewal potency, cellular transformation, differentiation patterns and some bioprocesses, like odontogenesis. Meanwhile, discovering the exact mechanisms of immortality, a major challenge for science yet, is believed to open new gateways toward understanding and treatment of cancer in the long term. This review summarises the methods involved in establishing immortality, its advantages and the challenges still being faced in this field. © 2013 International Federation for Cell Biology.

  9. DNA methylation and sensitivity to antimetabolites in cancer cell lines.

    Science.gov (United States)

    Sasaki, Shin; Kobunai, Takashi; Kitayama, Joji; Nagawa, Hirokazu

    2008-02-01

    The prediction of the cellular direction of metabolic pathways toward either DNA synthesis or DNA methylation is crucial for determining the susceptibility of cancers to anti-metabolites such as fluorouracil (5-FU). We genotyped the methylenetetrahydrofolate reductase (MTHFR) gene in NCI-60 cancer cell lines, and identified the methylation status of 24 tumor suppressor genes using methylation-specific multiplex ligation-dependent probe amplification. The susceptibility of the cancer cell lines to seven antimetabolites was then determined. Cells homozygous for CC at MTHFR-A1298C were significantly more sensitive to cyclocytidine, cytarabine (AraC) and floxuridine than those with AA or AC (p=0.0215, p=0.0166, and p=0.0323, respectively), and carried more methylated tumor suppressor genes (p=0.0313). Among the 12 tumor suppressor genes which were methylated in >25% of cancer cell lines, the methylation status of TIMP3, APC and IGSF4 significantly correlated with sensitivity to pyrimidine synthesis inhibitors. In particular, cells with methylated TIMP3 had reduced mRNA levels and were significantly more sensitive to aphidicolin-glycinate, AraC and 5-FU than cells with unmethylated TIMP3. We speculate that MTHFR-A1298C homozygous CC might direct the methylation rather than the synthesis of DNA, and result in the methylation of several tumor suppressor genes such as TIMP3. These genes could be useful biological markers for predicting the efficacy of antimetabolites.

  10. Apoptosis induction of epifriedelinol on human cervical cancer cell line

    African Journals Online (AJOL)

    Background: Present investigation evaluates the antitumor activity of epifriedelinol for the management of cervical cancer by inducing process of apoptosis. Methods: Human Cervical Cancer Cell Line, C33A and HeLa were selected for study and treated with epifriedelinol at a concentration of (50-1000 μg/ml). Cytotoxicity of ...

  11. Characterization of newly established colorectal cancer cell lines ...

    Indian Academy of Sciences (India)

    We have established a series of 20 colorectal cancer cell lines and performed cytogenetic and RFLP analyses to show that the recurrent genetic abnormalities of chromosomes 1, 5, 17 and 18 associated with multistep tumorigenesis in colorectal cancer, and frequently detected as recurrent abnormalities in primary tumours, ...

  12. Characterization of newly established colorectal cancer cell lines ...

    Indian Academy of Sciences (India)

    Unknown

    2000-12-19

    Dec 19, 2000 ... We have established a series of 20 colorectal cancer cell lines and performed cytogenetic and RFLP analyses to show that the recurrent genetic abnormalities of chromosomes 1, 5, 17 and 18 associated with multistep tumori- genesis in colorectal cancer, and frequently detected as recurrent abnormalities ...

  13. 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.

  14. Antibacterial and anti-breast cancer cell line activities of ...

    African Journals Online (AJOL)

    Purpose: To evaluate the activity of extracts of Sanghuangporus sp.1 fungus against pathogenic .... macro-dilution method. The effect of these extracts on a breast cancer cell line was also examined. EXPERIMENTAL. Fungal isolation. The wild fruiting body of the ..... cicada larva infected with entomopathogenic fungi in.

  15. Antibacterial and anti-breast cancer cell line activities of ...

    African Journals Online (AJOL)

    Purpose: To evaluate the activity of extracts of Sanghuangporus sp.1 fungus against pathogenic bacteria and a breast cancer cell line. Methods: The wild fruiting body and mycelium of Sanghuangporus sp.1 were extracted with water and ethanol by ultrasonication extraction. The activity of the extracts against pathogenic ...

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

    Science.gov (United States)

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

  17. 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.

  18. Regulation of transferrin receptor 2 in human cancer cell lines.

    Science.gov (United States)

    Calzolari, Alessia; Finisguerra, Veronica; Oliviero, Isabella; Deaglio, Silvia; Mariani, Gualtiero; Malavasi, Fabio; Testa, Ugo

    2009-01-01

    In a recent study we have explored TfR2 expression in a panel of cancer cell lines and we observed that about 40% of these cell lines clearly express TfR2. Taking advantage of this observation and considering the frequent overexpression of c-Myc in cancer cells we have explored the existence of a possible relationship between c-Myc and TfR2 in these cell lines. Our results provided evidence that TfR2(+) cell lines express low c-Myc levels and low TfR1 levels, while TfR2(-) cell lines express high c-Myc and TfR1 levels. Using the erythroleukemic K562 TfR2(+) cells as a model, we observed that agents that enhance c-Myc expression, such as iron, determine a decrease of TfR2 expression, while molecules that induce a decreased c-Myc expression, such as the iron chelator desferoxamine or the kinase inhibitor ST 1571, induce an enhanced TfR2 expression. On the other hand, we have evaluated a possible effect of hypoxia and nitric oxide on TfR2 expression in erythroleukemia K526 and hepatoma HepG2 cells, providing evidence that: (i) agents inducing cellular hypoxia, such as CoCl(2), elicited a marked upmodulation of TfR1, but a downmodulation of TfR2 expression; (ii) NO(+) donors, such as sodium nitroprusside (SNP), induced a moderate decrease of TfR1, associated with a marked decline of TfR2 expression; (iii) NO donors, such as S-Nitroso-N-Acetylpenicillamine (SNAP), induced a clear increase of TfR1, associated with a moderate upmodulation of TfR2 expression. The ensemble of these observations suggests that in cancer cell lines TfR2 expression can be modulated through stimuli similar to those known to act on TfR1 and these findings may have important implications for our understanding of the role of TfR2 in the regulation of iron homeostasis.

  19. AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

    Science.gov (United States)

    Luo, Yuxia; Frederick, Amy; Martin, John M; Scaria, Abraham; Cheng, Seng H; Armentano, Donna; Wadsworth, Samuel C; Vincent, Karen A

    2017-06-01

    Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

  20. [Isolation and characterization of side population cells in human gastric cancer cell line BGC-823].

    Science.gov (United States)

    Wu, Ting; Li, Jin-yi; Lu, Shi-xin

    2012-04-01

    Isolate and characterize the side population (SP) cells with potency of stem cells from human gastric carcinoma cell line BGC-823. SP and non-SP cells were sorted from BGC-823 cells by fluorescence-activated cell sorting (FACS) using Hoechst33342 staining. The tumorigenic ability of the SP cells was assessed by in vivo transplantation into non-obese diabetic/severe combined immunodeficiency mice. SP cells were isolated from BGC-823 cells in a proportion of 0.9% to 2.1% with respect to the whole cell population. The colony formation assay showed that the colony formation rate of the SP cells was significantly higher than that of the non-SP cells (72.56% vs. 49.00%, P line BGC-823 cells. Further characterization of this SP cell population may provide new insights for diagnosis and treatment of gastric cancer.

  1. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Science.gov (United States)

    2011-03-24

    ...; Identification of Human Cell Lines Project AGENCY: National Institute of Standards and Technology (NIST...) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All... in Designation: ASN-0002 Authentication of Human Cell Lines: Standardization of STR Profiling by the...

  2. Characterization of acylfulvene histiospecific toxicity in human tumor cell lines.

    Science.gov (United States)

    Kelner, M J; McMorris, T C; Montoya, M A; Estes, L; Uglik, S F; Rutherford, M; Samson, K M; Bagnell, R D; Taetle, R

    1998-01-01

    Acylfulvene derivatives demonstrate marked efficacy in xenograft carcinoma models as compared with the parent illudin compounds. To elucidate the increased therapeutic efficacy of acylfulvene analogs, we compared them with the illudin compounds in terms of their in vitro cytotoxicity, cellular accumulation and DNA incorporation. The cytotoxicity of various acylfulvene analogs was tested in vitro against a variety of tumor cell lines. Radiolabelled acylfulvene analog was prepared and used for cellular accumulation and DNA incorporation studies. The prototype acylfulvene analog retained selective histiospecific toxicity towards myeloid leukemia and various carcinoma cell lines. In vitro killing of tumor cells by acylfulvene required up to a 30-fold increase in molecules per cell, as compared with illudin S, indicating that acylfulvene was less toxic on a cellular level. At equitoxic concentrations, acylfulvene incorporation into genomic tumor cell DNA was equivalent to illudin S suggesting that cellular metabolism has a role in acylfulvene cytotoxicity. Analysis of cellular accumulation of acylfulvene into tumor cells revealed a markedly higher Vmax for tumor cells, and a lower Vd for diffusion accumulation into other cells. The combination of higher Vmax and lower Vd may explain the increased in vivo efficacy of acylfulvene.

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

    Science.gov (United States)

    Wei, Zhentong; Lv, Shuang; Wang, Yishu; Sun, Meiyu; Chi, Guangfan; Guo, Jun; Song, Peiye; Fu, Xiaoyu; Zhang, Songling; Li, Yulin

    2016-07-01

    The aim of the present study was to establish an ovarian cancer (OC) cell line from ascites of an ovarian serous cystadenocarcinoma patient and investigate the biological characteristics of its side population (SP) cells. The OC cell line was established by isolating, purifying and subculturing primary cells from ascites of an ovarian serous cystadenocarcinoma patient (stage IIIc; grade 3). SP and non-SP (NSP) cells were isolated by fluorescence-activated cell sorting and cultured in serum-free medium and soft agar to compare the tumorsphere and colony formation capacities. Furthermore, SP and NSP cell tumorigenesis was examined by subcutaneous and intraperitoneal injection of the cells to non-obese diabetic/severe combined immune deficiency (NOD/SCID) mice. Drug resistance to cisplatin was examined by cell counting kit-8. The OC cell line was successfully established from ascites of an ovarian serous cystadenocarcinoma patient, which exhibited properties similar to primary tumors subsequent to >50 passages and >2 years of culture. The SP cell ratio was 0.38% in the OC cell line, and a similar SP cell ratio (0.39%) was observed when sorted SP cells were cultured for 3 weeks. Compared with NSP cells, SP cells exhibited increased abilities in differentiation and tumorsphere and colony formation, in addition to the formation of xenografted tumors and ascites and metastasis of the tumors in NOD/SCID mice, even at low cell numbers (3.0×10 3 cells). The xenografted tumors demonstrated histological features similar to primary tumors and expressed the ovarian serous cystadenocarcinoma marker CA125. In addition, SP cells demonstrated a significantly stronger drug resistance to cisplatin compared with NSP and unsorted cells, while treatment with verapamil, an inhibitor of ATP-binding cassette transporters, potently abrogated SP cell drug resistance. In conclusion, the present study verified SP cells from an established OC cell line and characterized the cells with self

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

    Science.gov (United States)

    Silva, Dulcelena Ferreira; Vidal, Flávia Castello Branco; Santos, Debora; Costa, Maria Célia Pires; Morgado-Díaz, José Andrés; do Desterro Soares Brandão Nascimento, Maria; de Moura, Roberto Soares

    2014-05-29

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

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

    Directory of Open Access Journals (Sweden)

    Hilmar Quentmeier

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

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

    Directory of Open Access Journals (Sweden)

    Chen Lei

    2011-06-01

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

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

    NARCIS (Netherlands)

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

    2014-01-01

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

  8. Differences in radiosensitivity between three HER2 overexpressing cell lines

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-06-15

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

  9. Establishment of cell lines from adult T-cell leukemia cells dependent on negatively charged polymers.

    Science.gov (United States)

    Kagami, Yoshitoyo; Uchiyama, Susumu; Kato, Harumi; Okada, Yasutaka; Seto, Masao; Kinoshita, Tomohiro

    2017-07-05

    Growing adult T-cell leukemia/lymphoma (ATLL) cells in vitro is difficult. Here, we examined the effects of static electricity in the culture medium on the proliferation of ATLL cells. Six out of 10 ATLL cells did not proliferate in vitro and thus had to be cultured in a medium containing negatively charged polymers. In the presence of poly-γ-glutamic acid (PGA) or chondroitin sulfate (CDR), cell lines (HKOX3-PGA, HKOX3-CDR) were established from the same single ATLL case using interleukin (IL)-2, IL-4, and feeder cells expressing OX40L (OX40L+HK). Dextran sulfate inhibited growth in both HKOX3 cell lines. Both PGA and OX40L+HK were indispensable for HKOX3-PGA growth, but HKOX3-CDR could proliferate in the presence of CDR or OX40L+HK alone. Thus, the specific action of each negatively charged polymer promoted the growth of specific ATLL cells in vitro.

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

    Science.gov (United States)

    Bradbury, Andrew M.

    2012-07-24

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

  11. Characterization of side population cells isolated from the colon cancer cell line SW480.

    Science.gov (United States)

    Xiong, Binghong; Ma, Li; Hu, Xiang; Zhang, Caiquan; Cheng, Yong

    2014-09-01

    Side population (SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer. Many types of cell lines and tissues have demonstrated the presence of SP cells, including colon cancer cell lines. This study aimed to identify cancer stem cells (CSCs) in the SP of the colon cancer cell line SW480. SP cells were isolated by fluorescence-activated cell sorting (FACS), followed by serum-free medium (SFM) culture. The self-renewal, differentiated progeny, clone formation, proliferation, invasion ability, cell cycle, chemosensitivity and tumorigenic properties in SP and non-SP (NSP) cells were investigated through in vitro culture and in vivo serial transplantation. The expression profiles of ATP-binding cassette (ABC) protein transporters and stem cell-related genes were examined by RT-PCR and western blot analysis. The human colon cancer cell lines SW480, Lovo and HCT116 contain 1.1 ± 0.10, 0.93 ± 0.11 and 1.33 ± 0.05% SP cells, respectively. Flow cytometry analysis revealed that SP cells could differentiate into SP and NSP cells. SP cells had a higher proliferation potency and CFE than NSP cells. Compared to NSP cells, SP cells were also more resistant to CDDP and 5-FU, and were more invasive and displayed increased tumorigenic ability. Moreover, SP cells showed higher mRNA and protein expression of ABCG2, MDR1, OCT-4, NANOG, SOX-2, CD44 and CD133. SP cells isolated from human colon cancer cell lines harbor CSC properties that may be related to the invasive potential and therapeutic resistance of colon cancer.

  12. A preliminary study of side population cells in human gastric cancer cell line HGC-27.

    Science.gov (United States)

    Gao, Ganglong; Sun, Zhenliang; Wenyong, Liu; Dongxia, Ye; Zhao, Runjia; Zhang, Xueli

    2015-03-16

    Cancer stem cell-like side population (SP) cells, which may be responsible for recurrence, tumor metastasis, and resistance to cancer therapy, have been identified and characterized in several types of cell lines from gastric cancer. However, there is no report on isolation of SP cells from human gastric cancer cell line HGC-27. This study aims to analyze the proportion of SP cells in HGC-27 cell line, differentiate SP from non-side population (NSP) cells, and determine whether the SP cells have certain biological properties of stem cells. (1) HGC-27 suspension was prepared and stained with Hoechst33342 and PI for flow cytometric isolation of SP (2). Differences in proliferation and stemness-related gene expression profiles (CD133, CD44, OCT-4, MDR1, EpCAM, and ABCG2) between SP and NSP cells were detected by gastric formation assay and quantitative real-time PCR (3). Oncogenicity of SP and NSP cells was determined in nude mice in vivo. (1) SP cells accounted for 0.1-1.0% of HGC-27 cells, and decreased to 0% after verapamil inhibition. Using flow cytometry, we sorted 7.5×10⁵ SP cells and most HGC-27 cells were NSP cells (2). Gastric formation assay and MTT demonstrated that there was a significant difference in proliferation between SP and NSP cells. Gene expression analysis showed that the expression of genes was significantly higher in SP cells (3). The oncogenicity experiment in nude mice revealed that 105 SP cells were able to form tumors, which demonstrated higher tumorigenicity than non-SP cells. These results collectively suggested that SP cells from HGC-27 cell line have some cancer stem cell properties and could be used for studying the pathogenesis of gastric cancer, which may contribute to discovery of novel therapeutic targets.

  13. Studies of cloned Friend erythroleukemia tumor cells. Modulation of the tumor-specific cytologic T lymphocyte response by infectious Friend virus production in vitro

    OpenAIRE

    1980-01-01

    The HFL/b tumor cell line, induced by Friend erythroleukemia virus in BALB.B mice, was used to study the relation between virus production or nonproduction and the antigens recognized by Friend virus-specific cytolytic T lymphocytes (CTL). Analysis of clones and subclones of these tumor cells revealed a high degree of heterogeneity with respect to the production and release into culture fluids of infectious Friend virus in vitro, ranging from high levels to low or undetectable levels of virus...

  14. Epitope tagging of endogenous genes in diverse human cell lines.

    Science.gov (United States)

    Kim, Jung-Sik; Bonifant, Challice; Bunz, Fred; Lane, William S; Waldman, Todd

    2008-11-01

    Epitope tagging is a powerful and commonly used approach for studying the physical properties of proteins and their functions and localization in eukaryotic cells. In the case of Saccharomyces cerevisiae, it has been possible to exploit the high efficiency of homologous recombination to tag proteins by modifying their endogenous genes, making it possible to tag virtually every endogenous gene and perform genome-wide proteomics experiments. However, due to the relative inefficiency of homologous recombination in cultured human cells, epitope-tagging approaches have been limited to ectopically expressed transgenes, with the attendant limitations of their nonphysiological transcriptional regulation and levels of expression. To overcome this limitation, a modification and extension of adeno-associated virus-mediated human somatic cell gene targeting technology is described that makes it possible to simply and easily create an endogenous epitope tag in the same way that it is possible to knock out a gene. Using this approach, we have created and validated human cell lines with epitope-tagged alleles of two cancer-related genes in a variety of untransformed and transformed human cell lines. This straightforward approach makes it possible to study the physical and biological properties of endogenous proteins in human cells without the need for specialized antibodies for individual proteins of interest.

  15. Caffeine markedly sensitizes human mesothelioma cell lines to pemetrexed

    Science.gov (United States)

    Min, Sang Hee; Goldman, I. David; Zhao, Rongbao

    2013-01-01

    Pemetrexed is a new generation antifolate approved for the treatment of mesothelioma and non-small cell lung cancer. Caffeine is known to augment radiation or chemotherapeutic drug-induced cell killing. The current study addresses the impact of caffeine on the activity of pemetrexed in mesothelioma cell lines. Caffeine enhanced pemetrexed activity in all four mesothelioma cell lines tested (H2052, H2373, H28 and MSTO-211H). Caffeine sensitized H2052 cells in a dose- and schedule-dependent manner, and was associated with a markedly decreased clonogenic survival. Caffeine sensitization occurred only in cells subjected to pulse, but not continuous, exposure to pemetrexed. Similar pemetrexed sensitization was also observed with the clinically better tolerated caffeine analog, theobromine. Pemetrexed sensitization by caffeine was associated with an increase in pemetrexed-induced phosphorylation of ataxia-telangiectasia-mutated (ATM) and Chk1. These data indicate that caffeine and its analog, theobromine, may be a useful approach to enhance pemetrexed-based chemotherapy. PMID:17594092

  16. Automated solid-phase subcloning based on beads brought into proximity by magnetic force.

    Science.gov (United States)

    Hudson, Elton P; Nikoshkov, Andrej; Uhlen, Mathias; Rockberg, Johan

    2012-01-01

    In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications.

  17. [Isolation and in vitro characterization of CD133(+) side population cells from laryngeal cancer cell line].

    Science.gov (United States)

    Wu, Chun-ping; Zhou, Liang; Xie, Ming; Tao, Lei; Zhang, Ming; Tian, Jie

    2011-09-01

    To investigate an approach enriching cancer stem cells (CSCs) more effectively from laryngeal cancer cell line. CD133(+)SP and CD133(-)SP subpopulation was detected and isolated from Hep-2 cell line using Hoechst33342 dye and phycoerythrin (PE)-conjugated CD133 monoclonal antibody assisted by fluorescence activated cell sorting technology. Sorted CD133(+)SP and CD133(-)SP cells were compared in CSCs-related assays including proliferation, differentiation, spheroid formation and drug sensitivity. CD133(+)SP cells accounted for a very small fraction of (0.30 ± 0.12)% in Hep-2 cell line, far less than the proportion of CD133(+) subgroup and side population subgroup, which were (3.15 ± 0.83)% and (17.1 ± 2.0)% respectively. Intriguingly, CD133(+)SP cells proliferated much faster than CD133(-)SP cells in RPMI1640 and gave rise to CD133(-)SP cells and other heterogeneous cells that formed the bulk of the tumor. In contrast, CD133(-)SP cells were not able to differentiate into CD133(+)SP cells. In serum-free medium CD133(+)SP cells grew as spherical clusters and remained floating. In addition, CD133(+)SP cells manifested the marked resistance to chemotherapy than CD133(-)SP cells. Compared with CD133(-)SP cells, CD133(+)SP subpopulation exhibited extraordinary cancer stem-like properties, were enriched for cancer stem cells more effectively and might serve as an ideal putative candidate for CSCs research in laryngeal cancer.

  18. Imaging collective cell migration and hair cell regeneration in the sensory lateral line.

    Science.gov (United States)

    Venero Galanternik, M; Navajas Acedo, J; Romero-Carvajal, A; Piotrowski, T

    2016-01-01

    The accessibility of the lateral line system and its amenability to long-term in vivo imaging transformed the developing lateral line into a powerful model system to study fundamental morphogenetic events, such as guided migration, proliferation, cell shape changes, organ formation, organ deposition, cell specification and differentiation. In addition, the lateral line is not only amenable to live imaging during migration stages but also during postembryonic events such as sensory organ tissue homeostasis and regeneration. The robust regenerative capabilities of the mature, mechanosensory lateral line hair cells, which are homologous to inner ear hair cells and the ease with which they can be imaged, have brought zebrafish into the spotlight as a model to develop tools to treat human deafness. In this chapter, we describe protocols for long-term in vivo confocal imaging of the developing and regenerating lateral line. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    Zhang, Hai-hong; Cai, Ai-zhen; Wei, Xue-ming; Ding, Li; Li, Feng-zhi; Zheng, Ai-ming; Dai, Da-jiang; Huang, Rong-rong; Cao, Hou-jun; Zhou, Hai-yang; Wang, Jian-mei; Wang, Xue-jing; Shi, Wei; Zhu, Heng; Yuan, Xiao-ying; Chen, Lin

    2013-03-01

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

  20. Side population cells from HXO-Rb44 retinoblastoma cell line have cancer-initiating property.

    Science.gov (United States)

    She, Jun-Jun; Zhang, Peng-Ge; Che, Xiang-Ming; Wang, Xuan; Wang, Zi-Ming

    2011-01-01

    To ascertain whether side population (SP) cells in HXO-Rb44 retinoblastoma cell line have cancer stem cell-like property in vitro and in vivo. We analyzed and sorted SP from HXO-Rb44 retinoblastoma cell line by Hoechst 33342 staining on flow cytometry. SP and NSP cells were determined their ability of proliferation and self-renewal by SP reanalysis, soft agar assay and tumor sphere assay in vitro. Clone formation was detected by seeding HXO-Rb44 and HXO-Rb44 -RFP cells into soft agar. The expression of ABCG2, MDRI, Bmi-1 and Oct-4 was determined by RT-PCR between SP and non-SP (NSP) cells. Moreover, they were injected into nude mice to determine their tumorigency in vivo. SP from HXO-Rb44 retinoblastoma cell line could grow clonally in soft agar assays and form tumor spheres from single cells in conditioned media. The expressions of ABCG2, MDRI, Bmi-1 and Oct-4 were significantly higher in SP than NSP cells. As few as SP cells resulted in tumor formation in 6 of 12 injected sites, however, the injection of NSP cells failed to form new tumor. SP cells isolated by Hoechst 33342 from the HXO-Rb44 retinoblastoma cell line had property of high tumorigency in vivo and in vitro. Therefore, SP might be a target while developing retinoblastoma therapies.

  1. Designing of promiscuous inhibitors against pancreatic cancer cell lines

    Science.gov (United States)

    Kumar, Rahul; Chaudhary, Kumardeep; Singla, Deepak; Gautam, Ankur; Raghava, Gajendra P. S.

    2014-04-01

    Pancreatic cancer remains the most devastating disease with worst prognosis. There is a pressing need to accelerate the drug discovery process to identify new effective drug candidates against pancreatic cancer. We have developed QSAR models for predicting promiscuous inhibitors using the pharmacological data. Our models achieved maximum Pearson correlation coefficient of 0.86, when evaluated on 10-fold cross-validation. Our models have also successfully validated the drug-to-oncogene relationship and further we used these models to screen FDA approved drugs and tested them in vitro. We have integrated these models in a webserver named as DiPCell, which will be useful for screening and designing novel promiscuous drug molecules. We have also identified the most and least effective drugs for pancreatic cancer cell lines. On the other side, we have identified resistant pancreatic cancer cell lines, which need investigative scanner on them to put light on resistant mechanism in pancreatic cancer.

  2. Characterization of stem-like cells in a new astroblastoma cell line.

    Science.gov (United States)

    Coban, Esra Aydemir; Kasikci, Ezgi; Karatas, Omer Faruk; Suakar, Oznur; Kuskucu, Aysegul; Altunbek, Mine; Türe, Uğur; Sahin, Fikrettin; Bayrak, Omer Faruk

    2017-03-15

    Cell lines established from tumors are the most commonly used models in cancer research, and their use in recent years has enabled a greater understanding of the biology of cancer and the means to develop effective treatment strategies. Astroblastomas are uncommon neuroepithelial tumors of glial origin, predominantly affecting young people, mainly teenagers and children, predominantly females. To date, only a single study has reported that astroblastomas contain a large number of neural stem-like cells, which had only a partial proliferation capacity and differentiation. Our objective was to establish an astroblastoma cell line to investigate the presence of astroblastic cells and cancer stem-like cells. The migratory and invasion abilities of the cells were quantified with invasion and migration assays and compared to a glioblastoma cell line. The presence of stem cells was detected with surface-marker analysis by using flow cytometry, and measuring the differentiation ability with a differentiation assay and the self-renewal capacity with a sphere-forming assay. These characteristics may determine whether this novel cell line is a model for astroblastomas that may have stem-cell characteristics. With this novel cell line, scientists can investigate the molecular pathways underlying astroblastomas and develop new therapeutic strategies for patients with these tumors. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Monitoring cell line identity in collections of human induced pluripotent stem cells.

    Science.gov (United States)

    Sarafian, Raquel; Morato-Marques, Mariana; Borsoi, Juliana; Pereira, Lygia Veiga

    2018-01-31

    The ability to reprogram somatic cells into induced pluripotent stem cells (hiPSCs) has led to the generation of large collections of cell lines from thousands of individuals with specific phenotypes, many of which will be shared among different research groups as invaluable tools for biomedical research. As hiPSC-based research involves extensive culture of many cell lines, the issue periodic cell line identification is particularly important to ensure that cell line identity remains accurate. Here we analyzed the different commercially available genotyping methods considering ease of in-house genotyping, cost and informativeness, and applied one of them in our workflow for hiPSC generation. We show that the chosen STR method was able to establish a unique DNA profile for each of the 35 individuals/hiPSC lines at the examined sites, as well as identify two discrepancies resulting from inadvertently exchanged samples. Our results highlight the importance of hiPSC line genotyping by an in-house method that allows periodic cell line identification and demonstrate that STR is a useful approach to supplement less frequent karyotyping and epigenetic evaluations. Copyright © 2018. Published by Elsevier B.V.

  4. Proteomic analysis of cell lines to identify the irinotecan resistance ...

    Indian Academy of Sciences (India)

    MADHU

    [Peng X-C, Gong F-M, Wei M, Chen X, Chen Y, Cheng K, Gao F, Xu F, Bi F and Liu J-Y 2010 Proteomic analysis of cell lines to identify the irinotecan resistance proteins; J. Biosci. 35 557–564] DOI 10.1007/s12038-010-0064-9. Keywords. Colon cancer; 2-DE; drug resistance; irinotecan; proteomics. Abbreviations used: ACN ...

  5. Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines.

    Science.gov (United States)

    Tomiya, Noboru; Narang, Someet; Lee, Yuan C; Betenbaugh, Michael J

    2004-01-01

    In the past decades, a large number of studies in mammalian cells have revealed that processing of glycoproteins is compartmentalized into several subcellular organelles that process N-glycans to generate complex-type oligosaccharides with terminal N -acetlyneuraminic acid. Recent studies also suggested that processing of N-glycans in insect cells appear to follow a similar initial pathway but diverge at subsequent processing steps. N-glycans from insect cell lines are not usually processed to terminally sialylated complex-type structures but are instead modified to paucimannosidic or oligomannose structures. These differences in processing between insect cells and mammalian cells are due to insufficient expression of multiple processing enzymes including glycosyltransferases responsible for generating complex-type structures and metabolic enzymes involved in generating appropriate sugar nucleotides. Recent genomics studies suggest that insects themselves may include many of these complex transferases and metabolic enzymes at certain developmental stages but expression is lost or limited in most lines derived for cell culture. In addition, insect cells include an N -acetylglucosaminidase that removes a terminal N -acetylglucosamine from the N-glycan. The innermost N -acetylglucosamine residue attached to asparagine residue is also modified with alpha(1,3)-linked fucose, a potential allergenic epitope, in some insect cells. In spite of these limitations in N-glycosylation, insect cells have been widely used to express various recombinant proteins with the baculovirus expression vector system, taking advantage of their safety, ease of use, and high productivity. Recently, genetic engineering techniques have been applied successfully to insect cells in order to enable them to produce glycoproteins which include complex-type N-glycans. Modifications to insect N-glycan processing include the expression of missing glycosyltransferases and inclusion of the metabolic

  6. 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.

  7. Off-line test of the KISS gas cell

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-12-15

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

  8. Biomarkers in Tumorigenesis Using Cancer Cell Lines: A Systematic Review

    Science.gov (United States)

    Raju K, Lizbeth; Augustine, Dominic; Rao, Roopa S; S V, Sowmya; Haragannavar, Vanishri C; Nambiar, Shwetha; Prasad, Kavitha; Awan, Kamran Habib; Patil, Shankargouda

    2017-09-27

    Cancer is a leading cause of death worldwide. Despite many research advancements in the field, the genetic changes regulating the transformation of normal oral cells into malignant cells have not been fully elucidated. Several studies have evaluated carcinogenesis at the molecular level. Cancer cell lines are commonly used in biomedical research because they provide an unlimited source of cells and represent various stages of initiation and progression of carcinogenesis in vitro. Aims: The objective of the study was to review original research articles using cancer cell lines as a tool to understand carcinogenesis and to identify the genes involved in tumor development. Additionally, we also examined the application of the genes as predictive biomarkers. Methods and Materials: Several databases, including PubMed, Google Scholar, Ebsco, and Science Direct, were searched from 1985 to December 2016 using various combinations of the following key words: “mouth neoplasm”, “cell lines”, and “tumorigenesis”. Original experimental studies published in English were included. We excluded letters to the editor, historic reviews, and unpublished data from the analysis. Results: There were 17 studies (in vitro) included in the analysis. There were 14 genes and 4 miRNAs involved in malignant transformation of oral keratinocytes into cancer cells. The most commonly studied genes were p53, cyclin D1, and hTERT. Conclusion: Additional reviews and studies are needed to identify a panel of genes specific to various potentially malignant disorders and to aid in the early detection of oral squamous cell carcinoma (OSCC) because tumorigenesis involves the mutation of multiple genes. Furthermore, improving advanced cost-effective diagnostic methods may benefit the public health sector. Creative Commons Attribution License

  9. Cell-line dependent effects of hypoxia prior to irradiation in squamous cell carcinoma lines

    Directory of Open Access Journals (Sweden)

    Franziska Hauth

    2017-08-01

    Conclusion: We herein report a key role of ATM in the cellular fitness of cells exposed to prolonged moderate hypoxia prior to irradiation. While DNA damage response post-irradiation seem to be mainly driven by non-homologous end joining repair pathway in these conditions, our data suggest an important role for ATM kinase in hypoxia-driven modification of radiation response.

  10. 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.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  12. Innervation regulates synaptic ribbons in lateral line mechanosensory hair cells.

    Science.gov (United States)

    Suli, Arminda; Pujol, Remy; Cunningham, Dale E; Hailey, Dale W; Prendergast, Andrew; Rubel, Edwin W; Raible, David W

    2016-06-01

    Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse. © 2016. Published by The Company of Biologists Ltd.

  13. Ecdysone and The Cell Cycle: Investigations in a Mosquito Cell Line

    Science.gov (United States)

    Fallon, Ann M.; Gerenday, Anna

    2010-01-01

    Cell lines provide a tool for investigating basic biological processes that underlie the complex interactions among the tissues and organs of an intact organism. We compare the evolution of insect and mammalian populations as they progress from diploid cell strains to continuous cell lines, and review the history of the well-characterized Aedes albopictus mosquito cell line, C7-10. Like Kc and S3 cells from Drosophila melanogaster, C7-10 cells are sensitive to the insect steroid hormone, 20-hydroxyecdysone (20E), and express 20E-inducible proteins as well as the EcR and USP components of the ecdysteroid receptor. The decrease in growth associated with 20E treatment results in an accumulation of cells in the G1 phase of the cycle, and a concomitant decrease in levels of cyclin A. In contrast, 20E induces a G2 arrest in a well-studied imaginal disc cell line from the moth, Plodia interpunctella. We hypothesize that 20E-mediated events associated with molting and metamorphosis include effects on regulatory proteins that modulate the mitotic cell cycle and that differences between the 20E response in diverse insect cell lines reflect an interplay between classical receptor-mediated effects on gene expression and non-classical effects on signaling pathways similar to those recently described for the vertebrate steroid hormone, estrogen. PMID:20303973

  14. Cell Surface and Secreted Protein Profiles of Human Thyroid Cancer Cell Lines Reveal Distinct Glycoprotein Patterns

    Science.gov (United States)

    Arcinas, Arthur; Yen, Ten-Yang; Kebebew, Electron; Macher, Bruce A.

    2009-01-01

    Cell surface proteins have been shown to be effective therapeutic targets. In addition, shed forms of these proteins and secreted proteins can serve as biomarkers for diseases, including cancer. Thus, identification of cell surface and secreted proteins has been a prime area of interest in the proteomics field. Most cell surface and secreted proteins are known to be glycosylated and therefore, a proteomics strategy targeting these proteins was applied to obtain proteomic profiles from various thyroid cancer cell lines that represent the range of thyroid cancers of follicular cell origin. In this study, we oxidized the carbohydrates of secreted proteins and those on the cell surface with periodate and isolated them via covalent coupling to hydrazide resin. The glycoproteins obtained were identified from tryptic peptides and N-linked glycopeptides released from the hydrazide resin using 2-dimensional liquid chromatography-tandem mass spectrometry in combination with the gas phase fractionation. Thyroid cancer cell lines derived from papillary thyroid cancer (TPC-1), follicular thyroid cancer (FTC-133), Hürthle cell carcinoma (XTC-1), and anaplastic thyroid cancer (ARO and DRO-1) were evaluated. An average of 150 glycoproteins were identified per cell line, of which more than 57 percent are known cell surface or secreted glycoproteins. The usefulness of the approach for identifying thyroid cancer associated biomarkers was validated by the identification of glycoproteins (e.g. CD44, galectin 3 and metalloproteinase inhibitor 1) that have been found to be useful markers for thyroid cancer. In addition to glycoproteins that are commonly expressed by all of the cell lines, we identified others that are only expressed in the more well-differentiated thyroid cancer cell lines (follicular, Hürthle cell and papillary), or by cell lines derived from undifferentiated tumors that are uniformly fatal forms of thyroid cancer (i.e. anaplastic). Based on the results obtained, a

  15. Primed pluripotent cell lines derived from various embryonic origins and somatic cells in pig.

    Science.gov (United States)

    Park, Jin-Kyu; Kim, Hye-Sun; Uh, Kyung-Jun; Choi, Kwang-Hwan; Kim, Hyeong-Min; Lee, Taeheon; Yang, Byung-Chul; Kim, Hyun-Jong; Ka, Hak-Hyun; Kim, Heebal; Lee, Chang-Kyu

    2013-01-01

    Since pluripotent embryonic stem cell (ESC) lines were first derived from the mouse, tremendous efforts have been made to establish ESC lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to maintain an ESC-like state in pluripotent porcine cell lines due to the frequent occurrence of spontaneous differentiation into an epiblast stem cell (EpiSC)-like state during culture. We have been able to derive EpiSC-like porcine ESC (pESC) lines from blastocyst stage porcine embryos of various origins, including in vitro fertilized (IVF), in vivo derived, IVF aggregated, and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells (piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) into porcine fibroblast cells. In this study, we analyzed characteristics such as marker expression, pluripotency and the X chromosome inactivation status in female of our EpiSC-like pESC lines along with our piPSC line. Our results show that these cell lines demonstrate the expression of genes associated with the Activin/Nodal and FGF2 pathways along with the expression of pluripotent markers Oct4, Sox2, Nanog, SSEA4, TRA 1-60 and TRA 1-81. Furthermore all of these cell lines showed in vitro differentiation potential, the X chromosome inactivation in female and a normal karyotype. Here we suggest that the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines.

  16. Primed pluripotent cell lines derived from various embryonic origins and somatic cells in pig.

    Directory of Open Access Journals (Sweden)

    Jin-Kyu Park

    Full Text Available Since pluripotent embryonic stem cell (ESC lines were first derived from the mouse, tremendous efforts have been made to establish ESC lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to maintain an ESC-like state in pluripotent porcine cell lines due to the frequent occurrence of spontaneous differentiation into an epiblast stem cell (EpiSC-like state during culture. We have been able to derive EpiSC-like porcine ESC (pESC lines from blastocyst stage porcine embryos of various origins, including in vitro fertilized (IVF, in vivo derived, IVF aggregated, and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells (piPSCs via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4, and c-Myc into porcine fibroblast cells. In this study, we analyzed characteristics such as marker expression, pluripotency and the X chromosome inactivation status in female of our EpiSC-like pESC lines along with our piPSC line. Our results show that these cell lines demonstrate the expression of genes associated with the Activin/Nodal and FGF2 pathways along with the expression of pluripotent markers Oct4, Sox2, Nanog, SSEA4, TRA 1-60 and TRA 1-81. Furthermore all of these cell lines showed in vitro differentiation potential, the X chromosome inactivation in female and a normal karyotype. Here we suggest that the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines.

  17. Role of complex cadherins in cell-cell adhesion evaluated by spheroid formation in renal cell carcinoma cell lines.

    NARCIS (Netherlands)

    Shimazui, T.; Schalken, J.A.; Kawai, K.; Kawamoto, R.; Bockhoven, A. van; Oosterwijk, E.; Akaza, H.

    2004-01-01

    We have previously shown that renal cell carcinoma (RCC) cell lines expressed a complex set of cadherins, e.g. E-cadherin, N-cadherin and cadherin-6. It is also reported that E-cadherin and cadherin-6 have a predictive value for estimating a patient's prognosis in RCC. However, E-cadherin is

  18. The Subclonal Architecture of Metastatic Breast Cancer: Results from a Prospective Community-Based Rapid Autopsy Program “CASCADE”

    Science.gov (United States)

    Flensburg, Christoffer; Alsop, Kathryn; Mansour, Mariam; Francis, Prudence A.; Thorne, Heather A.; Silva, Maria Joao; Kanu, Nnennaya; Dietzen, Michelle; Bowtell, David D.; Speed, Terence P.; Swanton, Charles; Loi, Sherene

    2016-01-01

    Background Understanding the cancer genome is seen as a key step in improving outcomes for cancer patients. Genomic assays are emerging as a possible avenue to personalised medicine in breast cancer. However, evolution of the cancer genome during the natural history of breast cancer is largely unknown, as is the profile of disease at death. We sought to study in detail these aspects of advanced breast cancers that have resulted in lethal disease. Methods and Findings Three patients with oestrogen-receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer and one patient with triple negative breast cancer underwent rapid autopsy as part of an institutional prospective community-based rapid autopsy program (CASCADE). Cases represented a range of management problems in breast cancer, including late relapse after early stage disease, de novo metastatic disease, discordant disease response, and disease refractory to treatment. Between 5 and 12 metastatic sites were collected at autopsy together with available primary tumours and longitudinal metastatic biopsies taken during life. Samples underwent paired tumour-normal whole exome sequencing and single nucleotide polymorphism (SNP) arrays. Subclonal architectures were inferred by jointly analysing all samples from each patient. Mutations were validated using high depth amplicon sequencing. Between cases, there were significant differences in mutational burden, driver mutations, mutational processes, and copy number variation. Within each case, we found dramatic heterogeneity in subclonal structure from primary to metastatic disease and between metastatic sites, such that no single lesion captured the breadth of disease. Metastatic cross-seeding was found in each case, and treatment drove subclonal diversification. Subclones displayed parallel evolution of treatment resistance in some cases and apparent augmentation of key oncogenic drivers as an alternative resistance mechanism. We

  19. The Subclonal Architecture of Metastatic Breast Cancer: Results from a Prospective Community-Based Rapid Autopsy Program "CASCADE".

    Science.gov (United States)

    Savas, Peter; Teo, Zhi Ling; Lefevre, Christophe; Flensburg, Christoffer; Caramia, Franco; Alsop, Kathryn; Mansour, Mariam; Francis, Prudence A; Thorne, Heather A; Silva, Maria Joao; Kanu, Nnennaya; Dietzen, Michelle; Rowan, Andrew; Kschischo, Maik; Fox, Stephen; Bowtell, David D; Dawson, Sarah-Jane; Speed, Terence P; Swanton, Charles; Loi, Sherene

    2016-12-01

    Understanding the cancer genome is seen as a key step in improving outcomes for cancer patients. Genomic assays are emerging as a possible avenue to personalised medicine in breast cancer. However, evolution of the cancer genome during the natural history of breast cancer is largely unknown, as is the profile of disease at death. We sought to study in detail these aspects of advanced breast cancers that have resulted in lethal disease. Three patients with oestrogen-receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer and one patient with triple negative breast cancer underwent rapid autopsy as part of an institutional prospective community-based rapid autopsy program (CASCADE). Cases represented a range of management problems in breast cancer, including late relapse after early stage disease, de novo metastatic disease, discordant disease response, and disease refractory to treatment. Between 5 and 12 metastatic sites were collected at autopsy together with available primary tumours and longitudinal metastatic biopsies taken during life. Samples underwent paired tumour-normal whole exome sequencing and single nucleotide polymorphism (SNP) arrays. Subclonal architectures were inferred by jointly analysing all samples from each patient. Mutations were validated using high depth amplicon sequencing. Between cases, there were significant differences in mutational burden, driver mutations, mutational processes, and copy number variation. Within each case, we found dramatic heterogeneity in subclonal structure from primary to metastatic disease and between metastatic sites, such that no single lesion captured the breadth of disease. Metastatic cross-seeding was found in each case, and treatment drove subclonal diversification. Subclones displayed parallel evolution of treatment resistance in some cases and apparent augmentation of key oncogenic drivers as an alternative resistance mechanism. We also observed the role of

  20. 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.

  1. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-07-20

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

  3. Assessment of citalopram and escitalopram on neuroblastoma cell lines. Cell toxicity and gene modulation.

    Science.gov (United States)

    Sakka, Laurent; Delétage, Nathalie; Chalus, Maryse; Aissouni, Youssef; Sylvain-Vidal, Valérie; Gobron, Stéphane; Coll, Guillaume

    2017-06-27

    Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (pescitalopram.

  4. Assessment of citalopram and escitalopram on neuroblastoma cell lines: Cell toxicity and gene modulation

    Science.gov (United States)

    Sakka, Laurent; Delétage, Nathalie; Chalus, Maryse; Aissouni, Youssef; Sylvain-Vidal, Valérie; Gobron, Stéphane; Coll, Guillaume

    2017-01-01

    Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (pescitalopram. PMID:28467792

  5. Morphological study of the TK cholangiocarcinoma cell line with three-dimensional cell culture.

    Science.gov (United States)

    Akiyoshi, Kohei; Kamada, Minori; Akiyama, Nobutake; Suzuki, Masafumi; Watanabe, Michiko; Fujioka, Kouki; Ikeda, Keiichi; Mizuno, Shuichi; Manome, Yoshinobu

    2014-04-01

    Cholangiocarcinoma is an intractable carcinoma originating from the bile duct epithelium. To gain an understanding of the cell biology of cholangiocarcinoma, in vitro cell culture is valuable. However, well‑characterized cell lines are limited. In the present study, the morphology of the TK cholangiocarcinoma cell line was analyzed by three‑dimensional culture. Dispersed TK cells were injected into a gelatin mesh scaffold and cultivated for 3‑20 days. The morphology of the TK cells was investigated by phase‑contrast microscopy, optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TK cells were observed to proliferate three-dimensionally in the scaffold. The cells exhibited a globoid structure and attached to the scaffold. The SEM observation demonstrated typical microvilli and plicae on the surface of the structure. Light microscopy and TEM confirmed intercellular and cell‑to‑scaffold attachment in the three‑dimensional mesh. The culture also exhibited the formation of a duct-like structure covered by structured microvilli. In conclusion, three‑dimensional culture of TK cells demonstrated the morphological characteristics of cholangiocarcinoma in vitro. Production of high levels of carbohydrate antigen (CA)19‑9, CA50 and carcinoembryonic antigen was previously confirmed in the TK cell line. As a characteristic morphology was demonstrated in the present study, the TK cholangiocarcinoma cell line may be useful as an experimental model for further study of cholangiocarcinoma.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-10-19

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

  7. Aldehyde dehydrogenase 1 identifies cells with cancer stem cell-like properties in a human renal cell carcinoma cell line.

    Directory of Open Access Journals (Sweden)

    Kosuke Ueda

    Full Text Available Cancer stem cells (CSC or cancer stem cell-like cells (CSC-LCs have been identified in many malignant tumors. CSCs are proposed to be related with drug resistance, tumor recurrence, and metastasis and are considered as a new target for cancer treatment; however, there are only a few reports on CSCs or CSC-LCs in renal cell carcinoma (RCC. Different approaches have been reported for CSC identification, but there are no universal markers for CSC. We used two different approaches, the traditional side population (SP approach, and the enzymatic (aldehyde dehydrogenase 1 (ALDH1 approach to identify CSC-LC population in two RCC cell lines, ACHN and KRC/Y. We found that ACHN and KRC/Y contain 1.4% and 1.7% SP cells, respectively. ACHN SP cells showed a higher sphere forming ability, drug resistance, and a slightly higher tumorigenic ability in NOD/SCID mice than Non-SP (NSP cells, suggesting that cells with CSC-LC properties are included in ACHN SP cells. KRC/Y SP and NSP cells showed no difference in such properties. ALDH1 activity analysis revealed that ACHN SP cells expressed a higher level of activity than NSP cells (SP vs. NSP: 32.7% vs 14.6%. Analysis of ALDH1-positive ACHN cells revealed that they have a higher sphere forming ability, self-renewal ability, tumorigenicity and express higher mRNA levels of CSC-LC property-related genes (e.g., ABC transporter genes, self-replication genes, anti-apoptosis genes, and so forth than ALDH1-negative cells. Drug treatment or exposure to hypoxic condition induced a 2- to 3-fold increase in number of ALDH1-positive cells. In conclusion, the results suggest that the ALDH1-positive cell population rather than SP cells show CSC-LC properties in a RCC cell line, ACHN.

  8. Proteomic patterns of cervical cancer cell lines, a network perspective.

    Science.gov (United States)

    Higareda-Almaraz, Juan Carlos; Enríquez-Gasca, María del Rocío; Hernández-Ortiz, Magdalena; Resendis-Antonio, Osbaldo; Encarnación-Guevara, Sergio

    2011-06-22

    Cervical cancer is a major mortality factor in the female population. This neoplastic is an excellent model for studying the mechanisms involved in cancer maintenance, because the Human Papilloma Virus (HPV) is the etiology factor in most cases. With the purpose of characterizing the effects of malignant transformation in cellular activity, proteomic studies constitute a reliable way to monitor the biological alterations induced by this disease. In this contextual scheme, a systemic description that enables the identification of the common events between cell lines of different origins, is required to distinguish the essence of carcinogenesis. With this study, we sought to achieve a systemic perspective of the common proteomic profile of six cervical cancer cell lines, both positive and negative for HPV, and which differ from the profile corresponding to the non-tumourgenic cell line, HaCaT. Our objectives were to identify common cellular events participating in cancer maintenance, as well as the establishment of a pipeline to work with proteomic-derived results. We analyzed by means of 2D SDS-PAGE and MALDI-TOF mass spectrometry the protein extracts of six cervical cancer cell lines, from which we identified a consensus of 66 proteins. We call this group of proteins, the "central core of cervical cancer". Starting from this core set of proteins, we acquired a PPI network that pointed, through topological analysis, to some proteins that may well be playing a central role in the neoplastic process, such as 14-3-3ζ. In silico overrepresentation analysis of transcription factors pointed to the overexpression of c-Myc, Max and E2F1 as key transcription factors involved in orchestrating the neoplastic phenotype. Our findings show that there is a "central core of cervical cancer" protein expression pattern, and suggest that 14-3-3ζ is key to determine if the cell proliferates or dies. In addition, our bioinformatics analysis suggests that the neoplastic phenotype is

  9. Proteomic patterns of cervical cancer cell lines, a network perspective

    Directory of Open Access Journals (Sweden)

    Resendis-Antonio Osbaldo

    2011-06-01

    Full Text Available Abstract Background Cervical cancer is a major mortality factor in the female population. This neoplastic is an excellent model for studying the mechanisms involved in cancer maintenance, because the Human Papilloma Virus (HPV is the etiology factor in most cases. With the purpose of characterizing the effects of malignant transformation in cellular activity, proteomic studies constitute a reliable way to monitor the biological alterations induced by this disease. In this contextual scheme, a systemic description that enables the identification of the common events between cell lines of different origins, is required to distinguish the essence of carcinogenesis. Results With this study, we sought to achieve a systemic perspective of the common proteomic profile of six cervical cancer cell lines, both positive and negative for HPV, and which differ from the profile corresponding to the non-tumourgenic cell line, HaCaT. Our objectives were to identify common cellular events participating in cancer maintenance, as well as the establishment of a pipeline to work with proteomic-derived results. We analyzed by means of 2D SDS-PAGE and MALDI-TOF mass spectrometry the protein extracts of six cervical cancer cell lines, from which we identified a consensus of 66 proteins. We call this group of proteins, the "central core of cervical cancer". Starting from this core set of proteins, we acquired a PPI network that pointed, through topological analysis, to some proteins that may well be playing a central role in the neoplastic process, such as 14-3-3ζ. In silico overrepresentation analysis of transcription factors pointed to the overexpression of c-Myc, Max and E2F1 as key transcription factors involved in orchestrating the neoplastic phenotype. Conclusions Our findings show that there is a "central core of cervical cancer" protein expression pattern, and suggest that 14-3-3ζ is key to determine if the cell proliferates or dies. In addition, our

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

    Science.gov (United States)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M; Poulsen, H S

    1992-06-01

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

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

    DEFF Research Database (Denmark)

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

    1999-01-01

    Multidrug resistance (MDR) remains a major problem in the successful treatment of small cell lung cancer (SCLC). New treatment strategies are needed, such as gene therapy specifically targeting the MDR cells in the tumor. Retroviral LacZ gene-containing vectors that were either pseudotyped...... for the gibbon ape leukemia virus (GALV-1) receptor or had specificity for the amphotropic murine leukemia virus (MLV-A) receptor were used for transduction of five SCLC cell lines differing by a range of MDR mechanisms. Transduction efficiencies in these cell lines were compared by calculating the percentage...

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

    Directory of Open Access Journals (Sweden)

    Andreas Krieg

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

  13. Melatonin decreases cell proliferation, impairs myogenic differentiation and triggers apoptotic cell death in rhabdomyosarcoma cell lines.

    Science.gov (United States)

    Codenotti, Silvia; Battistelli, Michela; Burattini, Sabrina; Salucci, Sara; Falcieri, Elisabetta; Rezzani, Rita; Faggi, Fiorella; Colombi, Marina; Monti, Eugenio; Fanzani, Alessandro

    2015-07-01

    Melatonin is a small indole produced by the pineal gland and other tissues, and has numerous functions that aid in the maintenance of the whole body homeostasis, ranging from the regulation of circadian rhythms and sleep to protection from oxidative stress. Melatonin has also been reported to counteract cell growth and chemoresistance in different types of cancer. In the present study, we investigated the effects of exogenous melatonin administration on different human cell lines and primary mouse tumor cultures of rhabdomyosarcoma (RMS), the most frequent soft tissue sarcoma affecting childhood. The results showed that melatonin significantly affected the behavior of RMS cells, leading to inhibition of cell proliferation and impairment of myogenic differentiation followed by increased apoptotic cell death, as observed by immunoblotting analysis of apoptosis-related markers including Bax, Bcl-2 and caspase-3. Similar findings were observed using a combination of microscopy techniques, including scanning/transmission electron and confocal microscopy. Furthermore, melatonin in combination with doxorubicin or cisplatin, two compounds commonly used for the treatment of solid tumors, increased the sensitivity of RMS cells to apoptosis. These data indicated that melatonin may be effective in counteracting RMS tumor growth and chemoresistance.

  14. Epithelial cell adhesion molecule in human hepatocellular carcinoma cell lines: a target of chemoresistence.

    Science.gov (United States)

    Li, Yan; Farmer, Russell W; Yang, Yingbin; Martin, Robert C G

    2016-03-16

    The low survival rate of hepatocellular carcinoma (HCC) is partly attributable to its resistance to existing chemotherapeutic agents. Until now, there have been limited chemotherapeutic agents for liver cancer. Epithelial cell adhesion molecule (EpCAM) has been found to be over-expressed during stages of carcinogenesis and has been associated with poor overall survival in many cancers. The aim of this study was to evaluate EpCAM expression in HCC and evaluate the effects of EpCAM to established chemotherapy. Three human hepatocellular carcinoma cell lines--HepG2, Hep3B and HuH-7--were pre- and post-treated with doxorubicin, 5-fluorouracil (5-FU) and cisplatin. Cell viability and EpCAM protein expression were measured by MTT assay and Western Blotting respectively. EpCAM positive cells were analyzed by flow cytometry. To evaluate the effects of doxorubicin efficacy on EpCAM positive cells, a small interfering RNA (siRNA) specific to EpCAM was transfected into the cells and treated with doxorubicin. EpCAM was significantly down-regulated by doxorubicin treatment in all three HCC cell lines (P cells, however the EpCAM expression was up-regulated by 5-FU and cisplatin in Hep3B cell line. EpCAM expression was down-regulated by 5-FU, and up-regulated by cisplatin in Huh-7 cell line. Flow cytometry assay showed doxorubicin exposure decreased EpCAM positive cell quantities in three HCC cell lines. EpCAM siRNA knock-down attenuated cell mortality after doxorubicin exposure. All of these findings demonstrate that EpCAM is one of targets of chemoresistence.

  15. 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.

  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. [Effects of matrine on proliferation and apoptosis of human renal cell carcinoma cell line GRC-1].

    Science.gov (United States)

    Chong, Tie; Niu, Jian-Qiang; Wang, Zi-Ming; She, Jun-Jun; Huang, Chen

    2006-07-01

    To observe the effects of matrine on proliferation and apoptosis of human renal cell carcinoma cell line GRC-1 in vitro, and to explore its mechanism. The human renal cell carcinoma cell line GRC-1 was treated with matrine of different concentrations for 24, 48, 72 and 96 h respectively. The MTT assay was used to evaluate the cytotoxic effects of matrine on GRC-1 cells. The transmission electron microscope and flow cytometry were utilized to observe and detect the apoptosis of GRC-1 cells induced by matrine. The expression levels of Bcl-2 and Bax proteins were evaluated by streptavidin-biotin-peroxidase method. The matrine of different concentrations all have cytotoxic effects on GRC-1 cells, with obvious dose- and time-dependent effects. The apoptosis induced by matrine was confirmed in GRC-1 cells. With intervention of matrine (1.5 g/L) for 12 h, the expression level of Bcl-2 in GRC-1 cells was decreased while the expression level of Bax was increased as compared with those in the untreated group. The proliferation-inhibiting effects of matrine on human renal cell carcinoma cell line GRC-1 may be related to down-regulating the ratio of Bcl-2/Bax protein expression and promoting the apoptosis.

  18. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Science.gov (United States)

    2010-01-01

    ... each of at least one monolayer (at least 75 cm2) of: (i) Vero (African green monkey kidney) cell line... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of...

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

    Full Text Available Unc.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 Unclassified Blood Lymphoblastoid cell line... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Unc.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 Unclassified Blood Lymphoblastoid cell line... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  3. MDR Gene Expression Analysis of Six Drug-Resistant Ovarian Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Radosław Januchowski

    2013-01-01

    and protein levels. Cell lines resistant to agents used in ovarian cancer treatment remained sensitive to methotrexate. The main mechanisms of drug resistance were due to P-gp expression in the doxorubicin, vincristine, and paclitaxel resistant cell lines and BCRP expression in the topotecan resistant cell line.

  4. Characterization of side population cells isolated from the gastric cancer cell line SGC-7901.

    Science.gov (United States)

    Li, Rong; Wu, Xiaoling; Wei, Huang; Tian, Shangkun

    2013-03-01

    Side population (SP) cells are a subset of stem cells that have been isolated from several different gastrointestinal cancer cell lines. Using flow cytometry and the DNA-binding dye Hoechst 33342, we isolated SP cells from SGC-7901 human gastric tumor cell lines and found that they comprise 2.3±0.78% of the tumor cells. Using the Cell Counting Kit-8 (CCK-8) assay, we demonstrated that SP cells have a stronger proliferative activity than non-SP cells. Additionally, we observed tumor mass formation following the cultivation of SP cells in serum-free medium, indicating the capability of these cells for self-renewal. SP cells were observed to undergo non-symmetrical division, which is characteristic of stem cells. A drug resistance assay revealed that SP cells have a high survival rate when exposed to the chemotherapy drug 5-fluorouracil; the results of western blot analysis suggest that this stems from the abundant expression of the chemoresistance-associated proteins ABCG2 and Bcl-2. We also used fluorescence quantitative PCR to reveal that SP cells have relatively high expression levels of the stem cell-related genes Musashi-1 and CD44. In vivo experiments in mice revealed that the subcutaneous injection of 2×10(3) SP cells resulted in the formation of tumors, while the injection of 2×10(4) non-SP cells did not. Cumulatively, our results suggest that gastric tumorigenesis associated with SGC-7901 may partly be driven by the activity of SP cells, which exhibit certain biological characteristics of stem cells. Our results also show that the SP cell sorting method is an effective means for isolating and identifying gastric cancer stem cells during early screening.

  5. Pulsating electromagnetic field stimulation prevents cell death of puromycin treated U937 cell line.

    Science.gov (United States)

    Kaszuba-Zwoinska, J; Wojcik, K; Bereta, M; Ziomber, A; Pierzchalski, P; Rokita, E; Marcinkiewicz, J; Zaraska, W; Thor, P

    2010-04-01

    Aim of study was to verify whether pulsating electromagnetic field (PEMF) can affect cancer cells proliferation and death. U937 human lymphoid cell line at densities starting from 1 x 10(6) cells/ml to 0.0625 x 10(6) cells/ml, were exposed to a pulsating magnetic field 50 Hz, 45+/-5 mT three times for 3 h per each stimulation with 24 h intervals. Proliferation has been studied by counting number of cells stimulated and non-stimulated by PEMF during four days of cultivation. Viability of cells was analyzed by APC labeled Annexin V and 7-AAD (7-amino-actinomycin D) dye binding and flow cytometry. Growing densities of cells increase cell death in cultures of U937 cells. PEMF exposition decreased amount of cells only in higher densities. Measurement of Annexin V binding and 7-AAD dye incorporation has shown that density-induced cell death corresponds with decrease of proliferation activity. PEMF potentiated density-induced death both apoptosis and necrosis. The strongest influence of PEMF has been found for 1 x 10(6)cells/ml and 0.5 x 10(6) cells/ml density. To eliminate density effect on cell death, for further studies density 0.25 x 10(6) cells/ml was chosen. Puromycin, a telomerase inhibitor, was used as a cell death inducer at concentration 100 microg/ml. Combined interaction of three doses of puromycin and three fold PEMF interaction resulted in a reduced of apoptosis by 24,7% and necrosis by 13%. PEMF protects U937 cells against puromycin- induced cell death. PEMF effects on the human lymphoid cell line depends upon cell density. Increased density induced cells death and on the other hand prevented cells death induced by puromycin.

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

    LENUS (Irish Health Repository)

    O'Neill, Amanda J

    2011-10-07

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

  7. 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

  8. Cytotoxic effects of mistletoe (Viscum album L.) in head and neck squamous cell carcinoma cell lines.

    Science.gov (United States)

    Klingbeil, Ma Fátima G; Xavier, Flávia C A; Sardinha, Luiz R; Severino, Patricia; Mathor, Monica B; Rodrigues, Rodrigo V; Pinto, Décio S

    2013-11-01

    Head and neck squamous cell carcinoma is a complex disease with several etiologic factors and different molecular changes that may trigger certain events; it is also globally one of the most common malignancies in this topography. Extracts from Viscum album L. (VA) (mistletoe) have been used as adjuvant therapies with promising results in several types of cancer, mainly in European countries. In vitro studies have demonstrated that various types of VA may have cytotoxicity in carcinoma cells, activating the apoptotic cascade or leading cells to necrosis. This study aimed to verify the effects of three types of VA extracts (Iscador Qu Spezial, Iscador P and Iscador M) in squamous cell carcinoma of the tongue cell lines SCC9 and SCC25, not previously studied. A concentration of 0.3 mg/ml (IC50) of the drugs induced apoptosis, affecting gene expression and protein levels of AKT, PTEN and CYCLIN D1. It was concluded that VA extracts have a cytotoxic effect on SCC9 and SCC25 cell lines, but while SCC9 cell line was more resistant to the action of the drugs, Iscador Qu Spezial and Iscador M have higher cytotoxic potential in both cell lines compared to Iscador P.

  9. Cell lines derived from feline fibrosarcoma display unstable chromosomal aneuploidy and additionally centrosome number aberrations.

    Science.gov (United States)

    von Erichsen, J; Hecht, W; Löhberg-Gruene, C; Reinacher, M

    2012-07-01

    The purpose of the study was to evaluate clonality and presence of numerical chromosomal and centrosomal aberrations in 5 established feline fibrosarcoma cell lines and in a fetal dermal fibroblast cell line as a control. The clonality of all cell lines was examined using limited-dilution cloning. The number of chromosomes was counted in metaphase spreads. The immunocytochemical analysis of centrosome numbers was performed by indirect immunofluorescence using a monoclonal antibody that targets γ-tubulin, a well-characterized component of centrosomes. Monoclonal cell populations could be established from all cell lines. In all feline fibrosarcoma cell lines, the number of chromosomes deviated abnormally from the normal feline chromosome number of 2n = 38, ranging from 19 to 155 chromosomes per cell. Centrosome hyperamplification was observed in all 5 feline fibrosarcoma cell lines with a proportion of cells (5.7 to 15.2%) having more than 2 centrosomes. In the control cell line, only 0.6% of the cells had more than 2 centrosomes. In conclusion, the examinations revealed that centrosome hyperamplification occurs in feline fibrosarcoma cell lines. The feline fibrosarcoma cell lines possessed 10 to 25 times as many cells with centrosome hyperamplification as the control cell line. These observations suggest an association of numerical centrosome aberrations with karyotype instability by increasing the frequency of chromosome missegregation. The results of this study may be helpful for further characterization of feline fibrosarcomas and may contribute to the knowledge of cytogenetic factors that may be important for the pathogenesis of feline fibrosarcomas.

  10. Epigenetic alterations differ in phenotypically distinct human neuroblastoma cell lines

    Directory of Open Access Journals (Sweden)

    Salwen Helen R

    2010-06-01

    Full Text Available Abstract Background Epigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB. Seven cancer related genes (THBS-1, CASP8, HIN-1, TIG-1, BLU, SPARC, and HIC-1 that have been shown to have epigenetic changes in adult cancers and play important roles in the regulation of angiogenesis, tumor growth, and apoptosis were analyzed to investigate the role epigenetic alterations play in determining NB phenotype. Methods Two NB cell lines (tumorigenic LA1-55n and non-tumorigenic LA1-5s that differ in their ability to form colonies in soft agar and tumors in nude mice were used. Quantitative RNA expression analyses were performed on seven genes in LA1-5s, LA1-55n and 5-Aza-dC treated LA1-55n NB cell lines. The methylation status around THBS-1, HIN-1, TIG-1 and CASP8 promoters was examined using methylation specific PCR. Chromatin immunoprecipitation assay was used to examine histone modifications along the THBS-1 promoter. Luciferase assay was used to determine THBS-1 promoter activity. Cell proliferation assay was used to examine the effect of 5-Aza-dC on NB cell growth. The soft agar assay was used to determine the tumorigenicity. Results Promoter methylation values for THBS-1, HIN-1, TIG-1, and CASP8 were higher in LA1-55n cells compared to LA1-5s cells. Consistent with the promoter methylation status, lower levels of gene expression were detected in the LA1-55n cells. Histone marks associated with repressive chromatin states (H3K9Me3, H3K27Me3, and H3K4Me3 were identified in the THBS-1 promoter region in the LA1-55n cells, but not the LA1-5s cells. In contrast, the three histone codes associated with an active chromatin state (acetyl H3, acetyl H4, and H3K4Me3 were present in the THBS-1 promoter region in LA1-5s cells, but not the LA1-55n cells, suggesting that an accessible chromatin structure is important for THBS-1 expression. We also show that 5-Aza

  11. Evaluation of stem-like side population cells in a recurrent nasopharyngeal carcinoma cell line.

    Science.gov (United States)

    Hoe, Susan Ling Ling; Tan, Lu Ping; Jamal, Juliana; Peh, Suat Cheng; Ng, Ching Ching; Zhang, Wen Cai; Ahmad, Munirah; Khoo, Alan Soo Beng

    2014-01-01

    Side population (SP) assay identifies cells with dye/drug extrusion ability, a characteristic of stem cells. Here, we determined if SP cells exist in a verified cell line originating from recurrent nasopharyngeal carcinoma (NPC) and a xenograft established from recurrent metastatic NPC. These cells were evaluated for stem-like properties via functional assays as well as for tumourigenicity. We used Hoechst 33342 to identify the SP from non-SP (NSP) cells in HK1 NPC cell line and xeno-284 NPC xenograft. The cells were assayed for in vitro characteristics of cancer stem cells (CSC), gene expression and tumourigenicity ability. Student's t test was used to test for significance. Five to ten percent and less than 0.5% of HK1 and xeno-284 NPC cells, respectively, were SP cells. Fumitremorgin C (FTC), as opposed to verapamil, was effective in causing the cells to retain Hoechst 33342 dye. HK1 SP cells formed more holoclones, had more aldehyde dehydrogenase (ALDH) activity, divided asymmetrically and contained slow-proliferating cells. ABCG2, SOX2, TERT, MYC, Hedgehog, Notch, TGFβ and Wnt signalling pathway genes were significantly upregulated in the SP cells. However, despite these differences in vitro, both HK1 SP and NSP cells had an overall similar tumourigenic potential in vivo. HK1 SP cells were ABCG2-specific as confirmed by FTC inhibition and gene expression data. Despite data from in vitro and gene expression experiments suggesting stem-like features, there was no significant difference in tumourigenic potential between SP and NSP cells. We conclude that SP assay alone is not sufficient to identify CSCs in HK1 cells. Our work also suggests the presence of a stem-cell like population among NPC cells which do not display increased tumourigenicity.

  12. The telomerase inhibitor imetelstat depletes cancer stem cells in breast and pancreatic cancer cell lines.

    Science.gov (United States)

    Joseph, Immanual; Tressler, Robert; Bassett, Ekaterina; Harley, Calvin; Buseman, Christen M; Pattamatta, Preeti; Wright, Woodring E; Shay, Jerry W; Go, Ning F

    2010-11-15

    Cancer stem cells (CSC) are rare drug-resistant cancer cell subsets proposed to be responsible for the maintenance and recurrence of cancer and metastasis. Telomerase is constitutively active in both bulk tumor cell and CSC populations but has only limited expression in normal tissues. Thus, inhibition of telomerase has been shown to be a viable approach in controlling cancer growth in nonclinical studies and is currently in phase II clinical trials. In this study, we investigated the effects of imetelstat (GRN163L), a potent telomerase inhibitor, on both the bulk cancer cells and putative CSCs. When breast and pancreatic cancer cell lines were treated with imetelstat in vitro, telomerase activity in the bulk tumor cells and CSC subpopulations were inhibited. Additionally, imetelstat treatment reduced the CSC fractions present in the breast and pancreatic cell lines. In vitro treatment with imetelstat, but not control oligonucleotides, also reduced the proliferation and self-renewal potential of MCF7 mammospheres and resulted in cell death after telomerase activity expression levels or telomere length of CSCs and bulk tumor cells in these cell lines did not correlate with the increased sensitivity of CSCs to imetelstat, suggesting a mechanism of action independent of telomere shortening for the effects of imetelstat on the CSC subpopulations. Our results suggest that imetelstat-mediated depletion of CSCs may offer an alternative mechanism by which telomerase inhibition may be exploited for cancer therapy. Copyright © 2010 AACR.

  13. A Focused Microarray for Screening Rat Embryonic Stem Cell Lines

    Science.gov (United States)

    Hong, James; He, Hong; Bui, Phuoc; Ryba-White, Ben; Rumi, Mohammad A.K.; Soares, Michael J.; Dutta, Debasree; Paul, Soumen; Kawamata, Masaki; Ochiya, Takahiro; Ying, Qi-Long; Rajanahalli, Pavan

    2013-01-01

    Here, we describe a focused microarray for screening rat embryonic stem cells (ESCs) and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem (TS) cells, rat extraembryonic endoderm cells, mouse embryonic fibroblast feeder cells, and differentiated rat ESCs. Using this tool, genuine rat ESC lines, which have been expanded in a conventional rat ESC medium containing two inhibitors (2i), for example, glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (MEK) inhibitors, and leukemia inhibitory factor, and genuine rat ESCs, which have been expanded in rat ESC medium containing four inhibitors (4i), for example, GSK3, MEK, Alk5, and Rho-associated kinase inhibitors were compared; as were genuine rat ESCs from 4 different strains of rats. Expression of Cdx2, a gene associated with trophoblast determination, was observed in genuine, undifferentiated rat ESCs from 4 strains and from both 2i and 4i ESC derivation medium. This finding is in contrast to undifferentiated mouse ESCs that do not express Cdx2. The rat ESC focused microarray described in this report has utility for rapid screening of rat ESCs. This tool will enable optimization of culture conditions in the future. PMID:22889370

  14. Probenecid is a chemosensitizer in cancer cell lines.

    Science.gov (United States)

    Campos-Arroyo, Denise; Martínez-Lazcano, Juan Carlos; Melendez-Zajgla, Jorge

    2012-02-01

    Resistance and toxicity are the major barriers to successful cancer chemotherapies. Developing molecules that reduce drug resistance and improve antineoplastic effects is of great interest for cancer research; ideally, these substances should not affect the pharmacodynamics of the chemotherapeutic agent while providing a synergistic antineoplastic effect. In this study, we tested in vitro co-administration of the antineoplastic agents cisplatin or paclitaxel with probenecid, an anion channel inhibitor, in a panel of cancer cell lines to determine the cytotoxicity and synergistic effects of these drug combinations. In addition, we measured the clonogenicity and apoptotic index in these cells. We observed a synergistic interaction between probenecid and the chemotherapeutic agents, and increasing doses of probenecid resulted in a significant decrease in the effective doses of the chemotherapeutic agents. For the antineoplastic agent and probenecid combinations, we found increased cell death, reduced colony formation, and a higher number of apoptotic cells, compared with treatment of cisplatin or paclitaxel alone. Further research is necessary to elucidate the molecular mechanisms by which the synergistic effect occurs. If these synergistic effects can be reproduced in vivo, the co-administration of probenecid with different chemotherapeutic agents may provide a valid treatment in patients with chemotherapy resistance.

  15. Development and characterization of two cell lines PDF and PDH from Puntius denisonii (Day 1865).

    Science.gov (United States)

    Lakra, Wazir S; Goswami, M; Yadav, Kamalendra; Gopalakrishnan, A; Patiyal, R S; Singh, M

    2011-02-01

    The Puntius denisonii colloquially and more popularly referred to as Miss Kerala is a subtropical fish belonging to the genus Puntius (Barb) and family Cyprinidae. Two cell lines PDF and PDH were developed from the caudal fin and heart of P. denisonii, respectively. The cell lines were optimally maintained at 26°C in Leibovitz-15 medium supplemented with 10% fetal bovine serum. A diploid count of 50 chromosomes at passage 50 was observed in both the cell lines. The high growth potential of the cell lines was reflected from the cell doubling time of 28 and 30 h of PDF and PDH cell lines, respectively. The viability of the PDF and PDH cell lines was 70% and 76%, respectively, after 4 mo of storage in liquid nitrogen (-196°C). The origin of the cell lines was confirmed by the amplification of 653 bp fragments of cytochrome oxidase subunit I of mitochondrial DNA genes.

  16. PDGFRA Is Not Essential for the Derivation and Maintenance of Mouse Extraembryonic Endoderm Stem Cell Lines

    Directory of Open Access Journals (Sweden)

    Jiangwei Lin

    2017-10-01

    Full Text Available Extraembryonic endoderm stem (XEN cell lines can be derived and maintained in vitro and reflect the primitive endoderm lineage. Platelet-derived growth factor receptor alpha (PDGFRA is thought to be essential for the derivation and maintenance of mouse XEN cell lines. Here, we have re-evaluated this requirement for PDGFRA. We derived multiple PDGFRA-deficient XEN cell lines from postimplantation and preimplantation embryos of a PDGFRA-GFP knockout strain. We also converted PDGFRA-deficient embryonic stem cell lines into XEN cell lines chemically by transient culturing with retinoic acid and Activin A. We confirmed the XEN profile of our 12 PDGFRA-deficient cell lines by immunofluorescence with various markers, by NanoString gene expression analyses, and by their contribution to the extraembryonic endoderm of chimeric embryos produced by injecting these cells into blastocysts. Thus, PDGFRA is not essential for the derivation and maintenance of XEN cell lines.

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

    Directory of Open Access Journals (Sweden)

    Seyhan Turk

    2017-02-01

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

  18. Stable aneuploid tumors cells are more sensitive to TTK inhibition than chromosomally unstable cell lines.

    Science.gov (United States)

    Libouban, Marion A A; de Roos, Jeroen A D M; Uitdehaag, Joost C M; Willemsen-Seegers, Nicole; Mainardi, Sara; Dylus, Jelle; de Man, Jos; Tops, Bastiaan; Meijerink, Jules P P; Storchová, Zuzana; Buijsman, Rogier C; Medema, René H; Zaman, Guido J R

    2017-06-13

    Inhibition of the spindle assembly checkpoint kinase TTK causes chromosome mis-segregation and tumor cell death. However, high levels of TTK correlate with chromosomal instability (CIN), which can lead to aneuploidy. We show that treatment of tumor cells with the selective small molecule TTK inhibitor NTRC 0066-0 overrides the mitotic checkpoint, irrespective of cell line sensitivity. In stable aneuploid cells NTRC 0066-0 induced acute CIN, whereas in cells with high levels of pre-existing CIN there was only a small additional fraction of cells mis-segregating their chromosomes. In proliferation assays stable aneuploid cells were more sensitive than cell lines with pre-existing CIN. Tetraploids are thought to be an intermediate between diploid and unstable aneuploid cells. TTK inhibitors had the same potency on post-tetraploid and parental diploid cells, which is remarkable because the post-tetraploids are more resistant to mitotic drugs. Finally, we confirm that the reference compound reversine is a TTK inhibitor and like NTRC 0066-0, inhibits the proliferation of patient-derived colorectal cancer organoids. In contrast, treatment with TTK inhibitor did not reduce the viability of non-proliferating T cell acute lymphoblastic leukemia cells samples. Consequently, TTK inhibitor therapy is expected to spare non-dividing cells, and may be used to target stable aneuploid tumors.

  19. Extracellular vesicles from a muscle cell line (C2C12 enhance cell survival and neurite outgrowth of a motor neuron cell line (NSC-34

    Directory of Open Access Journals (Sweden)

    Roger D. Madison

    2014-02-01

    Full Text Available Introduction: 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. Background: 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. Methods: 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? Conclusion: 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.

  20. 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.

  1. Different toxic effects of YTX in tumor K-562 and lymphoblastoid cell lines

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    Andrea Fernández Araújo

    2015-06-01

    Full Text Available Yessotoxin (YTX modulates cellular phosphodiesterases (PDEs. In this regard, opposite effects had been described in the tumor model K-562 cell line and fresh human lymphocytes in terms of cell viability, cyclic adenosine 3´,5´-cyclic monophosphate (cAMP production and protein expression after YTX treatment. Studies in depth of the pathways activated by YTX in K-562 cell line, have demonstrated the activation of two different cell death types, apoptosis and autophagy after 24 and 48 hours of treatment, respectively. Furthermore, the key role of type 4A PDE (PDE4A in both pathways activated by YTX was demonstrated. Therefore, taking into account the differences between cellular lines and fresh cells, a study of cell death pathways activated by YTX in a non-tumor cell line with mitotic activity, was performed. The cellular model used was the lymphoblastoid cell line that represents a non-tumor model with normal apoptotic and mitotic machinery. In this context, cell viability and cell proliferation, expression of proteins involved in cell death activated by YTX and mitochondrial mass, were studied after the incubation with the toxin. Opposite to the tumor model, no cell death activation was observed in lymphoblastoid cell line in the presence of YTX. In this sense, variations in apoptosis hallmarks were not detected in the lymphoblastoid cell line after YTX incubation, whereas this type I of programmed cell death was observed in K-526 cells. On the other hand, autophagy cell death was triggered in this cellular line, while other autophagic process is suggested in lymphoblastoid cells. These YTX effects are related to PDE4A in both cellular lines. In addition, while cell death is triggered in K-526 cells after YTX treatment, in lymphoblastoid cells the toxin stops cellular proliferation. These results point to YTX as a specific toxic compound of tumor cells, since in the non-tumor lymphoblastoid cell line, no cell death hallmarks are observed.

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

    Science.gov (United States)

    2010-01-01

    Background The anti-infective agent Taurolidine (TRD) has been shown to have cell death inducing properties, but the mechanism of its action is largely unknown. The aim of this study was to identify potential common target genes modulated at the transcriptional level following TRD treatment in tumour cell lines originating from different cancer types. Methods Five different malignant cell lines (HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3) were incubated with TRD (100 μM, 250 μM and 1000 μM). Proliferation after 8 h and cell viability after 24 h were analyzed by BrdU assay and FACS analysis, respectively. Gene expression analyses were carried out using the Agilent -microarray platform to indentify genes which displayed conjoint regulation following the addition of TRD in all cell lines. Candidate genes were subjected to Ingenuity Pathways Analysis and selected genes were validated by qRT-PCR and Western Blot. Results TRD 250 μM caused a significant inhibition of proliferation as well as apoptotic cell death in all cell lines. Among cell death associated genes with the strongest regulation in gene expression, we identified pro-apoptotic transcription factors (EGR1, ATF3) as well as genes involved in the ER stress response (PPP1R15A), in ubiquitination (TRAF6) and mitochondrial apoptotic pathways (PMAIP1). Conclusions This is the first conjoint analysis of potential target genes of TRD which was performed simultaneously in different malignant cell lines. The results indicate that TRD might be involved in different signal transduction pathways leading to apoptosis. PMID:21034493

  3. [Radiosensitization effect of black garlic extract on lung cancer cell line Lewis cells].

    Science.gov (United States)

    Yang, Gui-qing; Wang, Dong; Wang, Yi-shan; Wang, Yuan-yuan; Yang, Ke

    2013-08-01

    To explore the radiosensitization effect of black garlic extract (BGE) on lung cancer cell line Lewis cells. The inhibition rate of lung cancer cells after BGE action was detected by MTT. Effect of BGE combined radiotherapy on the colony formation rate was observed by cloning formation assay. Changes of the cell morphology were observed by Hoechst staining. Changes of the cell cycle were detected by flow cytometry. Real time PCR was used to detect mRNA expressions of bcl-2 and bax. BGE could have significant inhibitory action on the growth of lung cancer Lewis cells. The combination of BGE and radiotherapy (by 60Co gamma) significantly induced Lewis cells' apoptosis in G2/M stage, obviously decreased the expression of bcl-2, and up-regulated the expression of bax. BGE could sensitize the lung cancer Lewis cells to ionizing irradiation. This effect might be probably caused by changing the cell cycles and affecting expressions of bax and bcl-2.

  4. [A PTHrP-producing cell line derived from human small cell lung carcinoma].

    Science.gov (United States)

    Iguchi, H

    1996-03-01

    We established a cell line, designated MS-1, from pleural effusion of a 54-yrs-old male patient with small cell lung carcinoma. MS-1 cells grew as a floating in RPMI-1640 medium supplemented with 10% FBS and the population doubling time was 45 hours. The chromosome number ranged from 49 to 52 and structural abnormalities of 1p+, 3q-, 6p-, 14p+ and 17p+ were observed in all the cells examined. MS-1 cells released PTHrP into the conditioned medium and heterogeneity of the PTHrP molecule produced in the cells was found in the gel permeation chromatography. Expression of the PTHrP gene as well as presence of the PTHrP protein in the cells were confirmed by reverse-transcriptase PCR(RT-PCR) and immunohistochemical staining. These findings indicate that MS-1 cells are derived from human small cell lung carcinoma, which produce PTHrP.

  5. Proteomics of cancer cell lines resistant to microtubule-stabilizing agents

    DEFF Research Database (Denmark)

    Albrethsen, Jakob; Angeletti, Ruth H; Horwitz, Susan Band

    2014-01-01

    resistance to the class of MIAs known as microtubule-stabilizing agents (MSA). The human lung cancer cell line A549 was compared with two drug-resistant daughter cell lines, a taxol-resistant cell line (AT12) and an epothilone B (EpoB)-resistant cell line (EpoB40). The ovarian cancer cell line Hey......Despite the clinical success of microtubule-interacting agents (MIA), a significant challenge for oncologists is the inability to predict the response of individual patients with cancer to these drugs. In the present study, six cell lines were compared by 2D DIGE proteomics to investigate cellular...... increased in EpoB- and ixabepilone-resistant cells and its suppression caused an increase in drug sensitivity in both drug-sensitive and -resistant Hey cells. Furthermore, the growth medium from resistant Hey cells contained higher levels of galectin-1, suggesting that galectin-1 could play a role...

  6. Tooth regeneration from newly established cell lines from a molar tooth germ epithelium.

    Science.gov (United States)

    Komine, Akihiko; Suenaga, Momoko; Nakao, Kazuhisa; Tsuji, Takashi; Tomooka, Yasuhiro

    2007-04-13

    In order to investigate tooth development, several cell lines of the dental epithelium and ectomesenchyme have been established. However, no attempt has been reported to regenerate teeth with cell lines. Here, we have established several clonal cell lines of the dental epithelium from a p53-deficient fetal mouse. They expressed specific markers of the dental epithelium such as ameloblastin and amelogenin. A new method has been developed to bioengineer tooth germs with dental epithelial and mesenchymal cells. Reconstructed tooth germs with cell lines and fetal mesenchymal cells were implanted under kidney capsule. The germs regenerated teeth with well-calcified structures as seen in natural tooth. Germs without the cell lines developed bone. This is the first success to regenerate teeth with dental epithelial cell lines. They are useful models in vitro for investigation of mechanisms in morphogenesis and of cell lineage in differentiation, and for clinical application for tooth regeneration.

  7. Extraintestinal pathogenic Escherichia coli sequence type 131 H30-R and H30-Rx subclones in retail chicken meat, Italy.

    Science.gov (United States)

    Ghodousi, Arash; Bonura, Celestino; Di Carlo, Paola; van Leeuwen, Willem B; Mammina, Caterina

    2016-07-02

    Extraintestinal pathogenic Escherichia coli sequence type 131 (ST131), typically fluoroquinolone-resistant (FQ-R) and/or extended-spectrum β-lactamase (ESBL)-producing, has emerged globally. Among clinical isolates, ST131, primarily its H30-R and H30-Rx subclones, accounts for most antimicrobial-resistant E. coli and is the dominant E. coli strain worldwide. We assessed its prevalence and characteristics among raw chicken meat samples on sale in Palermo, Italy. A collection of 237 fluoroquinolone resistant and ESBL/AmpC producing E. coli isolates, which had been isolated from processed retail chicken meat in the period May 2013-April 2015, was analyzed. Established polymerase chain reaction methods were used to define ST131 and its H30 subclones, ESBL, AmpC, and plasmid-mediated quinolone resistance (PMQR) determinants. Amplified Fragment Length Polymorphism (AFLP) was performed to assess the relatedness among ST131 isolates. Out of the 237 E. coli isolates, 12 isolates belonged to the phylogenetic group B2. Based on the molecular definition of ExPEC, all isolates were attributed with the status of ExPEC. SNP-PCR results confirmed that nine isolates were ST131. SNP-PCR for H30-R and H30-Rx subclones showed that six and three ExPEC ST131 were positive for H30-R and H30-Rx, respectively. The results of AFLP showed that, except for four isolates grouped into two clusters which proved to be indistinguishable, the isolates under study were genetically heterogeneous. To the best of our knowledge, this is the first report of H30-R and H30-Rx subclones in animal food samples. Our findings appear to support the role of food chain in their transmission to humans. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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    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.

  9. Effect of Quercetin on Cell Cycle and Cyclin Expression in Ovarian Carcinoma and Osteosarcoma Cell Lines.

    Science.gov (United States)

    Catanzaro, Daniela; Ragazzi, Eugenio; Vianello, Caterina; Caparrotta, Laura; Montopoli, Monica

    2015-08-01

    Resistance to chemotherapeutic drugs is a major problem in cancer treatment. The search for new interventions able to overcome this resistance may involve compounds of natural origin, such as flavonoids, ubiquitously present in many foods. In the present study, the cytotoxic effects and cell cycle modulation of the flavonoid quercetin were investigated in ovarian carcinoma (SKOV3) and osteosarcoma (U2OS) human cell lines and in their cisplatin (CDDP)-resistant counterparts (SKOV3/CDDP and U2OSPt cells, respectively). Quercetin (10-50 μM) caused evident changes in the distribution of cell cycle phases in the CDDP-resistant SKOV3/CDDP ovarian cell line. The levels of cyclin D1 and cyclin B1 were determined by means of Western blot in all cell lines incubated with quercetin (50 μM) for 48 hours. The cyclin D1 expression was significantly decreased following the treatment with quercetin in SKOV3 and U2OSPt cells, but not in SKOV3/CDDP and U2OS cells. The reduction of cyclin D1 level could be linked to the G1/S phase alteration found in quercetin-treated cells. Although cyclin B1 is required for G2/M phase, and despite our observation that quercetin influenced the G2/M phase of cell cycle, the flavonoid did not affect cyclin B1 levels in all cell lines, indicating the involvement of other possible mechanisms. These results suggest that quercetin, exceeding the resistance to CDDP, might become an interesting tool to evaluate cytotoxic activity in combination with chemotherapy drugs.

  10. Glypican4 modulates lateral line collective cell migration non cell-autonomously.

    Science.gov (United States)

    Venero Galanternik, Marina; Lush, Mark E; Piotrowski, Tatjana

    2016-11-15

    Collective cell migration is an essential process during embryonic development and diseases such as cancer, and still much remains to be learned about how cell intrinsic and environmental cues are coordinated to guide cells to their targets. The migration-dependent development of the zebrafish sensory lateral line proves to be an excellent model to study how proteoglycans control collective cell migration in a vertebrate. Proteoglycans are extracellular matrix glycoproteins essential for the control of several signaling pathways including Wnt/β-catenin, Fgf, BMP and Hh. In the lateral line primordium the modified sugar chains on proteoglycans are important regulators of cell polarity, ligand distribution and Fgf signaling. At least five proteoglycans show distinct expression patterns in the primordium; however, their individual functions have not been studied. Here, we describe the function of glypican4 during zebrafish lateral line development. glypican4 is expressed in neuromasts, interneuromast cells and muscle cells underlying the lateral line. knypek fr6 /glypican4 mutants show severe primordium migration defects and the primordium often U-turns and migrates back toward the head. Our analysis shows that Glypican4 regulates the feedback loop between Wnt/β-catenin/Fgf signaling in the primordium redundantly with other Heparan Sulfate Proteoglycans. In addition, the primordium migration defect is caused non-cell autonomously by the loss of cxcl12a-expressing muscle precursors along the myoseptum via downregulation of Hh. Our results show that glypican4 has distinct functions in primordium cells and cells in the environment and that both of these functions are essential for collective cell migration. Copyright © 2016. Published by Elsevier Inc.

  11. Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line

    Directory of Open Access Journals (Sweden)

    Behra Martine

    2012-01-01

    Full Text Available Abstract Background Because of the structural and molecular similarities between the two systems, the lateral line, a fish and amphibian specific sensory organ, has been widely used in zebrafish as a model to study the development/biology of neuroepithelia of the inner ear. Both organs have hair cells, which are the mechanoreceptor cells, and supporting cells providing other functions to the epithelium. In most vertebrates (excluding mammals, supporting cells comprise a pool of progenitors that replace damaged or dead hair cells. However, the lack of regenerative capacity in mammals is the single leading cause for acquired hearing disorders in humans. Results In an effort to understand the regenerative process of hair cells in fish, we characterized and cloned an egfp transgenic stable fish line that trapped tnks1bp1, a highly conserved gene that has been implicated in the maintenance of telomeres' length. We then used this Tg(tnks1bp1:EGFP line in a FACsorting strategy combined with microarrays to identify new molecular markers for supporting cells. Conclusions We present a Tg(tnks1bp1:EGFP stable transgenic line, which we used to establish a transcriptional profile of supporting cells in the zebrafish lateral line. Therefore we are providing a new set of markers specific for supporting cells as well as candidates for functional analysis of this important cell type. This will prove to be a valuable tool for the study of regeneration in the lateral line of zebrafish in particular and for regeneration of neuroepithelia in general.

  12. Thrombopoietin Receptor Levels in Tumor Cell Lines and Primary Tumors

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    Connie L. Erickson-Miller

    2010-01-01

    Full Text Available Thrombopoietin (TPO receptor agonists represent a new approach for the treatment of thrombocytopenia, which may develop as a consequence of immune thrombocytopenia, chemotherapy treatment, chronic hepatitis C infection, or myelodysplastic syndromes. There are concerns that use of certain growth factors can hasten disease progression in some types of hematologic malignancies and solid tumors. In this study, expression of MPL (TPO-R mRNA was examined in tumor cell lines, patient tumor samples (renal cell carcinoma, prostatic carcinoma, soft tissue and bony/cartilage sarcoma, colon cancer, and lymphoma, and normal tissues using microarray analysis and qRT-PCR. MPL mRNA is expressed at very low or undetectable levels compared with erythropoietin receptor (EPOR, human epidermal growth factor (ERBB2; HER2, and insulin-like growth factor-1 receptor (IGF1R in these patient samples. These data suggest TPO-R agonists will likely preferentially stimulate proliferation and differentiation of cells of megakaryocytic lineage, potentially demonstrating their utility for correcting thrombocytopenia in clinical settings.

  13. Diverse Hormone Response Networks in 41 Independent Drosophila Cell Lines

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    Marcus Stoiber

    2016-03-01

    Full Text Available Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. This atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly.

  14. Comprehensive characterization of genomic instability in pluripotent stem cells and their derived neuroprogenitor cell lines

    Directory of Open Access Journals (Sweden)

    Nestor Luis Lopez Corrales

    2012-12-01

    Full Text Available The genomic integrity of two human pluripotent stem cells and their derived neuroprogenitor cell lines was studied, applying a combination of high-resolution genetic methodologies. The usefulness of combining array-comparative genomic hybridization (aCGH and multiplex fluorescence in situ hybridization (M-FISH techniques should be delineated to exclude/detect a maximum of possible genomic structural aberrations. Interestingly, in parts different genomic imbalances at chromosomal and subchromosomal levels were detected in pluripotent stem cells and their derivatives. Some of the copy number variations were inherited from the original cell line, whereas other modifications were presumably acquired during the differentiation and manipulation procedures. These results underline the necessity to study both pluripotent stem cells and their differentiated progeny by as many approaches as possible in order to assess their genomic stability before using them in clinical therapies.

  15. Side population cells isolated from KATO III human gastric cancer cell line have cancer stem cell-like characteristics.

    Science.gov (United States)

    She, Jun-Jun; Zhang, Peng-Ge; Wang, Xuan; Che, Xiang-Ming; Wang, Zi-Ming

    2012-09-07

    To investigate whether the side population (SP) cells possess cancer stem cell-like characteristics in vitro and the role of SP cells in tumorigenic process in gastric cancer. We analyzed the presence of SP cells in different human gastric carcinoma cell lines, and then isolated and identified the SP cells from the KATO III human gastric cancer cell line by flow cytometry. The clonogenic ability and self-renewal were evaluated by clone and sphere formation assays. The related genes were determined by reverse transcription polymerase chain reaction. To compare tumorigenic ability, SP and non-side population (NSP) cells from the KATO III human gastric cancer cell line were subcutaneously injected into nude mice. SP cells from the total population accounted for 0.57% in KATO III, 1.04% in Hs-746T, and 0.02% in AGS (CRL-1739). SP cells could grow clonally and have self-renewal capability in conditioned media. The expression of ABCG2, MDRI, Bmi-1 and Oct-4 was different between SP and NSP cells. However, there was no apparent difference between SP and NSP cells when they were injected into nude mice. SP cells have some cancer stem cell-like characteristics in vitro and can be used for studying the tumorigenic process in gastric cancer.

  16. Single-cell printing to form three-dimensional lines of olfactory ensheathing cells

    Energy Technology Data Exchange (ETDEWEB)

    Othon, Christina M; Ringeisen, Bradley R [Naval Research Laboratory/Code 6113, 4555 Overlook Ave. SW, Washington, DC 20375 (United States); Wu Xingjia; Anders, Juanita J [Department of Anatomy, Physiology and Genetics, Uniformed Services University of Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814 (United States)], E-mail: ringeisen@nrl.navy.mil

    2008-09-01

    Biological laser printing (BioLP(TM)) is a unique tool capable of printing high resolution two- and three-dimensional patterns of living mammalian cells, with greater than 95% viability. These results have been extended to primary cultured olfactory ensheathing cells (OECs), harvested from adult Sprague-Dawley rats. OECs have been found to provide stimulating environments for neurite outgrowth in spinal cord injury models. BioLP is unique in that small load volumes ({approx}{mu}Ls) are required to achieve printing, enabling low numbers of OECs to be harvested, concentrated and printed. BioLP was used to form several 8 mm lines of OECs throughout a multilayer hydrogel scaffold. The line width was as low as 20 {mu}m, with most lines comprising aligned single cells. Fluorescent confocal microscopy was used to determine the functionality of the printed OECs, to monitor interactions between printed OECs, and to determine the extent of cell migration throughout the 3D scaffold. High-resolution printing of low cell count, harvested OECs is an important advancement for in vitro study of cell interactions and functionality. In addition, these cell-printed scaffolds may provide an alternative for spinal cord repair studies, as the single-cell patterns formed here are on relevant size scales for neurite outgrowth.

  17. [Studies on the interaction between interleukin-6 and human renal cell carcinoma cell line GRC-1].

    Science.gov (United States)

    Xi, Z; Yu, L; Guo, Y

    2000-04-01

    To demonstrate the effects of IL-6 on the renal cell carcinoma cell line GRC-1. Immunocytochemical staining and RT-PCR analysis indicated that this cell line could express IL-6 both on mRNA and protein levels. Secretion of IL-6 in this cell line was 465 pg/ml, which was identified by ELISA assay. By RT-PCR analysis, we found that GRC-1 expressed IL-6 receptor system including IL-6 mRNA and gp130 mRNA. The rhIL-6 did not stimulate the growth of GRC-1 while the neutralizing antibody did not inhibit its growth. For further identification of the effect of IL-6 on GRC-1 cells, we introduced an antisense IL-6 RNA into GRC-1 cells. Thereafter, the lowered expression of IL-6 mRNA was observed by Northern blot, and the secretion of IL-6 was reduced to 250 pg/ml. But there were no significant growth-inhibitory effects on GRC-1 cells. Although GRC-1 could express IL-6, IL-6 R and gp130, the rhIL-6 and IL-6 neutralizing antibody or transducing antisense IL-6 RNA could not change the growth of GRC-1 cells significantly. These results suggest that it is not likely for IL-6 functioning as an autocrine growth factor for GRC-1.

  18. Derivation and characterization of matched cell lines from primary and recurrent serous ovarian cancer

    Directory of Open Access Journals (Sweden)

    Létourneau Isabelle J

    2012-08-01

    Full Text Available Abstract Background Cell line models have proven to be effective tools to investigate a variety of ovarian cancer features. Due to the limited number of cell lines, particularly of the serous subtype, the heterogeneity of the disease, and the lack of cell lines that model disease progression, there is a need to further develop cell line resources available for research. This study describes nine cell lines derived from three ovarian cancer cases that were established at initial diagnosis and at subsequent relapse after chemotherapy. Methods The cell lines from three women diagnosed with high-grade serous ovarian cancer (1369, 2295 and 3133 were derived from solid tumor (TOV and ascites (OV, at specific time points at diagnosis and relapse (R. Primary treatment was a combination of paclitaxel/carboplatin (1369, 3133, or cisplatin/topotecan (2295. Second line treatment included doxorubicin, gemcitabine and topotecan. In addition to molecular characterization (p53, HER2, the cell lines were characterized based on cell growth characteristics including spheroid growth, migration potential, and anchorage independence. The in vivo tumorigenicity potential of the cell lines was measured. Response to paclitaxel and carboplatin was assessed using a clonogenic assay. Results All cell lines had either a nonsense or missense TP53 mutations. The ability to form compact spheroids or aggregates was observed in six of nine cell lines. Limited ability for migration and anchorage independence was observed. The OV3133(R cell line, formed tumors at subcutaneous sites in SCID mice. Based on IC50 values and dose response curves, there was clear evidence of acquired resistance to carboplatin for TOV2295(R and OV2295(R2 cell lines. Conclusion The study identified nine new high-grade serous ovarian cancer cell lines, derived before and after chemotherapy that provides a unique resource for investigating the evolution of this common histopathological subtype of ovarian

  19. In vitro comparative cytotoxic effect of Nimbolide: A limonoid from Azadirachta indica (Neem tree) on cancer cell lines and normal cell lines through MTT assay.

    Science.gov (United States)

    Kashif, Muhammad; Hwang, Yawon; Hong, Gyeongmi; Kim, Gonhyung

    2017-05-01

    The present study was conducted to find the cytotoxicity in vitro of nimbolide, limonoids derivative of flowers and leaves from Azadirachta indica (neem tree) on the selected cell lines of cancer (Du-145, PC-3, A-549) and normal fibroblast cell lines (NIH3T3, CCD-18Co) using MTT assay. The cells were seeded in 96 multi-well tissue plate using different concentrations of nimbolide for 24hrs and 48hrs. The percentage of viability of cell lines was calculated by optical density obtained by micro plate reader and cytotoxic effect in term of IC50 value was determined by using linear regression analysis. The percentages of viability of cells treated with different concentrations of nimbolide were significantly lower (P0.05) between treated and the non-treated cells was observed. Nimbolide exerted time and dose dependent cytotoxic effect on the cancer lines and mild effect on the normal cell lines. It was further confirmed through PKH 26. Results of the present study suggested nimbolide as a potent chemotherapeutic and chemopreventive agent as it exerted a more cytotoxic effect on cancer cell lines as compared with the normal cell lines. Nimbolide may be a new hope as an anticancer drug in future.

  20. Subclonal Genomic Architectures of Primary and Metastatic Colorectal Cancer Based on Intratumoral Genetic Heterogeneity.

    Science.gov (United States)

    Kim, Tae-Min; Jung, Seung-Hyun; An, Chang Hyeok; Lee, Sung Hak; Baek, In-Pyo; Kim, Min Sung; Park, Sung-Won; Rhee, Je-Keun; Lee, Sug-Hyung; Chung, Yeun-Jun

    2015-10-01

    The intratumoral heterogeneity (ITH) and the evolution of genomic architectures associated with the development of distant metastases are not well understood in colorectal cancers. We performed multiregion biopsies of primary and liver metastatic regions from five colorectal cancers with whole-exome sequencing and copy number profiling. In addition to a substantial level of genetic ITH, multiregion genetic profiling identifies the subclonal mutational architecture, leading to the region-based or spatial categorization of somatic mutations and the inference of intratumoral evolutionary history of cancers. The universal mutations (those observed in all the regional biopsies) are enriched in known cancer genes such as APC and TP53 with distinct mutational spectra compared with biopsy- or region-specific mutations, suggesting that major operative mutational mechanisms and their selective pressures are not constant across the metastatic progression. The phylogenies inferred from genomic data show branching evolutionary patterns where some primary biopsies are often segregated with metastastic lesions. Our analyses also revealed that copy number changes such as the chromosomal gains of c-MYC and chromothripsis can be region specific and the potential source of genetic ITH. Our data show that the genetic ITH is prevalent in colorectal cancer serving as a potential driving force to generate metastasis-initiating clones and also as a means to infer the intratumoral evolutionary history of cancers. The paucity of recurrent metastasis-clonal events suggests that colorectal cancer distant metastases may not follow a uniform course of genomic evolution, which should be considered in the genetic diagnosis and the selection of therapeutic targets for the advanced colorectal cancer. ©2015 American Association for Cancer Research.

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

    Science.gov (United States)

    Damstrup, L; Rude Voldborg, B; Spang-Thomsen, M; Brünner, N; Skovgaard Poulsen, H

    1998-09-01

    Formation of metastasis is a multistep process involving attachment to the basement membrane, local proteolysis and migration into surrounding tissues, lymph or bloodstream. In the present study, we have analysed the correlation between in vitro invasion and presence of the epidermal growth factor receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16% of the cells added to the upper chamber were able to traverse the Matrigel membrane. Expression of several matrix metalloproteases (MMP), of tissue inhibitor of MMP (TIMP) and of cathepsin B was evaluated by immunoprecipitation, Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition of this antibody resulted in a significant reduction of the in vitro invasion in three selected EGFR-positive cell lines. Our results show that only EGFR-positive SCLC cell lines had the in vitro invasive phenotype, and it is therefore suggested that the EGFR might play an important role for the invasion potential of SCLC cell lines.

  2. Single-cell states in the estrogen response of breast cancer cell lines.

    Science.gov (United States)

    Casale, Francesco Paolo; Giurato, Giorgio; Nassa, Giovanni; Armond, Jonathan W; Oates, Chris J; Corá, Davide; Gamba, Andrea; Mukherjee, Sach; Weisz, Alessandro; Nicodemi, Mario

    2014-01-01

    Estrogen responsive breast cancer cell lines have been extensively studied to characterize transcriptional patterns in hormone-responsive tumors. Nevertheless, due to current technological limitations, genome-wide studies have typically been limited to population averaged data. Here we obtain, for the first time, a characterization at the single-cell level of the states and expression signatures of a hormone-starved MCF-7 cell system responding to estrogen. To do so, we employ a recently proposed model that allows for dissecting single-cell states from time-course microarray data. We show that within 32 hours following stimulation, MCF-7 cells traverse, most likely, six states, with a faster early response followed by a progressive deceleration. We also derive the genome-wide transcriptional profiles of such single-cell states and their functional characterization. Our results support a scenario where estrogen promotes cell cycle progression by controlling multiple, sequential regulatory steps, whose single-cell events are here identified.

  3. Establishment of the mesodermal cell line QCE-6. A model system for cardiac cell differentiation.

    Science.gov (United States)

    Eisenberg, C A; Bader, D M

    1996-02-01

    The QCE-6 cell line was derived from precardiac mesoderm of the Japanese quail. As previously reported, these cells are able to differentiate into two distinct cardiac cell types with myocardial or endocardial endothelial cell properties. This present communication describes in detail the derivation of this cell line and further characterizes the nontreated and induced myocardial and endothelial phenotypes of these cells. The QCE-6 cells exhibit an epithelial morphology, as well as the pattern of protein expression, that is characteristic of precardiac mesoderm. Treatment with retinoic acid, basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-beta 2, and TGF-beta 3 induces these cells to differentiate and produce mixed cultures of epithelial and mesenchymal cells. The epithelial cells express myosin, desmin, and cardiac troponin I in a punctate pattern throughout the cytoplasm. These sarcomeric proteins become organized in a premyofibrillar pattern when TGF-beta 1, platelet-derived growth factor (PDGF)-BB, and insulin-like growth factor (IGF) II are added in combination along with retinoic acid, bFGF, TGF-beta 2, and TGF-beta 3. Also, these treatments induce Na+,K(+)-ATPase expression. When the QCE-6 cells are cultured on collagen type I, the mesenchymal cells that are promoted by retinoic acid, bFGF, TGF-beta 2, and TGF-beta 3 will invade the gel. These mesenchymal cells are positive for QH1 and JB3, which are both markers for presumptive endocardial cells within the early cardiogenic mesoderm. The addition of both PDGF-BB and IGF II to QCE-6 cell cultures will inhibit the ability of retinoic acid, bFGF, TGF-beta 2, and TGF-beta 3 to induce both the mesenchymal morphology and QH1 and JB3 expression. Collectively, these results suggest that the proces of cardiac cell differentiation is regulated by multiple signals and that early cardiogenic mesoderm contains a bipotential stem cell that can give rise to both the myocardial and endocardial

  4. Germline transmission of a novel rat embryonic stem cell line derived from transgenic rats.

    Science.gov (United States)

    Men, Hongsheng; Bauer, Beth A; Bryda, Elizabeth C

    2012-09-20

    Germline-competent rat embryonic stem (ES) cell lines are important resources for the creation of mutant rat models using ES-cell-based gene targeting technology. The ability to isolate germline-competent ES cell lines from any rat strain, including genetically modified strains, would allow for more sophisticated genetic manipulations without extensive breeding. Sprague Dawley (SD) males carrying an enhanced green fluorescent protein (EGFP) transgene were used as the founder animals for the derivation of ES cell lines. A number of ES cell lines were established and subjected to rigorous quality control testing that included assessment of pluripotency factor expression, karyotype analysis, and pathogen/sterility testing. Two male ES cell lines, SD-Tg.EC1/Rrrc and SD-Tg.EC8/Rrrc, were injected into blastocysts recovered from a cross of Dark Agouti (DA) males with SD females. Resulting chimeric animals were bred with wild-type SD mates to verify the germline transmissibility of the ES cell lines by identifying pups carrying the ES cell line-derived EGFP transgene. While both ES cell lines gave rise to chimeric animals, only SD-Tg.EC1 was germline competent. This confirms the feasibility of deriving germline-competent ES cell lines from transgenic rat strains and provides a novel ES cell line with a stable green fluorescent protein (GFP) reporter for future genetic manipulations to create new rat models.

  5. Heterogeneity in sarcoma cell lines reveals enhanced motility of tetraploid versus diploid cells

    Science.gov (United States)

    Jemaà, Mohamed; Abdallah, Samer; Lledo, Gwendaline; Perrot, Gaelle; Lesluyes, Tom; Teyssier, Catherine; Roux, Pierre; van Dijk, Juliette; Chibon, Frederic; Abrieu, Ariane; Morin, Nathalie

    2017-01-01

    Soft tissue sarcomas with complex genomics are very heterogeneous tumors lacking simple prognosis markers or targeted therapies. Overexpression of a subset of mitotic genes from a signature called CINSARC is of bad prognosis, but the significance of this signature remains elusive. Here we precisely measure the cell cycle and mitosis duration of sarcoma cell lines and we found that the mitotic gene products overexpression does not reflect variation in the time spent during mitosis or G2/M. We also found that the CINSARC cell lines, we studied, are composed of a mixture of aneuploid, diploid, and tetraploid cells that are highly motile in vitro. After sorting diploid and tetraploid cells, we showed that the tetraploid cell clones do not possess a proliferative advantage, but are strikingly more motile and invasive than their diploid counterparts. This is correlated with higher levels of mitotic proteins overexpression. Owing that mitotic proteins are almost systematically degraded at the end of mitosis, we propose that it is the abnormal activity of the mitotic proteins during interphase that boosts the sarcoma cells migratory properties by affecting their cytoskeleton. To test this hypothesis, we designed a screen for mitotic or cytoskeleton protein inhibitors affecting the sarcoma cell migration potential independently of cytotoxic activities. We found that inhibition of several mitotic kinases drastically impairs the CINSARC cell invasive and migratory properties. This finding could provide a handle by which to selectively inhibit the most invasive cells. PMID:28035071

  6. Alginate-matrigel microencapsulated schwann cells for inducible secretion of glial cell line derived neurotrophic factor.

    Science.gov (United States)

    de Guzman, Roche C; Ereifej, Evon S; Broadrick, Kristy M; Rogers, Richard A; VandeVord, Pamela J

    2008-10-01

    Controlled expression of glial cell line derived neurotrophic factor (Gdnf) can be integrated in the development of a system for repair of injured peripheral nerves. This delivery strategy was demonstrated via inducible Gdnf from microencapsulated cells in barium alginate. The Schwann cell line RT4-D6P2T was initially modified utilizing an ecdysone-based stable transfection system to produce RT4-Gdnf cells. During construct preparation, it was found that C6 cells (where Gdnf cDNA was isolated) make three Gdnf transcript variants. Additionally, the importance of 5' untranslated region to drive biologically-functional Gdnf synthesis was shown. Encapsulation of RT4-Gdnf in 1% alginate was then performed. It was determined that cells were able to survive at least 1 month in vitro using starting densities of 20, 200 and 2000 cells/capsule and barium ion concentrations of 10, 50, 100 and 200 mM. Most importantly, encapsulated cells secreted exogenous Gdnf upon ponasterone A induction. Mixture of basement membrane extract Matrigel to alginate promoted increased proliferation, cell spreading and Gdnf release. Finally, compression tests showed that cell-loaded microcapsules fractured at 75% diameter compression with 38 kPa of stress. Regulated Gdnf release from these microcapsules in vivo may potentially aid in the regeneration of damaged nerves.

  7. Side Population Cells From an Immortalized Human Liver Epithelial Cell Line Exhibit Hepatic Stem-Like Cell Properties.

    Science.gov (United States)

    Tokiwa, Takayoshi; Yamazaki, Taisuke; Enosawa, Shin

    2012-01-01

    The existence of hepatic stem cells in human livers is controversial. We investigated whether the side population (SP) cells derived from an immortalized human liver epithelial cell line THLE-5b possess the properties of hepatic stem-like cells. SP cells derived from THLE-5b were isolated using flow cytometry and were assayed for the expression of phenotypic markers by reverse transcription polymerase chain reaction and immunostaining. THLE-5b SP cells retained the capacity to generate both SP and non-SP cells, showed a capacity for self-renewal, and were more efficient in colony formation than non-SP cells. Neither the SP nor the non-SP cells formed tumors when transplanted into athymic nude mice or severe combined immunodeficient mice. The expression level of stem cell-associated markers such as an ATP-binding cassette membrane transporter, epithelial cell adhesion molecule, c-kit, Thy-1, and octomer binding transcription factor 4 was higher in SP cells than in non-SP cells. When cultivated as rotation-mediated aggregates, the expression of liver-specific genes including tryptophan oxygenase and CYP3A4 was up-regulated in SP cells, suggesting that THLE-5b SP cells have the ability to differentiate into a hepatocyte phenotype. One of the clonal cell lines derived from the SP cells expressed stem cell-associated markers. These results indicate that SP cells derived from THLE-5b possess hepatic stem-like cell properties and suggest that THLE-5b can be used as a model of normal human liver progenitor or stem cell line.

  8. 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.

  9. Molecular mechanism of action of opioids in human neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Yu, V.C.K.

    1987-01-01

    A series of human neuroblastoma cell lines was screened for the presence of opioid receptor sites. Of these cell lines, SK-N-SH was found to express approximately 50,000 ..mu.. and 10,000 delta opioid receptor sites/cell. In vitro characterization revealed that the binding properties of these receptor sites closely resembled those of human and rodent brain. Phosphatidylinositol turnover as a potential second messenger system for the ..mu.. receptor was examined in SK-N-SH cells. Neurotransmitter receptor systems were determined in the three sub-clones of SK-N-SH cells. Cells of the SH-SY5Y line, a phenotypically stable subclone of SK-N-SH cells, were induced to differentiate by treatment with various inducing agents, and changes of several neurotransmitter receptor systems were determined. Nerve growth factor (NGF) and retinoic acid (RA) up-regulated, while dBcAMP down-regulated opioid receptor sites. (/sup 3/H)Dopamine uptake was slightly enhanced only in RA-treated cells. Strikingly, the efficacy of PGE/sub 1/-stimulated accumulation of cAMP was enhanced by 15- to 30-fold upon RA treatment.

  10. Histamine as a Radiosensitizer of Malignant Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-07-01

    It has been established that the treatment with Histamine (Hi) produces a significant growth inhibition of different cell lines derived from human neoplasia. In a model of Knockout mice completely depleted of endogenous Hi, it was observed a significant delay in bone marroe repopulation after whole body irradiation. These results are in agreement with the hypothesis that histamine has a role in the regulation of haematopoiesis as well as an inhibitory effect on apoptosis. The objective of this paper was to study the possible effect of Hi as protector of normal cells and radiosensitizer of malignant ones. To study the effect of Hi on small-intestine and bone marrow, thirty made mice were randomly separeted into two groups: Control irradiated (C), and irradiated receiving Histamine (HI-group). All animals received a single dose of 10 Gy on whole-body employing a ''137Cs source of 189 TB{sub q} (Dose rate: 7.7 Gy/min) calibrated with TLD 700 dosimeter. Hi-group recieved a daily se injection (0.1 mg/kg) starting 20 hs before irradiation. Mice were sacrificed 5 days after irradiation. Histopathological analysis indicated that intestinal mucosae of C group showed important injury, whist mucosae of Hi-treated mice showed mild mucosal atrophy with conservation of villous projections and absence of vascular congestive changes. In order to investigate the effect of Hi on radiosensitivity of transformed cells, MDA-MB-231 (human breast carcinoma cells) were irradiated in vitro with doses ranging from 0 to 10 Gy. Results of radiobiological parameters indicate a significant increase on radiosensitivity of malignant cells. Employing specific fluorescent dyes and flow cytometric analysis we determined that the intracellular levels of hydrogen peroxide (H{sub 2}O{sub 2}) are significant increased by Hi 10 {mu}M in control and also in irradiated MDA-MB-231 cells, while the levels of superoxide (SO{sub 2}) were not significantly modified by Hi-treatment. (Author) 9 refs.

  11. Cytotoxicity screening of essential oils in cancer cell lines

    Directory of Open Access Journals (Sweden)

    Pollyanna Francielli de Oliveira

    Full Text Available Abstract This study evaluated the cytotoxicity activity of the essential oils of Tagetes erecta L., Asteraceae (TE-OE, Tetradenia riparia (Hochst. Codd, Lamiaceae (TR-OE, Bidens sulphurea (Cav. Sch. Bip., Asteraceae (BS-OE, and Foeniculum vulgare Mill., Apiaceae (FV-OE, traditionally used in folk medicine, against the tumor cell lines murine melanoma (B16F10, human colon carcinoma (HT29, human breast adenocarcinoma (MCF-7, human cervical adenocarcinoma (HeLa, human hepatocellular liver carcinoma (HepG2, and human glioblastoma (MO59J, U343, and U251. Normal hamster lung fibroblasts (V79 cells were included as control. The cells were treated with essential oil concentrations ranging from 3.12 to 400 µg/ml for 24 h. The cytotoxic activity was evaluated using the XTT assay; results were expressed as IC50, and the selectivity index was calculated. The results were compared with those achieved for classic chemotherapeutic agents. TE-OE was the most promising among the evaluated oils: it afforded the lowest IC50 values for B16F10 cells (7.47 ± 1.08 µg/ml and HT29 cells (6.93 ± 0.77 µg/ml, as well as selectivity indices of 2.61 and 2.81, respectively. The major BS-EO, FV-EO and TE-EO chemical constituents were identified by gas chromatography mass spectrometry as being (E-caryophyllene (10.5%, germacrene D (35.0% and 2,6-di-tert-butyl-4-methylphenol (43.0% (BS-EO; limonene (21.3% and (E-anethole (70.2% (FV-EO; limonene (10.4%, dihydrotagetone (11.8%, α-terpinolene (18.1% and (E-ocimenone (13.0% (TE-EO; and fenchone (6.1%, dronabinol (11.0%, aromadendrene oxide (14.7% and (E,E–farnesol (15.0% (TR-EO. 2,6-di-tert-butyl-4-methylphenol (43.0%, (E-anethole (70.2% and α-terpinolene (18.1%, respectively. These results suggest that TE-OE may be used to treat cancer without affecting normal cells.

  12. Biologic characteristics of the side population of human small cell lung cancer cell line H446.

    Science.gov (United States)

    Wang, Bo; Yang, Huan; Huang, Yu-Zheng; Yan, Ru-Hong; Liu, Fen-Ju; Zhang, Jun-Ning

    2010-03-01

    Recently, the theory of cancer stem cells (CSCs) has presented new targets and orientations for tumor therapy. The major difficulties in researching CSCs include their isolation and purification. The aim of this study is to identify and characterize the side population (SP) cells in small cell lung cancer (SCLC) cell line H446, which lays the foundation for the isolation and purification of CSCs. Fluorescence-activated cell sorting (FACS) was used to sort SP and non-SP (NSP) cells from H446. Both subgroups were cultivated to survey the capacity to form into suspended tumor cell spheres. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR were used to evaluate the expression levels of the mRNA of CD133, ABCG2, and nucleostemin in both subgroups. The capacity of proliferation and the differences in drug resistance of both subgroups and unsorted cells were tested by the MTT method. The differentiation ability of both subgroups was determined by FACS. Proliferation was determined by subcutaneous tumor formation in nude mice. The percent of Hoechst 33342 negative cells was about (5.1 +/- 0.2)% in H446 by fluorescence microscopy. The percent of SP cells was (6.3 +/- 0.1)% by flow cytometry. SP cells had a stronger capability of forming into tumor spheres than NSP cells. The mRNA expression levels of ABCG2, CD133, and nucleostemin in SP cells were 21.60 +/- 0.26, 7.10 +/- 0.14, and 1.02 +/- 0.08 folds higher than that in NSP cells (P 0.05, respectively). In vivo, SP cells showed better proliferative ability and tougher viability when treated with drugs. SP cells can differentiate into NSP cells, but NSP cells cannot differentiate into SP cells. SP cells had a greater ability to form tumors. The H446 cell line contained some SP cells with stem cell properties. CD133 and ABCG2 may be cancer stem cell markers of SCLC.

  13. Oxytocin stimulates cell proliferation in vaginal cell line Vk2E6E7.

    Science.gov (United States)

    Kallak, Theodora K; Uvnäs-Moberg, Kerstin

    2017-03-01

    Objective During and after menopause, the symptoms of vaginal atrophy cause great discomfort and necessitate effective treatment options. Currently, vaginally applied oxytocin is being investigated as a treatment for the symptoms of vaginal atrophy in postmenopausal women. To clarify the mechanisms behind oxytocins effects on vaginal atrophy, the present study investigated the effects of oxytocin on cell proliferation in the cells of the Vk2E6E7 line, a non-tumour vaginal cell line. The study also compared the effects of oxytocin with those of estradiol (E2). Study design The effects of both oxytocin and E2 on the proliferation of Vk2E6E7 cells were investigated using Cell Proliferation ELISA BrdU Colorimetric Assay. The expression of both oxytocin and oxytocin receptor was studied in Vk2E6E7 cells using quantitative real-time polymerase chain reaction and immunofluorescent staining. Main outcome measures Cell proliferation and gene expression. Results Oxytocin increased cell proliferation both time dependently and dose dependently. This differed from the effect pattern observed in cells treated with E2. In addition, in oxytocin-treated cells, the oxytocin receptor was found to be co-localized with caveolin-1, indicating pro-proliferative signalling within the cell. Conclusions Oxytocin stimulates cell proliferation and the co-localization of oxytocin receptor with caveolin-1 in oxytocin-treated cells, supporting the role of oxytocin signalling in cell proliferation. In addition, these findings suggest that increased cell proliferation is one mechanism by which local vaginal oxytocin treatment increases vaginal thickness and relieves vaginal symptoms in postmenopausal women with vaginal atrophy.

  14. LINES

    Directory of Open Access Journals (Sweden)

    Minas Bakalchev

    2015-10-01

    Full Text Available The perception of elements in a system often creates their interdependence, interconditionality, and suppression. The lines from a basic geometrical element have become the model of a reductive world based on isolation according to certain criteria such as function, structure, and social organization. Their traces are experienced in the contemporary world as fragments or ruins of a system of domination of an assumed hierarchical unity. How can one release oneself from such dependence or determinism? How can the lines become less “systematic” and forms more autonomous, and less reductive? How is a form released from modernistic determinism on the new controversial ground? How can these elements or forms of representation become forms of action in the present complex world? In this paper, the meaning of lines through the ideas of Le Corbusier, Leonidov, Picasso, and Hitchcock is presented. Spatial research was made through a series of examples arising from the projects of the architectural studio “Residential Transformations”, which was a backbone for mapping the possibilities ranging from playfulness to exactness, as tactics of transformation in the different contexts of the contemporary world.

  15. Opioid binding site in EL-4 thymoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Fiorica, E.; Spector, S.

    1988-01-01

    Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of (/sup 3/H) bremazocine indicated a single site with a K/sub D/ = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10/sup 6/ cells. To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of (/sup 3/H) bremazocine with an IC/sub 50/ value = 0.57..mu..M. The two steroisomers levorphanol and dextrorphan showed the same affinity for this site. While morphine, (D-Pen/sup 2/, D-Pen/sup 5/) enkephalin and ..beta..-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC/sub 50/ = 60..mu..M, that was similar to naloxone. 32 references, 3 figures, 2 tables.

  16. 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.

  17. Evaluation of Stem Cell Markers, CD44/CD24 in Breast Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Masoud Hashemi Arabi

    2014-05-01

    Four breast cancer cell lines, MCF-7 ، T47D ، MDA-MB231 and MDA-MB468 were purchased from National cell Bank of Iran based in Iran Pasture Institute and were cultured in high glucose DMEM supplemented with 10% FCS. Cells were stained with antiCD44-PE and antiCD24-FITC antibodies and Status of CD44 and CD24 as markers of breast cancer stem cells were evaluated using flow cytometer and fluorescent microscopy.Evaluation of CD44 and CD24 as markers of breast cancer stem cells showed that MDA-MB231 with 97±1.2% CD44+/CD24-/low cells is significantly different from the others that they were mainly CD44 and CD24 positive cells(p

  18. 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.

  19. Comparison of betanodavirus replication efficiency in ten Indian fish cell lines.

    Science.gov (United States)

    Sarath Babu, V; Abdul Majeed, S; Nambi, K S N; Taju, G; Madan, N; Sundar Raj, N; Sahul Hameed, A S

    2013-06-01

    Ten cell lines established from Indian marine, brackishwater and freshwater fish were tested for their susceptibility to fish nodavirus. In addition, the efficiency of betanodavirus replication was tested in these cell lines. Multiple vacuolation, a typical cytopathic effect for virus infection, was observed in infected SISK, SISS, SIGE and ICF cells. Infection of the different fish cell lines was confirmed by RT-PCR, immunodot blot assay and indirect ELISA. The virus concentration in culture supernatant collected from infected sea bass and grouper cell lines increased progressively from 10(3) at day 1 postinfection to 10(8) TCID50 ml(-1) at day 9. The amount of virus in different cell lines was also quantified by real-time PCR. These results indicate the suitability of the SISK, SISS, and SIGE cell lines for fish nodavirus propagation for developing viral diagnostics and vaccines.

  20. Cross-contamination of a UROtsa stock with T24 cells--molecular comparison of different cell lines and stocks.

    Science.gov (United States)

    Johnen, Georg; Rozynek, Peter; von der Gathen, Yvonne; Bryk, Oleksandr; Zdrenka, Ricarda; Johannes, Christian; Weber, Daniel G; Igwilo-Okuefuna, O Brien; Raiko, Irina; Hippler, Jörg; Brüning, Thomas; Dopp, Elke

    2013-01-01

    UROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity. UROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR) profiling was applied. All tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly. It is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments.

  1. Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y.

    Science.gov (United States)

    Attoff, K; Kertika, D; Lundqvist, J; Oredsson, S; Forsby, A

    2016-09-01

    Acrylamide is a well-known neurotoxic compound and people get exposed to the compound by food consumption and environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed resulting in a risk for developmental neurotoxicity. In this study, the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as alerting indicators for developmental neurotoxicity. For both cell lines, acrylamide reduced the number of viable cells by reducing proliferation and inducing cell death in undifferentiated cells. Acrylamide concentrations starting at 10fM attenuated the differentiation process in SH-SY5Y cells by sustaining cell proliferation and neurite outgrowth was reduced at concentrations from 10pM. Acrylamide significantly reduced the number of neurons starting at 1μM and altered the ratio between the different phenotypes in differentiating C17.2 cell cultures. Ten micromolar of acrylamide also reduced the expression of the neuronal and astrocyte biomarkers. Although the neurotoxic concentrations in the femtomolar range seem to be specific for the SH-SY5Y cell line, the fact that micromolar concentrations of acrylamide seem to attenuate the differentiation process in both cell lines raises the interest to further investigations on the possible developmental neurotoxicity of acrylamide. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Subpopulations of stem-like cells in side population cells from the human bladder transitional cell cancer cell line T24.

    Science.gov (United States)

    Ning, Z-F; Huang, Y-J; Lin, T-X; Zhou, Y-X; Jiang, C; Xu, K-W; Huang, H; Yin, X-B; Huang, J

    2009-01-01

    Cancer stem cells can be isolated from human tumours using specific cell surface markers. Bladder cancer cells, however, lack specific cell surface markers, making this approach impracticable. In this study an alternative method was used, involving isolation of side population cells to explore the stem cell characteristics of bladder cancer. Side population cells were isolated from the bladder transitional cell cancer cell line T24 and examined for potential stem cell characteristics related to proliferation, cell cycle distribution, self-renewal and differentiation. It was observed that T24 side population cells have stronger proliferative and colony formation abilities than non-side population cells. Side population cells were also more resistant to chemotherapy and radiotherapy, which may be due to expression of the ATP-binding cassette half-transporter, sub-family G, member 2 protein. Overall, the results suggest that side population cells from the human bladder transitional cell cancer cell line T24 harbour stem-like cells.

  3. HIV integration sites in latently infected cell lines: evidence of ongoing replication.

    Science.gov (United States)

    Symons, Jori; Chopra, Abha; Malatinkova, Eva; De Spiegelaere, Ward; Leary, Shay; Cooper, Don; Abana, Chike O; Rhodes, Ajantha; Rezaei, Simin D; Vandekerckhove, Linos; Mallal, Simon; Lewin, Sharon R; Cameron, Paul U

    2017-01-13

    Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position. We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells. Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.

  4. Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

    Science.gov (United States)

    Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

    2014-01-01

    We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

  5. Imiquimod activates p53-dependent apoptosis in a human basal cell carcinoma cell line.

    Science.gov (United States)

    Huang, Shi-Wei; Chang, Shu-Hao; Mu, Szu-Wei; Jiang, Hsin-Yi; Wang, Sin-Ting; Kao, Jun-Kai; Huang, Jau-Ling; Wu, Chun-Ying; Chen, Yi-Ju; Shieh, Jeng-Jer

    2016-03-01

    The tumor suppressor p53 controls DNA repair, cell cycle, apoptosis, autophagy and numerous other cellular processes. Imiquimod (IMQ), a synthetic toll-like receptor (TLR) 7 ligand for the treatment of superficial basal cell carcinoma (BCC), eliminates cancer cells by activating cell-mediated immunity and directly inducing apoptosis and autophagy in cancer cells. To evaluate the role of p53 in IMQ-induced cell death in skin cancer cells. The expression, phosphorylation and subcellular localization of p53 were detected by real-time PCR, luciferase reporter assay, cycloheximide chase analysis, immunoblotting and immunocytochemistry. Using BCC/KMC1 cell line as a model, the upstream signaling of p53 activation was dissected by over-expression of TLR7/8, the addition of ROS scavenger, ATM/ATR inhibitors and pan-caspase inhibitor. The role of p53 in IMQ-induced apoptosis and autophagy was assessed by genetically silencing p53 and evaluated by a DNA content assay, immunoblotting, LC3 puncta detection and acridine orange staining. IMQ induced p53 mRNA expression and protein accumulation, increased Ser15 phosphorylation, promoted nuclear translocation and up-regulated its target genes in skin cancer cells in a TLR7/8-independent manner. In BCC/KMC1 cells, the induction of p53 by IMQ was achieved through increased ROS production to stimulate the ATM/ATR-Chk1/Chk2 axis but was not mediated by inducing DNA damage. The pharmacological inhibition of ATM/ATR significantly suppressed IMQ-induced p53 activation and apoptosis. Silencing of p53 significantly decreased the IMQ-induced caspase cascade activation and apoptosis but enhanced autophagy. Mutant p53 skin cancer cell lines were more resistant to IMQ-induced apoptosis than wildtype p53 skin cancer cell lines. IMQ induced ROS production to stimulate ATM/ATR pathways and contributed to p53-dependent apoptosis in a skin basal cell carcinoma cell line BCC/KMC1. Copyright © 2015 Japanese Society for Investigative Dermatology

  6. [MYETS1 recombinant expression in prokaryotic cells and deletion analysis in multiple myeloma cell lines].

    Science.gov (United States)

    Wang, Jianjun; Hong, Liping; Pan, Yi; Liu, Shuiping; Wu, Kunlu; Tang, Lijun

    2012-01-01

    To explore the down-expression mechanism of MYETS1 gene in multiple myeloma cell lines ARH-77 or KM3, and express MYETS1 gene in prokaryotic express system. The region of chromosome 13q14.3 in ARH-77 and KM3 was detected by FISH. MYETS1 gene was amplified by RT-PCR and cloned into prokaryotic expression vector pGEX-4T. Positive consequence was acquired in 13q14.3 where MYETS1 located by FISH in ARH- 77 and KM3 cell lines. Bioinformatics indicated highly sequence homology between MYETS1 and LECT1, but excluded the homology of open reading frame between MYETS1 and that of LECT1 by RT-PCR. Myets1 protein was expressed and harvested successfully. The region of chromosome 13q14.3 ,where MYETS1 gene located, was not defected in ARH-77 and KM3 cell lines. Down-expression of MYETS1 might be regulated by other mechanisms in multiple myeloma cell lines.

  7. 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.

  8. Effect of arginase II on L-arginine depletion and cell growth in murine cell lines of renal cell carcinoma

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    Patterson John R

    2008-09-01

    Full Text Available Abstract Background L-arginine is the common substrate for the two isoforms of arginase. Arginase I, highly expressed in the liver and arginase II mainly expressed in the kidney. Arginase I-producing myeloid derived suppressor cells have been shown to inhibit T-cell function by the depletion of L-arginine. On the other hand, arginase II has been detected in patients with cancer and is thought to metabolize L-arginine to L-ornithine needed to sustain rapid tumor growth; however its role in L-arginine depletion is unclear. Thus, in tumor biology, L-arginine metabolism may play a dual role in tumor growth and in the induction of T cell dysfunction. Therefore, we studied in murine renal cell carcinoma (RCC cell lines, the effect of arginase II on tumor cell proliferation and L-arginine depletion. The effect of arginase inhibitors on cell proliferation was also tested. Methods Three murine renal cell carcinoma (mRCC cell lines were tested for the presence of arginase. nor-NOHA, an arginase inhibitor was used to substantiate the effect of arginase on cell growth and L-arginine depletion. Amino acid levels were tested by HPLC. Results Our results show that mRCC cell lines express only arginase II and were able to deplete L-arginine from the medium. Cell growth was independent of the amount of arginase activity expressed by the cells. nor-NOHA significantly (P = 0.01 reduced arginase II activity and suppressed cell growth in cells exhibiting high arginase activity. The depletion of L-arginine by mRCC induced the decrease expression of CD3ζ a key element for T-cell function. Conclusion The results of this study show for the first time that arginase II produced by RCC cell lines depletes L-arginine resulting in decreased expression of CD3ζ. These results indicate that RCC cell lines expressing arginase II can modulate the L-arginine metabolic pathway to regulate both cell growth and T-cell function. Blocking arginase may lead to a decrease in RCC cell

  9. Prediction of epigenetically regulated genes in breast cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Loss, Leandro A; Sadanandam, Anguraj; Durinck, Steffen; Nautiyal, Shivani; Flaucher, Diane; Carlton, Victoria EH; Moorhead, Martin; Lu, Yontao; Gray, Joe W; Faham, Malek; Spellman, Paul; Parvin, Bahram

    2010-05-04

    Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP). The pipeline (i) reduces the dimensionality of the methylation data, (ii) associates the reduced methylation data with gene expression data, and (iii) ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i) methylation sites are grouped across the genome to identify regions of interest, and (ii) methylation profles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fxed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically signifcant negative correlation between methylation profles and gene expression in the

  10. Prediction of epigenetically regulated genes in breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Lu Yontao

    2010-06-01

    Full Text Available Abstract Background Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP. The pipeline (i reduces the dimensionality of the methylation data, (ii associates the reduced methylation data with gene expression data, and (iii ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i methylation sites are grouped across the genome to identify regions of interest, and (ii methylation profles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fxed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. Results Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically signifcant negative correlation between

  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. Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.

    NARCIS (Netherlands)

    Rops, A.L.; Vlag, J. van der; Jacobs, C.W.M.; Dijkman, H.B.P.M.; Lensen, J.F.M.; Wijnhoven, T.J.M.; Heuvel, L.P.W.J. van den; Kuppevelt, A.H.M.S.M. van; Berden, J.H.M.

    2004-01-01

    BACKGROUND: The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has

  14. Genomic Landscape of Primary Mediastinal B-Cell Lymphoma Cell Lines.

    Science.gov (United States)

    Dai, Haiping; Ehrentraut, Stefan; Nagel, Stefan; Eberth, Sonja; Pommerenke, Claudia; Dirks, Wilhelm G; Geffers, Robert; Kalavalapalli, Srilaxmi; Kaufmann, Maren; Meyer, Corrina; Faehnrich, Silke; Chen, Suning; Drexler, Hans G; MacLeod, Roderick A F

    2015-01-01

    Primary mediastinal B-Cell lymphoma (PMBL) is a recently defined entity comprising ~2-10% non-Hodgkin lymphomas (NHL). Unlike most NHL subtypes, PMBL lacks recurrent gene rearrangements to serve as biomarkers or betray target genes. While druggable, late chemotherapeutic complications warrant the search for new targets and models. Well characterized tumor cell lines provide unlimited material to serve as preclinical resources for verifiable analyses directed at the discovery of new biomarkers and pathological targets using high throughput microarray technologies. The same cells may then be used to seek intelligent therapies directed at clinically validated targets. Four cell lines have emerged as potential PMBL models: FARAGE, KARPAS-1106P, MEDB-1 and U-2940. Transcriptionally, PMBL cell lines cluster near c(lassical)-HL and B-NHL examples showing they are related but separate entities. Here we document genomic alterations therein, by cytogenetics and high density oligonucleotide/SNP microarrays and parse their impact by integrated global expression profiling. PMBL cell lines were distinguished by moderate chromosome rearrangement levels undercutting cHL, while lacking oncogene translocations seen in B-NHL. In total 61 deletions were shared by two or more cell lines, together with 12 amplifications (≥4x) and 72 homozygous regions. Integrated genomic and transcriptional profiling showed deletions to be the most important class of chromosome rearrangement. Lesions were mapped to several loci associated with PMBL, e.g. 2p15 (REL/COMMD1), 9p24 (JAK2, CD274), 16p13 (SOCS1, LITAF, CIITA); plus new or tenuously associated loci: 2p16 (MSH6), 6q23 (TNFAIP3), 9p22 (CDKN2A/B), 20p12 (PTPN1). Discrete homozygous regions sometimes substituted focal deletions accompanied by gene silencing implying a role for epigenetic or mutational inactivation. Genomic amplifications increasing gene expression or gene-activating rearrangements were respectively rare or absent. Our findings

  15. Genomic Landscape of Primary Mediastinal B-Cell Lymphoma Cell Lines.

    Directory of Open Access Journals (Sweden)

    Haiping Dai

    Full Text Available Primary mediastinal B-Cell lymphoma (PMBL is a recently defined entity comprising ~2-10% non-Hodgkin lymphomas (NHL. Unlike most NHL subtypes, PMBL lacks recurrent gene rearrangements to serve as biomarkers or betray target genes. While druggable, late chemotherapeutic complications warrant the search for new targets and models. Well characterized tumor cell lines provide unlimited material to serve as preclinical resources for verifiable analyses directed at the discovery of new biomarkers and pathological targets using high throughput microarray technologies. The same cells may then be used to seek intelligent therapies directed at clinically validated targets. Four cell lines have emerged as potential PMBL models: FARAGE, KARPAS-1106P, MEDB-1 and U-2940. Transcriptionally, PMBL cell lines cluster near c(lassical-HL and B-NHL examples showing they are related but separate entities. Here we document genomic alterations therein, by cytogenetics and high density oligonucleotide/SNP microarrays and parse their impact by integrated global expression profiling. PMBL cell lines were distinguished by moderate chromosome rearrangement levels undercutting cHL, while lacking oncogene translocations seen in B-NHL. In total 61 deletions were shared by two or more cell lines, together with 12 amplifications (≥4x and 72 homozygous regions. Integrated genomic and transcriptional profiling showed deletions to be the most important class of chromosome rearrangement. Lesions were mapped to several loci associated with PMBL, e.g. 2p15 (REL/COMMD1, 9p24 (JAK2, CD274, 16p13 (SOCS1, LITAF, CIITA; plus new or tenuously associated loci: 2p16 (MSH6, 6q23 (TNFAIP3, 9p22 (CDKN2A/B, 20p12 (PTPN1. Discrete homozygous regions sometimes substituted focal deletions accompanied by gene silencing implying a role for epigenetic or mutational inactivation. Genomic amplifications increasing gene expression or gene-activating rearrangements were respectively rare or absent. Our

  16. Low dose perfluorooctanoate exposure promotes cell proliferation in a human non-tumor liver cell line

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hongxia; Cui, Ruina [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Guo, Xuejiang [State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029 (China); Hu, Jiayue [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Dai, Jiayin, E-mail: daijy@ioz.ac.cn [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China)

    2016-08-05

    Highlights: • Differential expression of proteins induced by PFOA in HL-7702 was identified. • Most of the differentially expressed proteins are related to cell proliferation. • A low dose of PFOA stimulates HL-7702 cell proliferation. • A high dose of PFOA inhibits HL-7702 cell proliferation. - Abstract: Perfluorooctanoate (PFOA) is a well-known persistent organic pollutant widely found in the environment, wildlife and humans. Medical surveillance and experimental studies have investigated the potential effects of PFOA on human livers, but the hepatotoxicity of PFOA on humans and its underlying mechanism remain to be clarified. We exposed a human liver cell line (HL-7702) to 50 μM PFOA for 48 h and 96 h, and identified 111 significantly differentially expressed proteins by iTRAQ analysis. A total of 46 proteins were related to cell proliferation and apoptosis. Through further analysis of the cell cycle, apoptosis and their related proteins, we found that low doses of PFOA (50–100 μM) promoted cell proliferation and numbers by promoting cells from the G1 to S phases, whereas high doses of PFOA (200–400 μM) led to reduced HL-7702 cell numbers compared with that of the control mainly due to cell cycle arrest in the G0/G1 phase. To our knowledge, this is the first report on the promotion of cell cycle progression in human cells following PFOA exposure.

  17. Mucous and ciliated cell metaplasia in epithelial linings of odontogenic inflammatory and developmental cysts.

    Science.gov (United States)

    Takeda, Yasunori; Oikawa, Yuko; Furuya, Izuru; Satoh, Masanobu; Yamamoto, Hirotsugu

    2005-06-01

    The incidence of mucous and ciliated cells in epithelial linings was examined among odontogenic inflammatory cysts (radicular cysts) and developmental cysts (dentigerous and primordial cysts). Mucous cells were found in 20.8% of all cysts examined, while ciliated cells were found in 11.4%; however, ciliated cells were always accompanied by mucous cells. The incidence of mucous cells in radicular cysts and dentigerous cysts and that of ciliated cells in radicular cysts was higher in the maxilla than in the mandible, while the incidence of mucous cells in primordial cysts and that of ciliated cells in dentigerous cysts and primordial cysts was higher in the mandible than in the maxilla. The present results regarding mucous cells and ciliated cells in the epithelial linings of intraosseous odontogenic cysts indicate a metaplasic origin, but the cause and biological significance of this phenomenon is not known. Mucous cells were present in the surface layer of epithelial linings, and intraepithelial gland-like structures lined with mucous cells were observed in the hyperplastic regions of epithelial linings of several radicular and dentigerous cysts. Such gland-like structures lined by mucous cells in the thickened epithelial lining, which have not been demonstrated previously, resembled the glandular structures of "glandular odontogenic cysts".

  18. Derivation of a germline competent transgenic Fischer 344 embryonic stem cell line.

    Directory of Open Access Journals (Sweden)

    Hongsheng Men

    Full Text Available Embryonic stem (ES cell-based gene manipulation is an effective method for the generation of mutant animal models in mice and rats. Availability of germline-competent ES cell lines from inbred rat strains would allow for creation of new genetically modified models in the desired genetic background. Fischer344 (F344 males carrying an enhanced green fluorescence protein (EGFP transgene were used as the founder animals for the derivation of ES cell lines. After establishment of ES cell lines, rigorous quality control testing that included assessment of pluripotency factor expression, karyotype analysis, and pathogen/sterility testing was conducted in selected ES cell lines. One male ES cell line, F344-Tg.EC4011, was further evaluated for germline competence by injection into Dark Agouti (DA X Sprague Dawley (SD blastocysts. Resulting chimeric animals were bred with wild-type SD mates and germline transmissibility of the ES cell line was confirmed by identification of pups carrying the ES cell line-derived EGFP transgene. This is the first report of a germline competent F344 ES cell line. The availability of a new germline competent ES cell line with a stable fluorescence reporter from an inbred transgenic rat strain provides an important new resource for genetic manipulations to create new rat models.

  19. 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.

  20. Bosutinib reduces the efficacy of Dasatinib in triple-negative breast cancer cell lines.

    Science.gov (United States)

    Tarpley, Mike; Abdissa, Temesgen T; Johnson, Gary L; Scott, John E

    2014-04-01

    Triple-negative breast cancer (TNBC) is an aggressive sub-type of breast cancer. Dasatinib and bosutinib are FDA-approved Src/Abl kinase inhibitor drugs. Dasatinib potently inhibits the proliferation of many TNBC cell lines. The cell viability/proliferation for a panel of 4 TNBC cell lines was measured by detection of cellular ATP levels and cell numbers were directly determined by automated cell counting. Bosutinib (≤1 μM) had little to no inhibitory activity on cell viability/proliferation, while dasatinib-alone generated potent IC50 values of bosutinib resulted in reduced efficacy of dasatinib in all four cell lines, with two of them displaying a dramatic loss of efficacy. Direct cell counting confirmed that bosutinib enhanced cell proliferation in the presence of dasatinib. Bosutinib potently reduced the in vitro anti-proliferative efficacy of dasatinib in TNBC cell lines. We, hereby, report on a novel drug-induced loss in dasatinib sensitivity.

  1. Rapid selection and proliferation of CD133+ cells from cancer cell lines: chemotherapeutic implications.

    Directory of Open Access Journals (Sweden)

    Sarah E Kelly

    2010-04-01

    Full Text Available Cancer stem cells (CSCs are considered a subset of the bulk tumor responsible for initiating and maintaining the disease. Several surface cellular markers have been recently used to identify CSCs. Among those is CD133, which is expressed by hematopoietic progenitor cells as well as embryonic stem cells and various cancers. We have recently isolated and cultured CD133 positive [CD133+] cells from various cancer cell lines using a NASA developed Hydrodynamic Focusing Bioreactor (HFB (Celdyne, Houston, TX. For comparison, another bioreactor, the rotary cell culture system (RCCS manufactured by Synthecon (Houston, TX was used. Both the HFB and the RCCS bioreactors simulate aspects of hypogravity. In our study, the HFB increased CD133+ cell growth from various cell lines compared to the RCCS vessel and to normal gravity control. We observed a +15-fold proliferation of the CD133+ cellular fraction with cancer cells that were cultured for 7-days at optimized conditions. The RCCS vessel instead yielded a (-4.8-fold decrease in the CD133+cellular fraction respect to the HFB after 7-days of culture. Interestingly, we also found that the hypogravity environment of the HFB greatly sensitized the CD133+ cancer cells, which are normally resistant to chemo treatment, to become susceptible to various chemotherapeutic agents, paving the way to less toxic and more effective chemotherapeutic treatment in patients. To be able to test the efficacy of cytotoxic agents in vitro prior to their use in clinical setting on cancer cells as well as on cancer stem cells may pave the way to more effective chemotherapeutic strategies in patients. This could be an important advancement in the therapeutic options of oncologic patients, allowing for more targeted and personalized chemotherapy regimens as well as for higher response rates.

  2. CELLULAR BASIS FOR DIFFERENTIAL SENSITIVITY TO CISPLATIN IN HUMAN GERM-CELL TUMOR AND COLON-CARCINOMA CELL-LINES

    NARCIS (Netherlands)

    SARK, MWJ; TIMMERBOSSCHA, H; MEIJER, C; UGES, DRA; SLUITER, WJ; PETERS, WHM; MULDER, NH; DEVRIES, EGE

    Cisplatin (CDDP) resistance mechanisms were studied in a model of three germ cell tumour and three colon carcinoma cell lines representing intrinsically CDDP-sensitive and -resistant tumours respectively. The CDDP sensitivity of the cell lines mimicked the clinical situation. The glutathione levels

  3. Micro-RNA expression in cisplatin resistant germ cell tumor cell lines

    Directory of Open Access Journals (Sweden)

    Meineke Viktor

    2011-05-01

    Full Text Available Abstract Background We compared microRNA expression patterns in three cisplatin resistant sublines derived from paternal cisplatin sensitive germ cell tumor cell lines in order to improve our understanding of the mechanisms of cisplatin resistance. Methods Three cisplatin resistant sublines (NTERA-2-R, NCCIT-R, 2102EP-R showing 2.7-11.3-fold increase in drug resistance after intermittent exposure to increasing doses of cisplatin were compared to their parental counterparts, three well established relatively cisplatin sensitive germ cell tumor cell lines (NTERA-2, NCCIT, 2102EP. Cells were cultured and total RNA was isolated from all 6 cell lines in three independent experiments. RNA was converted into cDNA and quantitative RT-PCR was run using 384 well low density arrays covering almost all (738 known microRNA species of human origin. Results Altogether 72 of 738 (9.8% microRNAs appeared differentially expressed between sensitive and resistant cell line pairs (NTERA-2R/NTERA-2 = 43, NCCIT-R/NCCIT = 53, 2102EP-R/2102EP = 15 of which 46.7-95.3% were up-regulated (NTERA-2R/NTERA-2 = 95.3%, NCCIT-R/NCCIT = 62.3%, 2102EP-R/2102EP = 46.7%. The number of genes showing differential expression in more than one of the cell line pairs was 34 between NTERA-2R/NTERA-2 (79% and NCCIT-R/NCCIT (64%, and 3 and 4, respectively, between these two cell lines and 2102EP-R/2102EP (about 27%. Only the has-miR-10b involved in breast cancer invasion and metastasis and has-miR-512-3p appeared to be up-regulated (2-3-fold in all three cell lines. The hsa-miR-371-373 cluster (counteracting cellular senescence and linked with differentiation potency, as well as hsa-miR-520c/-520h (inhibiting the tumor suppressor p21 were 3.9-16.3 fold up-regulated in two of the three cisplatin resistant cell lines. Several new micro-RNA species missing an annotation towards cisplatin resistance could be identified. These were hsa-miR-512-3p/-515/-517/-518/-525 (up to 8.1-fold up

  4. Micro-RNA expression in cisplatin resistant germ cell tumor cell lines.

    Science.gov (United States)

    Port, Matthias; Glaesener, Stephanie; Ruf, Christian; Riecke, Armin; Bokemeyer, Carsten; Meineke, Viktor; Honecker, Friedemann; Abend, Michael

    2011-05-15

    We compared microRNA expression patterns in three cisplatin resistant sublines derived from paternal cisplatin sensitive germ cell tumor cell lines in order to improve our understanding of the mechanisms of cisplatin resistance. Three cisplatin resistant sublines (NTERA-2-R, NCCIT-R, 2102EP-R) showing 2.7-11.3-fold increase in drug resistance after intermittent exposure to increasing doses of cisplatin were compared to their parental counterparts, three well established relatively cisplatin sensitive germ cell tumor cell lines (NTERA-2, NCCIT, 2102EP). Cells were cultured and total RNA was isolated from all 6 cell lines in three independent experiments. RNA was converted into cDNA and quantitative RT-PCR was run using 384 well low density arrays covering almost all (738) known microRNA species of human origin. Altogether 72 of 738 (9.8%) microRNAs appeared differentially expressed between sensitive and resistant cell line pairs (NTERA-2R/NTERA-2 = 43, NCCIT-R/NCCIT = 53, 2102EP-R/2102EP = 15) of which 46.7-95.3% were up-regulated (NTERA-2R/NTERA-2 = 95.3%, NCCIT-R/NCCIT = 62.3%, 2102EP-R/2102EP = 46.7%). The number of genes showing differential expression in more than one of the cell line pairs was 34 between NTERA-2R/NTERA-2 (79%) and NCCIT-R/NCCIT (64%), and 3 and 4, respectively, between these two cell lines and 2102EP-R/2102EP (about 27%). Only the has-miR-10b involved in breast cancer invasion and metastasis and has-miR-512-3p appeared to be up-regulated (2-3-fold) in all three cell lines. The hsa-miR-371-373 cluster (counteracting cellular senescence and linked with differentiation potency), as well as hsa-miR-520c/-520h (inhibiting the tumor suppressor p21) were 3.9-16.3 fold up-regulated in two of the three cisplatin resistant cell lines. Several new micro-RNA species missing an annotation towards cisplatin resistance could be identified. These were hsa-miR-512-3p/-515/-517/-518/-525 (up to 8.1-fold up-regulated) and hsa-miR-99a/-100/-145 (up to 10-fold

  5. [Isolation and in vivo tumorigenicity assay of CD133+ side population cells from laryngeal cancer cell line].

    Science.gov (United States)

    Wu, Chun-ping; Zhou, Liang; Xie, Ming; Tao, Lei; Zhang, Ming; Tian, Jie

    2012-03-01

    To investigate a valuable strategy for further purifying cancer stem cells (CSCs) from laryngeal cancer cell line. CD133+ side population (SP) and CD133-SP cells were detected and isolated from laryngeal cancer Hep-2 cell line with SP discrimination and CD133 surface marker, assisted by fluorescence activated cell sorting technology. Freshly sorted CD133+SP and CD133-SP cells were xenografted into the subcutaneous space of the right axillary fossa of NOD/SCID mice and tumorigenic capacity of the cells from two subgroups were examine. Cell cycle distributions of the two cell populations were detected. CD133+SP and CD133-SP cells accounted for (0.30±0.12)% and (17.52±1.59)% in Hep-2 cell line, respectively. CD133+SP cells formed tumor nodules in 15 of 16 mice and CD133-SP cells in 7 of 16 mice (Fisher's exact test, Pline.

  6. Induced Accelerated Aging in Induced Pluripotent Stem Cell Lines from Patients with Parkinson’s Disease

    Science.gov (United States)

    2014-07-01

    Induced Pluripotent Stem Cell Lines from Patients with Parkinson’s Disease PRINCIPAL INVESTIGATOR: Dr. Birgitt Schuele CONTRACTING...Aging in Induced Pluripotent Stem Cell Lines from Patients with Parkinson’s Disease 5b. GRANT NUMBER W81XWH-12-1-0003 5c. PROGRAM ELEMENT NUMBER...for grant W81XWH-12-1-0003 Title: “Induced accelerated aging in induced pluripotent stem cell lines from patients with

  7. CLImAT-HET: detecting subclonal copy number alterations and loss of heterozygosity in heterogeneous tumor samples from whole-genome sequencing data.

    Science.gov (United States)

    Yu, Zhenhua; Li, Ao; Wang, Minghui

    2017-03-15

    Copy number alterations (CNA) and loss of heterozygosity (LOH) represent a large proportion of genetic structural variations of cancer genomes. These aberrations are continuously accumulated during the procedure of clonal evolution and patterned by phylogenetic branching. This invariably results in the emergence of multiple cell populations with distinct complement of mutational landscapes in tumor sample. With the advent of next-generation sequencing technology, inference of subclonal populations has become one of the focused interests in cancer-associated studies, and is usually based on the assessment of combinations of somatic single-nucleotide variations (SNV), CNA and LOH. However, cancer samples often have several inherent issues, such as contamination of normal stroma, tumor aneuploidy and intra-tumor heterogeneity. Addressing these critical issues is imperative for accurate profiling of clonal architecture. We present CLImAT-HET, a computational method designed for capturing clonal diversity in the CNA/LOH dimensions by taking into account the intra-tumor heterogeneity issue, in the case where a reference or matched normal sample is absent. The algorithm quantitatively represents the clonal identification problem using a factorial hidden Markov model, and takes an integrated analysis of read counts and allele frequency data. It is able to infer subclonal CNA and LOH events as well as the fraction of cells harboring each event. The results on simulated datasets indicate that CLImAT-HET has high power to identify CNA/LOH segments, it achieves an average accuracy of 0.87. It can also accurately infer proportion of each clonal population with an overall Pearson correlation coefficient of 0.99 and a mean absolute error of 0.02. CLImAT-HET shows significant advantages when compared with other existing methods. Application of CLImAT-HET to 5 primary triple negative breast cancer samples demonstrates its ability to capture clonal diversity in the CAN

  8. Intestinal transport of Cylindrospermopsin using the Caco-2 cell line.

    Science.gov (United States)

    Pichardo, Silvia; Devesa, Vicenta; Puerto, María; Vélez, Dinoraz; Cameán, Ana M

    2017-02-01

    Cylindrospermopsin (CYN) is a cyanotoxin produced by various cyanobacterial species. It is a water soluble zwitterion, stable at extreme temperatures and pH. Despite the main route of exposure to CYN is through drinking water and food, there is a lack of data concerning its intestinal absorption and the mechanisms implicated. The aim of this study was to characterize the mechanisms involved in the intestinal absorption of CYN, using Caco-2 human cell line as a model of the intestinal epithelium. The results obtained in the present work increases the limited knowledge regarding CYN transport across the intestinal epithelium and identifies the paracellular route as an important pathway in CYN absorption. A minor carrier-mediated transcellular transport has been evidenced. This transport is not affected by low temperatures, suggesting that an active mechanism is not involved. Moreover, the transport through the intestinal monolayer is H(+) and GSH dependent and Na+independent. The transport characteristics elucidated in this study prepare the ground for future studies directed at identifying transporters involved in the intestinal absorption of this toxin. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. 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.

  10. Inhibition of geranylgeranylation mediates sensitivity to CHOP-induced cell death of DLBCL cell lines

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    Ageberg, Malin, E-mail: Malin.Ageberg@med.lu.se [Division of Hematology and Transfusion Medicine, Lund University, BMC C14, 221 84 Lund (Sweden); Rydstroem, Karin, E-mail: Karin.Rydstom@skane.se [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Linden, Ola, E-mail: Ola.Linden@skane.se [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Linderoth, Johan, E-mail: Johan.Linderoth@skane.se [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Jerkeman, Mats, E-mail: Mats.Jerkeman@skane.se [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Drott, Kristina, E-mail: Kristina.Drott@med.lu.se [Division of Hematology and Transfusion Medicine, Lund University, BMC C14, 221 84 Lund (Sweden)

    2011-05-01

    Prenylation is a post-translational hydrophobic modification of proteins, important for their membrane localization and biological function. The use of inhibitors of prenylation has proven to be a useful tool in the activation of apoptotic pathways in tumor cell lines. Rab geranylgeranyl transferase (Rab GGT) is responsible for the prenylation of the Rab family. Overexpression of Rab GGTbeta has been identified in CHOP refractory diffuse large B cell lymphoma (DLBCL). Using a cell line-based model for CHOP resistant DLBCL, we show that treatment with simvastatin, which inhibits protein farnesylation and geranylgeranylation, sensitizes DLBCL cells to cytotoxic treatment. Treatment with the farnesyl transferase inhibitor FTI-277 or the geranylgeranyl transferase I inhibitor GGTI-298 indicates that the reduction in cell viability was restricted to inhibition of geranylgeranylation. In addition, treatment with BMS1, a combined inhibitor of farnesyl transferase and Rab GGT, resulted in a high cytostatic effect in WSU-NHL cells, demonstrated by reduced cell viability and decreased proliferation. Co-treatment of BMS1 or GGTI-298 with CHOP showed synergistic effects with regard to markers of apoptosis. We propose that inhibition of protein geranylgeranylation together with conventional cytostatic therapy is a potential novel strategy for treating patients with CHOP refractory DLBCL.

  11. Glioma cells on the run – the migratory transcriptome of 10 human glioma cell lines

    Directory of Open Access Journals (Sweden)

    Holz David

    2008-01-01

    Full Text Available Abstract Background Glioblastoma multiforme (GBM is the most common primary intracranial tumor and despite recent advances in treatment regimens, prognosis for affected patients remains poor. Active cell migration and invasion of GBM cells ultimately lead to ubiquitous tumor recurrence and patient death. To further understand the genetic mechanisms underlying the ability of glioma cells to migrate, we compared the matched transcriptional profiles of migratory and stationary populations of human glioma cells. Using a monolayer radial migration assay, motile and stationary cell populations from seven human long term glioma cell lines and three primary GBM cultures were isolated and prepared for expression analysis. Results Gene expression signatures of stationary and migratory populations across all cell lines were identified using a pattern recognition approach that integrates a priori knowledge with expression data. Principal component analysis (PCA revealed two discriminating patterns between migrating and stationary glioma cells: i global down-regulation and ii global up-regulation profiles that were used in a proband-based rule function implemented in GABRIEL to find subsets of genes having similar expression patterns. Genes with up-regulation pattern in migrating glioma cells were found to be overexpressed in 75% of human GBM biopsy specimens compared to normal brain. A 22 gene signature capable of classifying glioma cultures based on their migration rate was developed. Fidelity of this discovery algorithm was assessed by validation of the invasion candidate gene, connective tissue growth factor (CTGF. siRNA mediated knockdown yielded reduced in vitro migration and ex vivo invasion; immunohistochemistry on glioma invasion tissue microarray confirmed up-regulation of CTGF in invasive glioma cells. Conclusion Gene expression profiling of migratory glioma cells induced to disperse in vitro affords discovery of genomic signatures; selected

  12. Screening for chemicals that affect hair cell death and survival in the zebrafish lateral line.

    Science.gov (United States)

    Ou, Henry; Simon, Julian A; Rubel, Edwin W; Raible, David W

    2012-06-01

    The zebrafish lateral line is an efficient model system for the evaluation of chemicals that protect and damage hair cells. Located on the surface of the body, lateral line hair cells are accessible for manipulation and visualization. The zebrafish lateral line system allows rapid screens of large chemical libraries, as well as subsequent thorough evaluation of interesting compounds. In this review, we focus on the results of our previous screens and the evolving methodology of our screens for chemicals that protect hair cells, and chemicals that damage hair cells using the zebrafish lateral line. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Development, characterization, conservation and storage of fish cell lines: a review.

    Science.gov (United States)

    Lakra, W S; Swaminathan, T Raja; Joy, K P

    2011-03-01

    Cell lines provide an important biological tool for carrying out investigations into physiology, virology, toxicology, carcinogenesis and transgenics. Teleost fish cell lines have been developed from a broad range of tissues such as ovary, fin, swim bladder, heart, spleen, liver, eye muscle, vertebrae, brain, skin. One hundred and twenty-four new fish cell lines from different fish species ranging from grouper to eel have been reported since the last review by Fryer and Lannan (J Tissue Culture Methods 16: 87-94, 1994). Among the cell lines listed, more than 60% were established from species from Asia, which contributes more than 80% of total fish production. This includes 59 cell lines from 19 freshwater, 54 from 22 marine and 11 from 3 brackish water fishes. Presently, about 283 cell lines have been established from finfish around the world. In addition to the listing and a scientific update on new cell lines, the importance of authentication, applications, cross-contamination and implications of overpassaged cell lines has also been discussed in this comprehensive review. The authors feel that the review will serve an updated database for beginners and established researchers in the field of fish cell line research and development.

  14. Establishment and characterization of a unique 1 microm diameter liver-derived progenitor cell line.

    Science.gov (United States)

    Aravalli, Rajagopal N; Behnan Sahin, M; Cressman, Erik N K; Steer, Clifford J

    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 microm 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. Copyright 2009 Elsevier Inc. All rights reserved.

  15. The DG75 B-cell lymphoma line exhibits biclonal immunoglobulin gene rearrangement.

    Science.gov (United States)

    Qi, Zongli; Li, Yuan; Hu, Jun; Guo, Hua; Zhao, Xiangrong; Wang, Guanghua; Gao, Jinwei; Hu, Qiaoxia

    2013-01-01

    Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement (GR) studies have been successfully employed to investigate the clonality and cell lineage of various lymphoid malignancies. Several lymphoma cell lines, such as BJAB, RAJI, DG75 and Jurkat cell lines, were often used as the positive controls in GR detection assays. Of those, the DG75 B-cell lymphoma line was found to exhibit biclonality [two or more homoduplex and heteroduplex bands in a polymerase chain reaction (PCR) product of clonality assay] in the PCR of GR detection assays. To further explore these characteristics of the biclonal phenomenon, the PCR products were purified and cloned into a pEGM-T clone vector. The sequences were analyzed using DNA analysis software. The results demonstrated that the two bands originated from two forms of GR of DG75 cell lines, i.e., DG75 is a biclonal cell line in Ig GRs, which has not been reported before.

  16. Genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia cell line.

    Science.gov (United States)

    Stoczynska-Fidelus, Ewelina; Piaskowski, Sylwester; Pawlowska, Roza; Szybka, Malgorzata; Peciak, Joanna; Hulas-Bigoszewska, Krystyna; Winiecka-Klimek, Marta; Rieske, Piotr

    2016-01-01

    Thorough examination of genetic heterogeneity of cell lines is uncommon. In order to address this issue, the present study analyzed the genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia (T-ALL) cell line. For this purpose, traditional techniques such as fluorescence in situ hybridization and immunocytochemistry were used, in addition to more advanced techniques, including cell sorting, Sanger sequencing and massive parallel sequencing. The results indicated that the RPMI-8402 cell line consists of several genetically different cell subpopulations. Furthermore, massive parallel sequencing of RPMI-8402 provided insight into the evolution of T-ALL carcinogenesis, since this cell line exhibited the genetic heterogeneity typical of T-ALL. Therefore, the use of cell lines for drug testing in future studies may aid the progress of anticancer drug research.

  17. Genomic characterization of high-count MBL cases indicates that early detection of driver mutations and subclonal expansion are predictors of adverse clinical outcome.

    Science.gov (United States)

    Barrio, S; Shanafelt, T D; Ojha, J; Chaffee, K G; Secreto, C; Kortüm, K M; Pathangey, S; Van-Dyke, D L; Slager, S L; Fonseca, R; Kay, N E; Braggio, E

    2017-01-01

    High-count monoclonal B-cell lymphocytosis (MBL) is an asymptomatic expansion of clonal B cells in the peripheral blood without other manifestations of chronic lymphocytic leukemia (CLL). Yearly, 1% of MBLs evolve to CLL requiring therapy; thus being critical to understand the biological events that determine which MBLs progress to intermediate/advanced CLL. In this study, we performed targeted deep sequencing on 48 high-count MBLs, 47 of them with 2-4 sequential samples analyzed, exploring the mutation status of 21 driver genes and evaluating clonal evolution. We found somatic non-synonymous mutations in 25 MBLs (52%) at the initial time point analyzed, including 12 (25%) with >1 mutated gene. In cases that subsequently progressed to CLL, mutations were detected 41 months (median) prior to progression. Excepting NOTCH1, TP53 and XPO1, which showed a lower incidence in MBL, genes were mutated with a similar prevalence to CLL, indicating the early origin of most driver mutations in the MBL/CLL continuum. MBLs with mutations at the initial time point analyzed were associated with shorter time-to-treatment (TTT). Furthermore, MBLs showing subclonal expansion of driver mutations on sequential evaluation had shorter progression time to CLL and shorter TTT. These findings support that clonal evolution has prognostic implications already at the pre-malignant MBL stage, anticipating which individuals will progress earlier to CLL.

  18. Establishment and characterization of a new cell line derived from feline mammary tumor.

    Science.gov (United States)

    Muleya, J S; Nakaichi, M; Sugahara, J; Taura, Y; Murata, T; Nakama, S

    1998-08-01

    A new cell line designated FRM was established from pleural effusion of a 13-year-old female cat with mammary adenocarcinoma. The cell line exhibited irregular round and polygonal shaped epithelial cells and demonstrated cell growth in a monolayer fashion with a doubling time of 22.4 hr. It possessed a modal chromosome number of 79. The immortality of this cell line was demonstrated using the TRAP assay which revealed a high telomeric activity of these cells. Scatchard analysis revealed quite low levels of estrogen receptors in both tumor mass produced in nude mice and FRM cells. Subcutaneous transplantation of the cells produced localized palpable masses in athymic nude mice within two weeks. This cell line may provide a good model for in vivo and in vitro studies on feline mammary tumors.

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

    Science.gov (United States)

    Boso, Guney; Somia, Nikunj V

    2015-01-01

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

  20. Fish cell culture characteristics of a cell line from the silver perch Bairdiella chrysura.

    Science.gov (United States)

    Wharton, J H; Ellender, R D; Middlebrooks, B L; Stocks, P K; Lawler, A R; Howse, H D

    1977-06-01

    A cell designated SP-1 was established from tissue of the silver perch, Bairdiella chrysura. Cells were fibroblast-like and grew best at 26 degrees C in Leibovitz medium (L-15) containing 15% fetal bovine serum and 0.150 M sodium chloride. Passage 1 to passage 9 SP-1 cells contained a chromosome number of 48; at passages 27 and 50 the modal numbers were 51 and 54, respectively. Confirmation of the origin of SP-1 cells was made by the cytotoxic antibody dye-exclusion test. This cell line supported the growth of lymphocystis virus from the silver perch but was not found to replicate various other fish and mammalian viruses.

  1. Ethanolic Extract Cytotoxic Effect of Zingiber Afficinale in Breast Cancer (MCF7 Cell Line

    Directory of Open Access Journals (Sweden)

    J Tavakkol Afshari

    2010-07-01

    Full Text Available Introduction & Objective: Biological activities of Zingiber afficieale plants have been reported as possessing anticancer, antibacterial, anti ulcer, antifungal, and insecticidal properties. However, its antitumor effects haven't been studied in cancer cell lines. The aim of this study was to investigate the antitumor effect of zingiber afficieale on breast cancer cell lines. Materials & Methods: This experimental study was conducted in 2010 at Mashhad University of medical Sciences. Breast cancer cell line (MCF7 and normal connective tissue cell line (L929 were cultured in DMEM medium. Ethanolic extract of Zingiber afficinale was prepared and cell lines were treated with different concentration of extract (5000 to 78 µg. Cell viability was measured by MTT assay after 24, 48, and 72 hours. The collected data were statistically analyzed by SPSS software. Results: The effects of Zingiber afficinale on cell viability were observed after 48 hours on cell lines. Ginger doses in 2500 µg concentration inhibited 50% of cell growth (IC50 in cell lines after 48 hours. Conclusion: Our study revealed that fresh ginger extract has cytotoxic effects on tumor cells, but it doesn’t have any cytotoxic effect on normal cells. It seems that ginger could be considered as a promising chemotherapeutic agent in cancer treatment.

  2. Epigenetic inactivation and aberrant transcription of CSMD1 in squamous cell carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Scholnick Steven B

    2005-09-01

    Full Text Available Abstract Background The p23.2 region of human chromosome 8 is frequently deleted in several types of epithelial cancer and those deletions appear to be associated with poor prognosis. Cub and Sushi Multiple Domains 1 (CSMD1 was positionally cloned as a candidate for the 8p23 suppressor but point mutations in this gene are rare relative to the frequency of allelic loss. In an effort to identify alternative mechanisms of inactivation, we have characterized CSMD1 expression and epigenetic modifications in head and neck squamous cell carcinoma cell lines. Results Only one of the 20 cell lines examined appears to express a structurally normal CSMD1 transcript. The rest express transcripts which either lack internal exons, terminate abnormally or initiate at cryptic promoters. None of these truncated transcripts is predicted to encode a functional CSMD1 protein. Cell lines that express little or no CSMD1 RNA exhibit DNA methylation of a specific region of the CpG island surrounding CSMD1's first exon. Conclusion Correlating methylation patterns and expression suggests that it is modification of the genomic DNA preceding the first exon that is associated with gene silencing and that methylation of CpG dinucleotides further 3' does not contribute to inactivation of the gene. Taken together, the cell line data suggest that epigenetic silencing and aberrant splicing rather than point mutations may be contributing to the reduction in CSMD1 expression in squamous cancers. These mechanisms can now serve as a focus for further analysis of primary squamous cancers.

  3. Treatment of prostate cancer cell lines and primary cells using low temperature plasma

    Science.gov (United States)

    O'Connell, Deborah; Hirst, Adam; Frame, Fiona F.; Maitland, Norman J.

    2014-10-01

    The mechanisms of cell death after plasma treatment of both benign and cancerous prostate epithelial cells are investigated. Prostate cancer tissue was obtained with patient consent from targeted needle core biopsies following radical prostatectomy. Primary cells were cultured from cancer tissue and plated onto a chamber slide at a density of 10,000 cells per well in 200 microliter of stem cell media (SCM). The treated sample was previously identified as Gleason grade 7 cancer through tissue histo-pathology. A dielectric barrier discharge (DBD) jet configuration, with helium as a carrier gas, and 0.3% O2 admixture was used for treating the cells. Reactive oxygen and nitrogen species (RONS) produced by the plasma are believed to be the main mediators of the plasma-cell interaction and response. We found the concentration of reactive oxygen species (ROS) induced inside the cells increased with plasma exposure. Exposure to the plasma for >3 minutes showed high levels of DNA damage compared to untreated and hydrogen peroxide controls. Cell viability and cellular recovery are also investigated and will be presented. All findings were common to both cell lines, suggesting the potential of LTP therapy for both benign and malignant disease.

  4. Real-time cell analysis of human cancer cell lines after chemotherapy with functionalized magnetic nanoparticles.

    Science.gov (United States)

    Dürr, Stephan; Lyer, Stefan; Mann, Jenny; Janko, Christina; Tietze, Rainer; Schreiber, Eveline; Herrmann, Martin; Alexiou, Christoph

    2012-05-01

    Magnetic drug targeting is a new and innovative approach in cancer treatment. In order to avoid the adverse effects of chemotherapy, the therapeutic agent is linked to superparamagnetic nanoparticles which are injected into a tumour-supporting artery and is focused by an external magnetic field to the tumour region in order to provoke maximum local impact. Analysis of nanoparticles and chemotherapeutic substances in human cancer cell culture is necessary to provide respective information for in vivo applications. The effect of pure mitoxantrone and mitoxantrone bound to nanoparticles was tested on human cancer cell lines using real-time cell analysis (RTCA) and lactate dehydrogenase (LDH) assays. RTCA was performed by impedance measuring. The impedance is expressed as the cell index (CI), which is a parameter of cell viability. RTCA showed that mitoxantrone when bound to nanoparticles was more toxic than the drug alone. The CI clearly decreased faster after adding the chemotherapeutic bound to nanoparticles than when adding the pure drug alone. However, in the first experiments, the particles themselves showed no toxicity at therapeutically relevant concentrations. These results were confirmed by LDH assays. The toxic effects of chemotherapeutic agents (e.g. mitoxantrone) on human cancer cell lines (e.g. MCF-7) can be enhanced if these drugs are bound to magnetic nanoparticles. These preliminary data show a dependency on the different application modes of RTCA. The results presented here are a first step for a better understanding of the effectiveness of magnetic drug targeting as a new and innovative cancer treatment.

  5. Establishment and Characterization of Novel Cell Lines from Sinonasal Undifferentiated Carcinoma

    Science.gov (United States)

    Takahashi, Yoko; Kupferman, Michael E.; Bell, Diana; Jiffar, Tilahun; Lee, June Goo; Xie, Tong-Xin; Li, Ning-Wei; Zhao, Mei; Frederick, Mitchell J.; Gelbard, Alexander; Myers, Jeffrey N.; Hanna, Ehab Y.

    2012-01-01

    Purpose Sinonasal undifferentiated carcinoma (SNUC) is a rare and aggressive cancer. Despite the use of multi-modality treatment, the overall prognosis remains poor. To better understand the biological features of SNUC and help develop new therapies for the disease, we established SNUC cell lines and characterized their biological behaviors. Methods Cell lines were established from a patient with a T4N0M0 SNUC of the right maxillary sinus who was treated with surgical resection at our center. Tumor colonies were harvested and were sequentially replated onto larger plates. Two populations were developed and labeled MDA8788-6 and MDA8788-7. These cell lines were characterized with molecular, biomarker, functional and histologic analyses. Results Short tandem repeat genotyping revealed that the cell line is isogenic to the parental tumor, and cytogenetic analysis identified 12 chromosomal translocations. The SNUC cell lines do not form colonies in soft agar, but are tumorigenic and non-metastatic in an orthotopic mouse model of sinonasal cancer. Western blot analysis revealed that both MDA8788 cell lines express epithelial markers, but do not express mesenchymal markers or the endocrine marker synaptophysin. Conclusions This is the first report of the establishment of stable human-derived SNUC cell lines. The lines were highly tumorigenic and maintain the histologic and molecular features of the original tumor. These cell lines should serve as useful tools for the future study of SNUC biology and the development and testing of novel therapies for this deadly disease. PMID:23032744

  6. Comparative Membranome expression analysis in primary tumors and derived cell lines.

    Directory of Open Access Journals (Sweden)

    Paolo Uva

    Full Text Available Despite the wide use of cell lines in cancer research, the extent to which their surface properties correspond to those of primary tumors is poorly characterized. The present study addresses this problem from a transcriptional standpoint, analyzing the expression of membrane protein genes--the Membranome--in primary tumors and immortalized in-vitro cultured tumor cells. 409 human samples, deriving from ten independent studies, were analyzed. These comprise normal tissues, primary tumors and tumor derived cell lines deriving from eight different tissues: brain, breast, colon, kidney, leukemia, lung, melanoma, and ovary. We demonstrated that the Membranome has greater power than the remainder of the transcriptome when used as input for the automatic classification of tumor samples. This feature is maintained in tumor derived cell lines. In most cases primary tumors show maximal similarity in Membranome expression with cell lines of same tissue origin. Differences in Membranome expression between tumors and cell lines were analyzed also at the pathway level and biological themes were identified that were differentially regulated in the two settings. Moreover, by including normal samples in the analysis, we quantified the degree to which cell lines retain the Membranome up- and down-regulations observed in primary tumors with respect to their normal counterparts. We showed that most of the Membranome up-regulations observed in primary tumors are lost in the in-vitro cultured cells. Conversely, the majority of Membranome genes down-regulated upon tumor transformation maintain lower expression levels also in the cell lines. This study points towards a central role of Membranome genes in the definition of the tumor phenotype. The comparative analysis of primary tumors and cell lines identifies the limits of cell lines as a model for the study of cancer-related processes mediated by the cell surface. Results presented allow for a more rational use of

  7. The necessity of identity assessment of animal intestinal cell lines: A case report.

    Science.gov (United States)

    Steube, Klaus G; Koelz, Anne-Leena; Uphoff, Cord C; Drexler, Hans G; Kluess, Jeannette; Steinberg, Pablo

    2012-08-01

    Eight intestinal cell lines, established from different animal species were submitted to DSMZ (German Collection of Microorganisms and Cell Cultures) in order to analyze their species of origin and their microbial contamination. Species identity was determined by PCR targeting mitochondrial genes and hence confirmed by sequencing the amplified PCR products. For three cell lines (CIEB, CLAB, PSI-1) we confirmed the species identity, whereas the species of origin of the three other cell lines (B6, B10XI and IPEC) was not the expected one: B6 and B10XI cells, which were supposed to be of chicken origin were identified as porcine cells. IPEC, allegedly a sub clone of the well-known porcine intestinal cell line IPEC-J2, was of bovine instead of porcine origin. However, two further IPEC-clones, namely IPEC-1 and IPEC-J2, provided by another source were shown to be derived from the correct species (i.e. pig). Furthermore, six out of these eight cell lines turned out to be highly contaminated with mycoplasma. Alerted by this high incidence of infected and false specified cell lines, we feel obliged to inform all those working with animal intestinal cell lines and we strongly recommend verifying the species identity before using them. Also, the presence of mycoplasma should be tested when taking the cells in culture for the first time, and this mycoplasma control should be repeated at regular time intervals (e.g. every 4 weeks).

  8. A common carp (Cyprinus carpio L.) leucocyte cell line shares morphological and functional characteristics with macrophages.

    NARCIS (Netherlands)

    Weyts, F.A.A.; Rombout, J.H.W.M.; Flik, G.; Verburg-van Kemenade, B.M.L.

    1997-01-01

    A carp leucocyte cell line (CLC), originating from peripheral blood, was characterised to assess its suitability for studies into carp macrophage functions. The cells reacted with a monoclonal antibody raised against carp head kidney macrophages. Other macrophage characteristics observed were:

  9. Effect of sirolimus on urinary bladder cancer T24 cell line.

    Science.gov (United States)

    Pinto-Leite, Rosario; Botelho, Pedro; Ribeiro, Eufemia; Oliveira, Paula A; Santos, Lucios

    2009-01-07

    Sirolimus is recently reported to have antitumour effects on a large variety of cancers. The present study was performed to investigate sirolimus's ability to inhibit growth in T24 bladder cancer cells. T24 bladder cancer cells were treated with various concentrations of sirolimus. MTT assay was used to evaluate the proliferation inhibitory effect on T24 cell line. The viability of T24 cell line was determined by Trypan blue exclusion analysis. Sirolimus inhibits the growth of bladder carcinoma cells and decreases their viability. Significant correlations were found between cell proliferation and sirolimus concentration (r = 0.830; p T24 bladder carcinoma cell line. The information from our results is useful for a better understanding sirolimus's anti-proliferative activity in the T24 bladder cancer cell line.

  10. Effect of sirolimus on urinary bladder cancer T24 cell line

    Directory of Open Access Journals (Sweden)

    Oliveira Paula A

    2009-01-01

    Full Text Available Abstract Background Sirolimus is recently reported to have antitumour effects on a large variety of cancers. The present study was performed to investigate sirolimus's ability to inhibit growth in T24 bladder cancer cells. Methods T24 bladder cancer cells were treated with various concentrations of sirolimus. MTT assay was used to evaluate the proliferation inhibitory effect on T24 cell line. The viability of T24 cell line was determined by Trypan blue exclusion analysis. Results Sirolimus inhibits the growth of bladder carcinoma cells and decreases their viability. Significant correlations were found between cell proliferation and sirolimus concentration (r = 0.830; p Conclusion Sirolimus has an anti-proliferation effect on the T24 bladder carcinoma cell line. The information from our results is useful for a better understanding sirolimus's anti-proliferative activity in the T24 bladder cancer cell line.

  11. Alginate-based microencapsulation of retinal pigment epithelial cell line for cell therapy.

    Science.gov (United States)

    Wikström, Jonna; Elomaa, Matti; Syväjärvi, Heli; Kuokkanen, Johanna; Yliperttula, Marjo; Honkakoski, Paavo; Urtti, Arto

    2008-03-01

    The goals of this study were to evaluate human retinal pigment epithelial cell line (ARPE-19) for cell encapsulation and to optimize the alginate-based microencapsulation. We used immortalized ARPE-19 cells and the transfected sub-line that expresses secreted alkaline phosphatase (SEAP) reporter enzyme. Alginate was cross-linked with different divalent cations (Ca(2+), Ba(2+), Sr(2+) and combination of Ca(2+) and Ba(2+)), coated first with poly-l-lysine (PLL), and then with alginate. Microcapsules with different pore sizes and stability were generated. The pore size of the microcapsules was assessed by the release of encapsulated fluorescein isothiocyanate (FITC)-dextrans. The viability of the cells in the microcapsules was studied in vitro by assessing the secretion rates of SEAP and oxygen consumption by the cells. The best microcapsule morphology, durability and cellular viability were obtained with alginate microcapsules that were cross-linked with Ca(2+) and Ba(2+) ions and then coated with PLL and alginate. Based on FITC-dextran release these microcapsules have porous wall that enables the rapid contents release. The ARPE-19 cells maintained viability in the Ca(2+) and Ba(2+) cross-linked microcapsules for at least 110 days. The alginate microcapsules cross-linked with Ca(2+) and Ba(2+) have sufficiently large pore size for prolonged cell viability and for the release of secreted SEAP model protein (Mw 50 kDa; radius of gyration of 3 nm). ARPE-19 cells show long-term viability and protein secretion within alginate microcapsules cross-linked with Ca(2+) and Ba(2+). This combination may be useful in cell therapy.

  12. Cytotoxic Effect of Thiabendazole on Hn5 Head and Neck Squamous Cell Carcinoma Cell Line.

    Science.gov (United States)

    Abassi, Amir Jalal; Mohamadnia, Abdolreza; Parhiz, Seyed Alireza; Azizi Moghadam, Nahid; Bahrami, Naghmeh

    2017-09-01

    Evidence shows thiabendazole has the potential to inhibit angiogenesis in melanoma and fibrosarcoma; however, its effect on oral squamous cell carcinoma has not been previously studied. This study sought to assess the cytotoxic effects of thiabendazole on HN5 head and neck squamous carcinoma cell line. HN5 cell lines were exposed to different concentrations of thiabendazole (prepared from 99% pure powder) for 24, 48 and 72 hours. Cell viability was assessed by the methyl thiazol tetrazolium assay, and IC50 of thiabendazole was calculated. Cells were also exposed to different concentrations of thiabendazole for 48 hours to determine its effect on expression and transcription of vascular endothelial growth factor gene. Expression of vascular endothelial growth factor mRNA was assessed by real-time polymerase chain reaction. The vascular endothelial growth factor release was assessed by the enzyme-linked immunosorbent assay test. In all concentrations of thiabendazole except for 200 and 550μM, cell viability was significantly different at different time points (p< 0.05). At 48 and 72 hours, cell viability at all concentrations of thiabendazole (100-650μM) significantly decreased compared to the control group (zero concentration). In addition, cell viability significantly decreased with an increase in thiabendazole concentration. At 48 hours, expression of vascular endothelial growth factor mRNA was significantly lower in presence of 500μM thiabendazole compared to the control group (p< 0.001) and release of vascular endothelial growth factor was inhibited in a dose-dependent manner. Thiabendazole inhibited the proliferation of HN5 cells in a dose-dependent and time-dependent manner. It also inhibited the expression of vascular endothelial growth factor gene.

  13. Susceptibility to cytotoxic T cell lysis of cancer stem cells derived from cervical and head and neck tumor cell lines.

    Science.gov (United States)

    Liao, Tian; Kaufmann, Andreas M; Qian, Xu; Sangvatanakul, Voramon; Chen, Chao; Kube, Tina; Zhang, Guoyou; Albers, Andreas E

    2013-01-01

    To explore cancer stem cell susceptibility to a host's cytotoxic T lymphocyte (CTL)-mediated immune response. We compared the susceptibility of putative CSC generated from cancer cell lines to immunologic recognition and killing by alloantigen-specific CD8(+) CTL. CSC-enriched spheroid culture-derived cells (SDC) exhibited higher expression of ALDH, ICAM1 and of stem/progenitor cell markers on all 3 tumor cell lines investigated and lower MHC class I on the cervical cancer cell line as compared to their monolayer-derived cells (MDC). The expression of ICAM1 and MHCI was upregulated by IFN-γ treatment. CSC populations were less sensitive to MHC class I-restricted alloantigen-specific CD8(+) CTL lysis as compared to matched MDC. IFN-γ pretreatment resulted in over-proportionally enhanced lysis of SDC. Finally, the subset of ALDH(high) expressing SDC presented more sensitivity toward CD8(+) CTL killing than the ALDH(low) SDC. Tumor therapy resistance has been attributed to cancer stem cells (CSC). We show in vitro susceptibility of CSC to CTL-mediated lysis. Immunotherapy targeting of ALDH(+) CSC may therefore be a promising approach. Our results and method may be helpful for the development and optimization of adjuvants, as here exemplified for INF-γ, for CSC-targeted vaccines, independent of the availability of CSC-specific antigens.

  14. Improved retroviral suicide gene transfer in colon cancer cell lines after cell synchronization with methotrexate

    Directory of Open Access Journals (Sweden)

    Nordlinger Bernard

    2011-10-01

    Full Text Available Abstract Background Cancer gene therapy by retroviral vectors is mainly limited by the level of transduction. Retroviral gene transfer requires target cell division. Cell synchronization, obtained by drugs inducing a reversible inhibition of DNA synthesis, could therefore be proposed to precondition target cells to retroviral gene transfer. We tested whether drug-mediated cell synchronization could enhance the transfer efficiency of a retroviral-mediated gene encoding herpes simplex virus thymidine kinase (HSV-tk in two colon cancer cell lines, DHDK12 and HT29. Methods Synchronization was induced by methotrexate (MTX, aracytin (ara-C or aphidicolin. Gene transfer efficiency was assessed by the level of HSV-TK expression. Transduced cells were driven by ganciclovir (GCV towards apoptosis that was assessed using annexin V labeling by quantitative flow cytometry. Results DHDK12 and HT29 cells were synchronized in S phase with MTX but not ara-C or aphidicolin. In synchronized DHDK12 and HT29 cells, the HSV-TK transduction rates were 2 and 1.5-fold higher than those obtained in control cells, respectively. Furthermore, the rate of apoptosis was increased two-fold in MTX-treated DHDK12 cells after treatment with GCV. Conclusions Our findings indicate that MTX-mediated synchronization of target cells allowed a significant improvement of retroviral HSV-tk gene transfer, resulting in an increased cell apoptosis in response to GCV. Pharmacological control of cell cycle may thus be a useful strategy to optimize the efficiency of retroviral-mediated cancer gene therapy.

  15. Effect of sirolimus on urinary bladder cancer T24 cell line

    OpenAIRE

    Oliveira Paula A; Ribeiro Eufemia; Botelho Pedro; Pinto-Leite Rosario; Santos Lucios

    2009-01-01

    Abstract Background Sirolimus is recently reported to have antitumour effects on a large variety of cancers. The present study was performed to investigate sirolimus's ability to inhibit growth in T24 bladder cancer cells. Methods T24 bladder cancer cells were treated with various concentrations of sirolimus. MTT assay was used to evaluate the proliferation inhibitory effect on T24 cell line. The viability of T24 cell line was determined by Trypan blue exclusion analysis. Results Sirolimus in...

  16. Radiosensitization of non-small cell lung cancer cell lines by ionizing irradiation and caffeine

    OpenAIRE

    Schulze, Joern

    2010-01-01

    The radiosensitising effect of caffeine was investigated for NSCLC cell lines. Radiosensitization of A549 and H460 of the same p53 wild-type status and different acute radiosensitivity was achieved by the addition of 2 mM caffeine. At maximum G1 synchronisation, the methylxanthine resulted in an increase in the temporary arrest of G1-S transition in both the irradiated and unirradiated fractions. The percentage of S phase cells was also reduced by caffeine. 5 mM caffeine resulted in an increa...

  17. Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning.

    Science.gov (United States)

    Sun, Changhong; Fan, Yu; Li, Juan; Wang, Gancheng; Zhang, Hanshuo; Xi, Jianzhong Jeff

    2015-02-01

    Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line. © 2014 Society for Laboratory Automation and Screening.

  18. The Tribolium castaneum cell line TcA: a new tool kit for cell biology.

    Science.gov (United States)

    Silver, Kristopher; Jiang, Hongbo; Fu, Jinping; Phillips, Thomas W; Beeman, Richard W; Park, Yoonseong

    2014-10-30

    The red flour beetle, Tribolium castaneum, is an agriculturally important insect pest that has been widely used as a model organism. Recently, an adherent cell line (BCIRL-TcA-CLG1 or TcA) was developed from late pupae of the red flour beetle. Next generation transcriptome sequencing of TcA cells demonstrated expression of a wide variety of genes associated with specialized functions in chitin metabolism, immune responses and cellular and systemic RNAi pathways. Accordingly, we evaluated the sensitivity of TcA cells to dsRNA to initiate an RNAi response. TcA cells were highly sensitive to minute amounts of dsRNA, with a minimum effective dose of 100 pg/mL resulting in significant suppression of gene expression. We have also developed a plasmid containing two TcA-specific promoters, the promoter from the 40S ribosomal protein subunit (TC006550) and a bi-directional heat shock promoter (TcHS70) from the intergenic space between heat shock proteins 68a and b. These promoters have been employed to provide high levels of either constitutive (TC006550) or inducible (TcHS70) gene expression of the reporter proteins. Our results show that the TcA cell line, with its sensitivity to RNAi and functional TcA-specific promoters, is an invaluable resource for studying basic molecular and physiological questions.

  19. Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines.

    Science.gov (United States)

    Yu, Vicky; Rahimy, Mehran; Korrapati, Avinaash; Xuan, Yinan; Zou, Angela E; Krishnan, Aswini R; Tsui, Tzuhan; Aguilera, Joseph A; Advani, Sunil; Crotty Alexander, Laura E; Brumund, Kevin T; Wang-Rodriguez, Jessica; Ongkeko, Weg M

    2016-01-01

    Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines. HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 h to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Application of low level laser on skin cell lines

    CSIR Research Space (South Africa)

    Ndhundhuma, IM

    2010-01-01

    Full Text Available Lasers have emerged as powerful tools for tissue engineering. To examine cellular growth, and cell to cell interactions, in vitro skin models have been developed combining two major cell types of skin, keratinocytes and fibroblasts. The main...

  1. Generation of mouse ES cell lines engineered for the forced induction of transcription factors.

    Science.gov (United States)

    Correa-Cerro, Lina S; Piao, Yulan; Sharov, Alexei A; Nishiyama, Akira; Cadet, Jean S; Yu, Hong; Sharova, Lioudmila V; Xin, Li; Hoang, Hien G; Thomas, Marshall; Qian, Yong; Dudekula, Dawood B; Meyers, Emily; Binder, Bernard Y; Mowrer, Gregory; Bassey, Uwem; Longo, Dan L; Schlessinger, David; Ko, Minoru S H

    2011-01-01

    Here we report the generation and characterization of 84 mouse ES cell lines with doxycycline-controllable transcription factors (TFs) which, together with the previous 53 lines, cover 7-10% of all TFs encoded in the mouse genome. Global gene expression profiles of all 137 lines after the induction of TFs for 48 hrs can associate each TF with the direction of ES cell differentiation, regulatory pathways, and mouse phenotypes. These cell lines and microarray data provide building blocks for a variety of future biomedical research applications as a community resource.

  2. Susceptibility and production of a feline endogenous retrovirus (RD-114 virus) in various feline cell lines.

    Science.gov (United States)

    Okada, Masaya; Yoshikawa, Rokusuke; Shojima, Takayuki; Baba, Kenji; Miyazawa, Takayuki

    2011-01-01

    RD-114 virus is a replication-competent feline endogenous retrovirus that has been classified as a xenotropic virus. In this study, we examined the expression of the receptors for RD-114 virus in feline cell lines by conducting a pseudotype virus infection assay. Six out of eight feline cell lines were susceptible to the RD-114 pseudotype virus and two cell lines (MCC and FER cells) were resistant. The two resistant cell lines and one cell line (CRFK cells) weakly sensitive to the RD-114 pseudotype virus were found to produce replication-competent RD114-like viruses by the LacZ marker rescue assay and the interference assay. These data strongly suggest that RD-114 virus is polytropic and resistance to RD-114 virus in certain cell lines is due to receptor interference but not polymorphism of the RD-114 receptors. In addition, we determined the amino acid sequences of the envelope region of RD-114-like viruses produced from MCC, FER and CRFK cells. The sequences were identical with the authentic RD-114 virus. Because many feline cell lines are used to manufacture live attenuated vaccines for companion animals, attention should be paid to contamination of the RD-114 virus in vaccines. Copyright © 2010. Published by Elsevier B.V.

  3. MS-HRM assay identifies high levels of epigenetic heterogeneity in human immortalized cell lines.

    Science.gov (United States)

    Putnik, Milica; Wojdacz, Tomasz K; Pournara, Angeliki; Vahter, Marie; Wallberg, Annika E

    2015-04-15

    Immortalized cell lines are widely used in genetic and epigenetic studies, from exploration of basic molecular pathways to evaluation of disease-specific cellular properties. They are also used in biotechnology, e.g., in drug toxicity tests and vaccine production. Cellular and genetic uniformity is the main feature of immortalized cell lines and it has been particularly advantageous in functional genomic research, which has in recent years been expanded to include epigenetic mechanisms of gene expression regulation. Using the MS-HRM technique, we demonstrated heterogeneity in locus-specific methylation patterns in different cell cultures of four human cell lines: HEK293, HEK293T, LCL and DU145. Our results show that some human immortalized cell lines consist of cells that differ in the methylation status of specific loci, i.e., that they are epigenetically heterogeneous. We show that even two cultures of the same cell line obtained from different laboratories can differ in the methylation status of the specific loci. The results indicated that epigenetic uniformity of the cell lines cannot be assumed in experiments which utilize cell cultures and that the methylation status of the specific loci in the immortalized cell lines should be re-characterized and carefully profiled before epigenetic studies are performed. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Establishment and culture optimization of a new type of pituitary immortalized cell line

    Energy Technology Data Exchange (ETDEWEB)

    Kokubu, Yuko [Graduate School of Life and Environmental Sciences, The University of Tsukuba, Tsukuba, Ibaraki 305-8562 (Japan); Asashima, Makoto [Graduate School of Life and Environmental Sciences, The University of Tsukuba, Tsukuba, Ibaraki 305-8562 (Japan); Life Science Center of TARA, The University of Tsukuba, Ibaraki-ken 305-8577 (Japan); Kurisaki, Akira, E-mail: akikuri@hotmail.com [Graduate School of Life and Environmental Sciences, The University of Tsukuba, Tsukuba, Ibaraki 305-8562 (Japan); Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8562 (Japan)

    2015-08-07

    The pituitary gland is a center of the endocrine system that controls homeostasis in an organism by secreting various hormones. The glandular anterior pituitary consists of five different cell types, each expressing specific hormones. However, their regulation and the appropriate conditions for their in vitro culture are not well defined. Here, we report the immortalization of mouse pituitary cells by introducing TERT, E6, and E7 transgenes. The immortalized cell lines mainly expressed a thyrotroph-specific thyroid stimulating hormone beta (Tshb). After optimization of the culture conditions, these immortalized cells proliferated and maintained morphological characteristics similar to those of primary pituitary cells under sphere culture conditions in DMEM/F12 medium supplemented with N2, B27, basic FGF, and EGF. These cell lines responded to PKA or PKC pathway activators and induced the expression of Tshb mRNA. Moreover, transplantation of the immortalized cell line into subcutaneous regions and kidney capsules of mice further increased Tshb expression. These results suggest that immortalization of pituitary cells with TERT, E6, and E7 transgenes is a useful method for generating proliferating cells for the in vitro analysis of pituitary regulatory mechanisms. - Highlights: • Mouse pituitary cell lines were immortalized by introducing TERT, E6, and E7. • The immortalized cell lines mainly expressed thyroid stimulating hormone beta. • The cell lines responded to PKA or PKC pathway activators, and induced Tshb.

  5. Cardamom extract induces cell proliferation by increasing potassium currents in NIH3T3 cell line.

    Science.gov (United States)

    Siddiqui, Sonia; Hassan, Sohail; Imran, Sumaira; Khan, Faisal; Ahmed, Faheem; Dar, Asim

    2017-11-01

    Amommum subulatum (Roxb.) or Cardamom extract is known to have anti-inflammatory and neuroprotective effects towards many gastrointestinal related problems. However, uptill now different fractions of cardamom extract on fibroblasts with respect to potassium channel activity have not been investigated. Therefore, present study investigated the effects of different fractions of cardamom extract on potassium channels in non-tumor NIH3T3 cell line. Phytochemical analysis of hydroalcoholic, n-hexane, butane and ethyl acetate fractions of cardamom extracts were purified and isolated by thin layer chromatography (TLC). 3T3 cells were cultured and incubated with hydroalcohol (1-2 μ/ml), n-hexane (1 μ/ml), butane (2 μ/ml) and ethyl acetate (1-2 μ/ml) for 5 hrs at 37°C. Modulation in potassium currents were recorded by whole-cell patch clamp method. The data showed two constituents Cineol (C 10 H 18 O) and Terpinyl acetate (C10H17OOCCH3) by TLC method. The present study shows that the constituents in n-hexane, hydro alcohol (1 μ/ml) and ethyl acetate (2 μ/ml) significantly increased (p<0.01) the potassium outward rectifying currents from NIH3T3 cells when compared to untreated controls cells. Whereas, butanol fraction (2 μ/ml) significantly decreased (p<0.01) the inward rectifying currents when compared to controls. Moreover hydroalcoholic and n-hexane fractions have increased the proliferation in 3T3 cell line. On the other hand butanol and ethyl acetate did not induce proliferation in 3T3 cells. Taken together, our data suggested that cardamom extract contains constituents that increased K+ currents, cell migration and proliferation and are involved in wound healing.

  6. Effects of simulated weightlessness on cellular morphology and biological characteristics of cell lines SGC-7901 and HFE-145

    National Research Council Canada - National Science Library

    Zhu, M; Jin, X W; Wu, B Y; Nie, J L; Li, Y H

    2014-01-01

    We investigated the effects of simulated weightlessness on cellular morphology, proliferation, cell cycle, and apoptosis of the human gastric carcinoma cell line SGC-7901 and the human gastric normal cell line HFE-145...

  7. Characterization of P-glycoprotein and multidrug resistance proteins in rat kidney and intestinal cell lines.

    NARCIS (Netherlands)

    Water, F.M. van de; Boleij, J.M.; Peters, J.G.P.; Russel, F.G.M.; Masereeuw, R.

    2007-01-01

    The activity of P-glycoprotein (Pgp/MDR1/ABCB1) and multidrug resistance proteins (MRP/ABCC) influence the pharmacokinetics and bioavailability of many drugs. Few suitable cell lines for the study of drug transport exist. Additional non-human cell lines may help clarify species differences and

  8. 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

  9. Identifying anti-growth factors for human cancer cell lines through genome-scale metabolic modeling

    DEFF Research Database (Denmark)

    Ghaffari, Pouyan; Mardinoglu, Adil; Asplund, Anna

    2015-01-01

    Human cancer cell lines are used as important model systems to study molecular mechanisms associated with tumor growth, hereunder how genomic and biological heterogeneity found in primary tumors affect cellular phenotypes. We reconstructed Genome scale metabolic models (GEMs) for eleven cell line...

  10. Characterization of the alkaline phosphatase expressed on the surface of a Hodgkin's lymphoma cell line

    NARCIS (Netherlands)

    Belland, L; Visser, L; Poppema, S; Stinson, R A

    1993-01-01

    Alkaline phosphatase solubilized from a human Hodgkin's lymphoma cell line (L428) was compared with purified amphiphilic and hydrophilic forms of the enzyme from human liver, and with the enzyme solubilized from a cultured osteosarcoma cell line (Saos-2). Purified hydrophilic alkaline phosphatases

  11. Cytotoxicity of arctigenin and matairesinol against the T-cell lymphoma cell line CCRF-CEM.

    Science.gov (United States)

    Su, Shan; Cheng, Xinlai; Wink, Michael

    2015-09-01

    Arctigenin and matairesinol possess a diversity of bioactivities. Here we investigated the cytotoxicity of arctigenin and matairesinol against a T-cell lymphoma cell line CCRF-CEM and the underlying mechanisms that have not been explored before. The cytotoxic activity was investigated using MTT assay. The cell cycle arrest and reactive oxygen species (ROS) accumulation were determined by flow cytometric analysis. The apoptosis induction was assessed using Annexin V/Propidium Iodide assay. The gene quantification analysis was measured through real-time polymerase chain reaction. Arctigenin and matairesinol exhibited significant antiproliferative activity against CCRF-CEM cells after 72 h treatment with IC50 values of 1.21 ± 0.15 μm and 4.27 ± 0.41 μm, respectively. In addition, both lignans arrest CCRF-CEM cells in the S phase. Furthermore, they could induce apoptosis in CCRF-CEM cells in a concentration- and time-dependent manner. Interestingly, the lignans differentially regulated the expression of several key genes involved in apoptosis pathways, including Bax, Bad and caspase-9. Moreover, both lignans could increase ROS levels in CCRF-CEM cells. Our study provides an insight into the potential of arctigenin and matairesinol as good candidates for the development of novel agents against T-cell lymphoma. © 2015 Royal Pharmaceutical Society.

  12. Single-cell states in the estrogen response of breast cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Francesco Paolo Casale

    Full Text Available Estrogen responsive breast cancer cell lines have been extensively studied to characterize transcriptional patterns in hormone-responsive tumors. Nevertheless, due to current technological limitations, genome-wide studies have typically been limited to population averaged data. Here we obtain, for the first time, a characterization at the single-cell level of the states and expression signatures of a hormone-starved MCF-7 cell system responding to estrogen. To do so, we employ a recently proposed model that allows for dissecting single-cell states from time-course microarray data. We show that within 32 hours following stimulation, MCF-7 cells traverse, most likely, six states, with a faster early response followed by a progressive deceleration. We also derive the genome-wide transcriptional profiles of such single-cell states and their functional characterization. Our results support a scenario where estrogen promotes cell cycle progression by controlling multiple, sequential regulatory steps, whose single-cell events are here identified.

  13. Inhibition of cell motility by troglitazone in human ovarian carcinoma cell line

    Directory of Open Access Journals (Sweden)

    Chen Show-Li

    2007-11-01

    Full Text Available Abstract Background Troglitazone (TGZ is a potential anticancer agent. Little is known about the effect of this agent on cancer cell migration. Methods Human ovarian carcinoma cell line, ES-2 cells were treated with various concentrations of TGZ. Cell migration was evaluated by wound-healing and Boyden chamber transwell experiments. PPARγ expression was blocked by PPARγ small interfering RNA. The effects of TGZ on phosphorylation of FAK, PTEN, Akt were assessed by immunoblotting using phospho-specific antibodies. The cellular distribution of paxillin, vinculin, stress fiber and PTEN was assessed by immunocytochemistry. Results TGZ dose- and time-dependently impaired cell migration through a PPARγ independent manner. TGZ treatment impaired cell spreading, stress fiber formation, tyrosine phosphorylation of focal adhesion kinase (FAK, and focal adhesion assembly in cells grown on fibronectin substratum. TGZ also dose- and time-dependently suppressed FAK autophosphorylation and phosphorylation of the C-terminal of PTEN (a phosphatase. At concentration higher than 10 μM, TGZ caused accumulation of PTEN in plasma membrane, a sign of PTEN activation. Conclusion These results indicate that TGZ can suppress cultured ES-2 cells migration. Our data suggest that the anti-migration potential of TGZ involves in regulations of FAK and PTEN activity.

  14. Amygdalin induces apoptosis in human cervical cancer cell line HeLa cells.

    Science.gov (United States)

    Chen, Yu; Ma, Jinshu; Wang, Fang; Hu, Jie; Cui, Ai; Wei, Chengguo; Yang, Qing; Li, Fan

    2013-02-01

    Amygdalin, a naturally occurring substance, has been suggested to be efficacious as an anticancer substance. The effect of amygdalin on cervical cancer cells has never been studied. In this study, we found that the viability of human cervical cancer HeLa cell line was significantly inhibited by amygdalin. 4,6-Diamino-2-phenyl indole (DAPI) staining showed that amygdalin-treated HeLa cells developed typical apoptotic changes. The development of apoptosis in the amygdalin-treated HeLa cells were confirmed by double staining of amygdalin-treated HeLa cells with annexin V-FITC and propidium iodide (PI) along with increase in caspase-3 activity in these cells. Further studies indicated that antiapoptotic protein Bcl-2 was downregulated whereas proapoptotic Bax protein was upregulated in the amygdalin-treated HeLa cells implying involvement of the intrinsic pathway of apoptosis. In vivo, amygdalin administration inhibited the growth of HeLa cell xenografts through a mechanism of apoptosis. The results in the present study suggest that amygdalin may offer a new therapeutic option for patients with cervical cancer.

  15. False and mycoplasma-contaminated leukemia-lymphoma cell lines: time for a reappraisal.

    Science.gov (United States)

    Drexler, Hans G; Dirks, Wilhelm G; MacLeod, Roderick A F; Uphoff, Cord C

    2017-03-01

    Leukemia-lymphoma cell lines are important research tools in a variety of fields. To represent adequate model systems it is of utmost importance that cell lines faithfully model the primary tumor material and are not cross-contaminated with unrelated cell material (or contaminated with mycoplasma). As it has been previously reported that cross-contaminated cell lines represent a significant problem, it is of interest to know whether any improvement in the prevalence of such "false cell lines" had occurred since we called the alert in 1999. A retrospective review of our data archives covered 848 cell lines received from 1990 to 2014 from 290 laboratories in 23 countries spanning the spectrum of leukemia-lymphoma entities. Two variables were considered: authenticity and freedom from mycoplasma infection. Regarding provenance, we separately considered primary sources (original investigators having established the cell lines or reference repositories) and secondary sources. The percentages of mycoplasma-contaminated cell lines decreased significantly over the 25-year timespan. Among primary sourced material: mycoplasma-contamination fell from 23% to 0%; among secondary sourced: from 48% to 21%. The corresponding figures for cross-contamination declined from 15% to 6%, while among material obtained from secondary sources prevalence remained remarkably high, throughout the time periods at 14-18%. Taken together, our data indicate that using non-authenticated cell lines from secondary sources carries a risk of about 1:6 for obtaining a false cell line. The use of authentic leukemia-lymphoma cell lines holds important translational value for their model character and the reproducibility of the laboratory data in the clinical arena. © 2016 UICC.

  16. Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives.

    Science.gov (United States)

    Dumont, Jennifer; Euwart, Don; Mei, Baisong; Estes, Scott; Kshirsagar, Rashmi

    2016-12-01

    Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s. Although most biotherapeutic proteins developed to date have been produced using the mammalian Chinese hamster ovary and murine myeloma (NS0, Sp2/0) cell lines, there has been a recent shift toward the use of human cell lines. One of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Although other mammalian cell lines can produce PTMs similar to human cells, they also produce non-human PTMs, such as galactose-α1,3-galactose and N-glycolylneuraminic acid, which are potentially immunogenic. In addition, human cell lines are grown easily in a serum-free suspension culture, reproduce rapidly and have efficient protein production. A possible disadvantage of using human cell lines is the potential for human-specific viral contamination, although this risk can be mitigated with multiple viral inactivation or clearance steps. In addition, while human cell lines are currently widely used for biopharmaceutical research, vaccine production and production of some licensed protein therapeutics, there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins (compared with other types of cell lines). With additional research investment, human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins.

  17. Isolation of cancer stem cells from three human glioblastoma cell lines: characterization of two selected clones.

    Directory of Open Access Journals (Sweden)

    Fortunata Iacopino

    Full Text Available Cancer stem cells (CSC were isolated via a non-adherent neurosphere assay from three glioma cell lines: LI, U87, and U373. Using a clonal assay, two clones (D2 and F11 were selected from spheres derived from LI cells and were characterized for the: expression of stem cell markers (CD133, Nestin, Musashi-1 and Sox2; proliferation; differentiation capability (determined by the expression of GalC, βIII-Tubulin and GFAP; Ca(2+ signaling and tumorigenicity in nude mice. Both D2 and F11 clones expressed higher levels of all stem cell markers with respect to the parental cell line. Clones grew more slowly than LI cells with a two-fold increase in duplication time. Markers of differentiation (βIII-Tubulin and GFAP were expressed at high levels in both LI cells and in neurospheres. The expression of Nestin, Sox2, and βIII-Tubulin was down-regulated in D2 and F11 when cultured in serum-containing medium, whereas Musashi-1 was increased. In this condition, duplication time of D2 and F11 increased without reaching that of LI cells. D2, F11 and parental cells did not express voltage-dependent Ca(2+-channels but they exhibited increased intracellular Ca(2+ levels in response to ATP. These Ca(2+ signals were larger in LI cells and in spheres cultured in serum-containing medium, while they were smaller in serum-free medium. The ATP treatment did not affect cell proliferation. Both D2 and F11 induced the appearance of tumors when ortotopically injected in athymic nude mice at a density 50-fold lower than that of LI cells. All these data indicate that both clones have characteristics of CSC and share the same stemness properties. The findings regarding the expression of differentiation markers and Ca(2+-channels show that both clones are unable to reach the terminal differentiation. Both D2 and F11 might represent a good model to improve the knowledge on CSC in glioblastoma and to identify new therapeutic approaches.

  18. Production Process for Stem Cell Based Therapeutic Implants: Expansion of the Production Cell Line and Cultivation of Encapsulated Cells

    Science.gov (United States)

    Weber, C.; Pohl, S.; Poertner, R.; Pino-Grace, Pablo; Freimark, D.; Wallrapp, C.; Geigle, P.; Czermak, P.

    Cell based therapy promises the treatment of many diseases like diabetes mellitus, Parkinson disease or stroke. Microencapsulation of the cells protects them against host-vs-graft reactions and thus enables the usage of allogenic cell lines for the manufacturing of cell therapeutic implants. The production process of such implants consists mainly of the three steps expansion of the cells, encapsulation of the cells, and cultivation of the encapsulated cells in order to increase their vitality and thus quality. This chapter deals with the development of fixed-bed bioreactor-based cultivation procedures used in the first and third step of production. The bioreactor system for the expansion of the stem cell line (hMSC-TERT) is based on non-porous glass spheres, which support cell growth and harvesting with high yield and vitality. The cultivation process for the spherical cell based implants leads to an increase of vitality and additionally enables the application of a medium-based differentiation protocol.

  19. Rice callus suspension culture inhibits growth of cell lines of multiple cancer types and induces apoptosis in lung cancer cell line.

    Science.gov (United States)

    Rahman, Nafeesa; Dhadi, Surendar Reddy; Deshpande, Aparna; Ramakrishna, Wusirika

    2016-11-02

    Cancer is one of the leading cause of mortality. Even though efficient drugs are being produced to treat cancer, conventional medicines are costly and have adverse effects. As a result, alternative treatments are being tried due to their low cost and little or no adverse effects. Our previous study identified one such alternative in rice callus suspension culture (RCSC) which was more efficient than Taxol® and Etoposide, in reducing the viability of human colon and renal cancer cells in culture with minimal or no effect on a normal cell line. In this study, we tested the effect of RCSC by studying the dynamics of lactate dehydrogenase (LDH) in lung cancer cell lines (NCI-H460 and A549), breast cancer cell lines (MDA-MB-231 and MCF-7) and colorectal cancer cell lines (SW620 and Caco-2) as well as their normal-prototypes. Complementary analysis for evaluating membrane integrity was performed by estimating LDH release in non-lysed cells and cell viability with WST-1 assay. Fluorescence microscopy with stains targeting nucleus and cell membrane as well as caspase 3/7 and Annexin V assays were performed. Real-time quantitative RT-PCR was performed to evaluate expression of 92 genes associated with molecular mechanisms of cancer in RCSC treated ling cancer cell line, NCI-H460 and its normal prototype, MRC-5. High performance liquid chromatography (HPLC) was used to collect RCSC fractions, which were evaluated on NCI-H460 for their anti-cancer activity. Lower dilutions of RCSC showed maximum reduction in total LDH indicating reduced viability in majority of the cancer cell lines tested with minimal or no effect on normal cell lines compared to the control. Complementary analysis based on LDH release in non-lysed cells and WST-1 assay mostly supported total LDH results. RCSC showed the best effect on the lung non-small carcinoma cell line, NCI-H460. Fluorescence microscopy analyses suggested apoptosis as the most likely event in NCI-H460 treated with RCSC. Gene expression

  20. Propagation of Asian isolates of canine distemper virus (CDV in hamster cell lines

    Directory of Open Access Journals (Sweden)

    Yamaguchi Ryoji

    2009-10-01

    Full Text Available Abstract Backgrounds The aim of this study was to confirm the propagation of various canine distemper viruses (CDV in hamster cell lines of HmLu and BHK, since only a little is known about the possibility of propagation of CDV in rodent cells irrespective of their epidemiological importance. Methods The growth of CDV in hamster cell lines was monitored by titration using Vero.dogSLAMtag (Vero-DST cells that had been proven to be susceptible to almost all field isolates of CDV, with the preparations of cell-free and cell-associated virus from the cultures infected with recent Asian isolates of CDV (13 strains and by observing the development of cytopathic effect (CPE in infected cultures of hamster cell lines. Results Eleven of 13 strains grew in HmLu cells, and 12 of 13 strains grew in BHK cells with apparent CPE of cell fusion in the late stage of infection. Two strains and a strain of Asia 1 group could not grow in HmLu cells and BHK cells, respectively. Conclusion The present study demonstrates at the first time that hamster cell lines can propagate the majority of Asian field isolates of CDV. The usage of two hamster cell lines suggested to be useful to characterize the field isolates biologically.

  1. Establishment of an agamid cell line and isolation of adenoviruses from central bearded dragons (Pogona vitticeps).

    Science.gov (United States)

    Ball, Inna; Hoferer, Marc; Marschang, Rachel E

    2014-03-01

    A cell line was established from whole 6-8-week-old central bearded dragon (Pogona vitticeps) embryos. Cells were mid-sized and showed an elongated and polymorphic form. The cell line grew in a monolayer and has been serially passaged for 17 passages at time of publication. This cell line has been used with samples from adenovirus polymerase chain reaction (PCR)-positive bearded dragons, and 2 virus isolates have been obtained so far. The isolates show a clear cytopathic effect in inoculated cells. Both virus isolates have been serially passaged on this cell line, and have been identified by PCR amplification and sequencing of a portion of the DNA-dependent DNA polymerase gene and show 100% nucleotide identity to the corresponding region of an agamid adenovirus. Electron microscopic examination of supernatant from infected cells demonstrated the presence of nonenveloped particles, with a diameter of approximately 80 nm in both virus isolates.

  2. The transcriptional diversity of 25 Drosophila cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Cherbas, Lucy [Indiana Univ., Bloomington, IN (United States); Willingham, Aarron [Affymetrix Inc., Santa Clara, CA (United States); Zhang, Dayu [Indiana Univ., Bloomington, IN (United States); Yang, Li [University of Connecticut Health Center, Farmington, Connecticut (United States); Zou, Yi [Indiana Univ., Bloomington, IN (United States); Eads, Brian D. [Indiana Univ., Bloomington, IN (United States); Carlson, Joseph W. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Landolin, Jane M. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Kapranov, Philipp [Affymetrix Inc., Santa Clara, CA (United States); Dumais, Jacqueline [Affymetrix Inc., Santa Clara, CA (United States); Samsonova, Anastasia [Harvard Medical School, Boston, MA (United States); Choi, Jeong-Hyeon [Indiana Univ., Bloomington, IN (United States); Roberts, Johnny [Indiana Univ., Bloomington, IN (United States); Davis, Carrie A. [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Tang, Haixu [Indiana Univ., Bloomington, IN (United States); van Baren, Marijke J. [Washington Univ., St. Louis, MO (United States); Ghosh, Srinka [Affymetrix Inc., Santa Clara, CA (United States); Dobin, Alexander [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Bell, Kim [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Lin, Wei [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Langton, Laura [Washington Univ., St. Louis, MO (United States); Duff, Michael O. [University of Connecticut Health Center, Farmington, Connecticut (United States); Tenney, Aaron E. [Washington Univ., St. Louis, MO (United States); Zaleski, Chris [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Brent, Michael R. [Washington Univ., St. Louis, MO (United States); Hoskins, Roger A. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Kaufman, Thomas C. [Indiana University, Bloomington, Indiana (United States); Andrews, Justen [Indiana University, Bloomington, Indiana (United States); Graveley, Brenton R. [University of Connecticut Health Center, Farmington, Connecticut (United States); Perrimon, Norbert [Harvard Medical School, Boston, MA (United States); Howard Hughes Medical Institute, Boston, MA (United States); Celniker, Susan E. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Gingeras, Thomas R. [Affymetrix Inc., Santa Clara, CA (United States); Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Cherbas, Peter [Indiana Univ., Bloomington, IN (United States)

    2010-12-22

    Drosophila melanogaster cell lines are important resources for cell biologists. In this article, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. We report the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25

  3. Generation of a predictive melphalan resistance index by drug screen of B-cell cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Martin Boegsted

    Full Text Available BACKGROUND: Recent reports indicate that in vitro drug screens combined with gene expression profiles (GEP of cancer cell lines may generate informative signatures predicting the clinical outcome of chemotherapy. In multiple myeloma (MM a range of new drugs have been introduced and now challenge conventional therapy including high dose melphalan. Consequently, the generation of predictive signatures for response to melphalan may have a clinical impact. The hypothesis is that melphalan screens and GEPs of B-cell cancer cell lines combined with multivariate statistics may provide predictive clinical information. MATERIALS AND METHODS: Microarray based GEPs and a melphalan growth inhibition screen of 59 cancer cell lines were downloaded from the National Cancer Institute database. Equivalent data were generated for 18 B-cell cancer cell lines. Linear discriminant analyses (LDA, sparse partial least squares (SPLS and pairwise comparisons of cell line data were used to build resistance signatures from both cell line panels. A melphalan resistance index was defined and estimated for each MM patient in a publicly available clinical data set and evaluated retrospectively by Cox proportional hazards and Kaplan-Meier survival analysis. PRINCIPAL FINDINGS: Both cell line panels performed well with respect to internal validation of the SPLS approach but only the B-cell panel was able to predict a significantly higher risk of relapse and death with increasing resistance index in the clinical data sets. The most sensitive and resistant cell lines, MOLP-2 and RPMI-8226 LR5, respectively, had high leverage, which suggests their differentially expressed genes to possess important predictive value. CONCLUSION: The present study presents a melphalan resistance index generated by analysis of a B-cell panel of cancer cell lines. However, the resistance index needs to be functionally validated and correlated to known MM biomarkers in independent data sets in order to

  4. Comparison of the rat and mouse cell lines commercially available for CALUX bioassays

    Energy Technology Data Exchange (ETDEWEB)

    Goeyens, L.; Windal, I.; Wouwe, N. van [Institute of Public Health, Brussels (Belgium); Scippo, M.L.; Eppe, G.; Pauw, E. de; Maghin-Rogister, G. [Liege Univ. (Belgium)

    2004-09-15

    CALUX (Chemical-activated luciferase gene expression) is nowadays more and more widely used, both for the control of the norms applied to the food chain and to environmental contamination evaluation. In that purpose, two cell lines are commercially available: one rat cell line, commercialized by Bio Detection System (BDS, The Netherlands) and one mouse cell line, commercialized by Xenobiotic Detection System (XDS, USA). Both suppliers propose different clean-up methods and a slightly different method in the preparation, dosage and reading of the plates. Until now, almost no comparison of the cell lines has been performed, or the comparison includes many variables (extraction, purification, method of preparation, dosage and reading of the plate) so that it is difficult to evaluate which variables are mainly responsible of the observed differences. The objective of the research presented here is to perform a direct comparison of the 2 cell lines, and evaluate which variables can be responsible of the discrepancy observed between the results.

  5. Spheroid body-forming cells in the human gastric cancer cell line MKN-45 possess cancer stem cell properties.

    Science.gov (United States)

    Liu, Jianming; Ma, Lilin; Xu, Junfei; Liu, Chun; Zhang, Jianguo; Liu, Jie; Chen, Ruixin; Zhou, Youlang

    2013-02-01

    The cancer stem cell theory hypothesizes that cancer stem cells (CSCs), which possess self-renewal and other stem cell properties, are regarded as the cause of tumor formation, recurrence and metastasis. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. In this study, we enriched gastric cancer stem cells through spheroid body formation by cultivating the human gastric cancer cell line MKN-45 in defined serum-free medium. The stemness characteristics of spheroid body-forming cells, including self-renewal, proliferation, chemoresistance, tumorigenicity of the MKN-45 spheroid body-forming cells were evaluated, and the expression levels of stemness genes and related proteins in the MKN-45 spheroid body-forming cells were assessed. Furthermore, immunofluorescence staining for the stem cell markers on spheroid body-forming cells was examined to evaluate the association between stemness factors (Oct4, Sox2, Nanog) and the proposed CSC marker CD44. Our data demonstrated that non-adherent spheroid body-forming cells from the gastric cancer cell line MKN-45 cultured in stem cell-conditioned medium possessed gastric CSC properties, such as persistent self-renewal, extensive proliferation, drug resistance, high tumorigenic capacity and overexpression of CSC-related genes and proteins (Oct4, Sox2, Nanog and CD44), compared with the parental cells. More importantly, CD44-positive cells co-expressing the pluripotency genes Oct4, Sox2 and Nanog may represent gastric CSCs. Further experiments using more refined selection criteria such as a combination of two or multiple markers would be useful to specifically identify and purify CSCs.

  6. 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.

  7. Distinct population of highly malignant cells in a head and neck squamous cell carcinoma cell line established by xenograft model

    Directory of Open Access Journals (Sweden)

    Jan Chia-Ing

    2009-11-01

    Full Text Available Abstract The progression and metastasis of solid tumors, including head and neck squamous cell carcinoma (HNSCC, have been related to the behavior of a small subpopulation of cancer stem cells. Here, we have established a highly malignant HNSCC cell line, SASVO3, from primary tumors using three sequential rounds of xenotransplantation. SASVO3 possesses enhanced tumorigenic ability both in vitro and in vivo. Moreover, SASVO3 exhibits properties of cancer stem cells, including that increased the abilities of sphere-forming, the number of side population cells, the potential of transplanted tumor growth and elevated expression of the stem cell marker Bmi1. Injection of SASVO3 into the tail vein of nude mice resulted in lung metastases. These results are consistent with the postulate that the malignant and/or metastasis potential of HNSCC cells may reside in a stem-like subpopulation.

  8. Interactions between osteosarcoma cell lines and dendritic cells immune function: An in vitro study.

    Science.gov (United States)

    Muraro, Michela; Mereuta, Oana M; Saglio, Francesco; Carraro, Francesca; Berger, Massimo; Madon, Enrico; Fagioli, Franca

    2008-01-01

    Dendritic cells (DCs) might be partly responsible for the defective immune response in tumor bearing hosts, but no study in osteosarcoma patients is still available. Therefore, we investigated in vitro whether human osteosarcoma cell lines have an inhibitor effect on different types of DCs: CD14+DCs, DC1 and DC2. DCs derived from healthy donors were cultured with osteosarcoma cell lines and appropriate cytokine cocktails and analysed for the expression of co-stimulatory molecules (CD40, CD80, CD83, CD86, HLA-DR). Each interaction resulted in a lower phenotypic expression of the DCs maturation markers, especially on DC2. Moreover, the addition of various cytokines and compounds (rhIL-12, CD40L, Indometacin) induced the DC1 and DC2 subsets towards the Th1 pattern as shown by ELISA. Osteosarcoma highly interferes with an in vitro DCs immune function as antigen presenting cells. The understanding of tumor biology underlines the need for a specific osteosarcoma immunotherapy able to reverse this immune-surveillance inhibition.

  9. Human thymic epithelial cells present superantigens to T-cell lines and thymocytes

    DEFF Research Database (Denmark)

    Jlrgensen, A; Nielsen, M; Svejgaard, A

    1996-01-01

    It is generally accepted that thymic epithelial cells (TEC) act as accessory cells in positive selection of pre-T cells. However, our knowledge of the antigen presentation and accessory cell function to human TEC is limited. Here we present results obtained by the use of serum-free cultured human...... TEC, showing that IFN-gamma-treated TEC are able to support T-cell-mediated responses to the bacterial superantigens (Sag) SEA and SEB, even at very low Sag concentrations. T-cell responses to TEC-presented Sags were dependent on the presence of the adhesion molecules ICAM-1, ICAM-2, LFA-1, and LFA-3......, but not on CD4 and CD8 molecules. There is a low but significant expression of B7 molecules on human TEC, and treatment of TEC with anti-B7.1 and anti-B7.2 antibodies before Sag pulsing leads to decreased Sag responses, indicating a significant importance of B7 molecules on TEC. Both CD4+ T-cell lines and CD4...

  10. 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.

  11. Induction of delayed-type hypersensitivity by the T cell line specific to bacterial peptidoglycans

    Energy Technology Data Exchange (ETDEWEB)

    Katsuki, M.; Kakimoto, K.; Kawata, S.; Kotani, S.; Koga, T.

    1987-12-01

    A T cell line specific for the chemically well-defined peptidoglycan of bacterial cell wall, disaccharide tetrapeptide, was established from Lewis rats immunized with the antigen covalently linked to the autologous rat serum albumin. The antigen specificity was examined with various analogues or derivatives of the peptidoglycan. The cell line was reactive to analogues with the COOH-terminal D-amino acid, but least reactive to those with L-amino acid as COOH terminus. Transferring of the T cell line into X-irradiated normal Lewis rats induced delayed-type hypersensitivity in an antigen specific manner.

  12. Derivation of Trisomy 21 affected human embryonic stem cell line Genea021

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea021 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Trisomy 21, indicative of Down Syndrome. Following ICM outgrowth on inactivated human feeders, CGH and STR analyses demonstrated a 47, XY, +21 karyotype and male allele pattern. The hESC line had pluripotent cell morphology, 71% of cells expressed Nanog, 84% Oct4, 23% Tra1–60 and 95% SSEA4, gave a Pluritest Pluripotency score of 21.85, Novelty of 1.42, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  13. Robust regeneration of adult zebrafish lateral line hair cells reflects continued precursor pool maintenance.

    Science.gov (United States)

    Cruz, Ivan A; Kappedal, Ryan; Mackenzie, Scott M; Hailey, Dale W; Hoffman, Trevor L; Schilling, Thomas F; Raible, David W

    2015-06-15

    We have examined lateral line hair cell and support cell maintenance in adult zebrafish when growth is largely complete. We demonstrate that adult zebrafish not only replenish hair cells after a single instance of hair cell damage, but also maintain hair cells and support cells after multiple rounds of damage and regeneration. We find that hair cells undergo continuous turnover in adult zebrafish in the absence of damage. We identify mitotically-distinct support cell populations and show that hair cells regenerate from underlying support cells in a region-specific manner. Our results demonstrate that there are two distinct support cell populations in the lateral line, which may help explain why zebrafish hair cell regeneration is extremely robust, retained throughout life, and potentially unlimited in regenerative capacity. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Low levels of Her2/neu expressed by Ewing's family tumor cell lines can redirect cytokine-induced killer cells.

    Science.gov (United States)

    Verneris, Michael R; Arshi, Arash; Edinger, Matthias; Kornacker, Martin; Natkunam, Yaso; Karimi, Mobin; Karami, Mobin; Cao, Yu-An; Marina, Neyssa; Contag, Christopher H; Negrin, Robert S

    2005-06-15

    To identify novel treatments for pediatric solid tumors and/or for malignancies with low-level Her2/neu expression. Using fluorescence-activated cell sorting and immunohistochemistry, Her2/neu expression was determined on cell lines derived vfrom Ewing's family tumors (EFT) and neuroblastoma. Sensitivity to trastuzumab treatment was investigated using an in vitro proliferation assay. Cytotoxicity against EFT cell lines was done with either freshly isolated or ex vivo activated and expanded T cells (cytokine-induced killer cells, CIK cells), with or without addition of a CD3xHer2/neu bispecific antibody. The effects of either trastuzumab, CIK cells alone, or CD3xHer2/neu bispecific antibody redirected CIK cells was determined using a SCID/hu model of EFTs and serial, noninvasive bioluminescent imaging. EFT cell lines express 5- to 10-fold lower levels of her2/neu than either breast (BT-474) or ovarian (SK-OV-3) cell lines. Treatment of EFT cell lines with trastuzumab did not induce growth inhibition either in vitro or in vivo. In contrast, Her2/neu could be used to redirect CIK cell to mediate cytotoxicity against EFTs both in vitro and in vivo (using two different treatment schemas). CD3xHer2/neu bispecific antibody and CIK cells may be a suitable approach to treat malignancies with low-level Her2/neu expression not responsive to trastuzumab.

  15. Action spectra (254-302 nm) for four human photosensitive cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Coohill, T.P.; Moore, S.P.; Grider, R.A. (Western Kentucky Univ., Bowling Green (USA))

    1983-07-01

    The inactivation of cellular viral capacity for Herpes simplex type I growth in six separate wavelengths (254-302 nm) was measured in five human cell lines. These consisted of one ''normal''-skin fibroblast line (KD), and four photosensitive lines. Two lines of Xeroderma pigmentosum, one of Bloom's syndrome, and one of Cockayne's syndrome cells were used. Similar relative sensitivity were observed for the Bloom's syndrome. Xeroderma pigmentosum, and normal cell lines. The Cockayne's syndrome-line became relatively more sensitive at 289 nm and longer wavelengths. Absolute sensitivities varied. Some divergence in response was noted at the longest wavelength tested, 302 nm.

  16. Establishment of a Vero cell line persistently infected with African swine fever virus.

    OpenAIRE

    Salas, J; Viñuela, E

    1986-01-01

    A Vero cell line persistently infected with African swine fever virus was established by infecting the cells in the presence of 10 mM NH4Cl (Vero-P cell line). The virus derived from the Vero-P cultures infected Vero cells, and virus titers were comparable to those obtained in Vero cells acutely infected with African swine fever virus. The structural proteins of the virus from Vero-P cells were similar to those of the virus produced in lytic infections. Virus production was low when the Vero-...

  17. Cell lines from grc congenic strains of rats having different susceptibilities to chemical carcinogens.

    Science.gov (United States)

    Lu, D; Kunz, H W; Melhem, M F; Gill, T J

    1993-09-01

    The growth and reproduction complex (grc-) strains of rats have a 70-kilobase deletion in the major histocompatibility complex (MHC)-linked grc-G/C region that is associated with embryonic death, developmental defects, and an increased susceptibility to chemical carcinogens. To study further the effects associated with the deletion, fibroblastic cell lines from grc-, grc+, and grc+/- rat embryos were developed: BIL-derived cell lines are congenic for the MHC and grc, whereas R16-derived cell lines are congenic for the grc alone. In early passages, all cell lines expressed the MHC class I antigen RT1.A, had a diploid chromosome number, and did not display anchorage-independent growth or in vivo tumorigenicity. The grc- cells [median population doubling time (PDT), 47 h] grew more slowly than the grc+ (PDT, 30.5 h) and grc+/- (PDT, 33 h) cells. All cells underwent crisis, but the crisis stage began earlier and lasted longer in the grc- cells. The established grc- cell lines (PDT, 32.5 h) grew faster than the grc+ (PDT, 48.5 h) and grc+/- (PDT, 54 h) cell lines. Two of the three BIL-derived grc- lines that survived crisis became anchorage independent in tissue culture and tumorigenic in histocompatible F1 rats (highly malignant fibrosarcomas) at passages 33 and 48, respectively; by contrast, none of the R16-derived grc- cell lines transformed. None of 8 grc+ or 8 grc+/- cell lines that survived crisis displayed anchorage-independent growth or tumorigenicity under the same conditions up to passage 50. All of the established cell lines, including the two tumorigenic ones, expressed MHC class I antigens. Southern and Northern blot analyses of BIL-derived cell lines before and after crisis showed that they all constitutively expressed H-ras and Rb and that no cell line showed rearrangement, amplification, or overexpression of c-myc, H-ras, Rb, and p53 either before or after crisis. These observations indicate that: (a) the homozygous grc- deletion is necessary but not

  18. BETULINIC ACID WAS MORE CYTOTOXIC TOWARDS THE HUMAN BREAST CANCER CELL LINE MDA-MB-231 THAN THE HUMAN PROMYELOCYTIC LEUKAEMIA CELL LINE HL-60

    Directory of Open Access Journals (Sweden)

    LATIFAH SAIFUL YAZAN

    2009-01-01

    Full Text Available Betulinic acid (BA is a pentacyclic triterpene found in several botanical sources that has been shown to cause apoptosis in a number of cell lines. This study was undertaken to determine the in vitro cytotoxic properties of BA towards the human mammary carcinoma cell line MDA-MB-231 and the human promyelocytic leukaemia cell line HL-60 and the mode of the induced cell death. The cytotoxicity and mode of cell death of BA were determined using the MTT assay and DNAfragmentation analysis, respectively. In our study, the compound was found to be cytotoxic to MDA-MB-231 and HL-60 cells with IC50 values of 58 μg/mL and 134 μg/mL, respectively. Cells treated with high concentrations of BA exhibited features characteristic of apoptosis such as blebbing, shrinking and a number of small cytoplasm body masses when viewed under an inverted light microscope after 24 h. The incidence of apoptosis in MDA-MB-231 was further confirmed bythe DNA fragmentation analysis, with the formation of DNA fragments of oligonucleosomal size (180-200 base pairs, giving a ladder-like pattern on agarose gel electrophoresis. BA was more cytotoxic towards MDA-MB-231 than HL-60 cells, and induced apoptosis in MDA-MB-231 cells.

  19. Biological characteristics and chemosensitivity profile of four human anaplastic thyroid carcinoma cell lines.

    Science.gov (United States)

    Sekiguchi, M; Shiroko, Y; Arai, T; Kishino, T; Sugawara, I; Kusakabe, T; Suzuki, T; Yamashita, T; Obara, T; Ito, K; Hasumi, K

    2001-10-01

    Anaplastic thyroid carcinoma is a rapidly growing, aggressive neoplasm affecting the elderly which does not respond to most of the therapies. We established cultured cell lines from four untreated tumors. The cultures grew in a monolayer of spindle-shaped cells in three cell lines and of small polygonal cells in one line, having relatively long doubling times and chromosomal abnormalities. The xenotransplantation of the lines in athymic nude mice produced tumors with a histology similar to the original tumors. The immunocytochemical staining showed the expression of PCNA, HLA-class 1, cytokeratin, vimentin and FAS (fatty acid synthase) but not CEA, desmin or P-glycoprotein. The lines secreted TPA, IL-6, IL-8 and few or no thyroid-related hormones in the culture supernatant. One cell line produced G-CSF. The chemosensitivity assay revealed intrinsic drug resistance to nine out of 11 antineoplastic agents. The reverse transcriptase-polymerase chain reaction (RT-PCR) detected MRP (multidrug resistance-associated protein) mRNA but not mdr (multidrug resistance protein)-1 and mdr-3 mRNAs. This finding indicates that the multidrug resistance of these lines is mediated by a P-glycoprotein-unrelated mechanism. The RT-PCR also presented FAS mRNA in all the lines, and IL-6 and IL-8 mRNAs in some of the lines.

  20. Comparative proteomic analysis of cell lines and scrapings of the human intestinal epithelium

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    Renes Johan

    2007-04-01

    Full Text Available Abstract Background In vitro models are indispensable study objects in the fields of cell and molecular biology, with advantages such as accessibility, homogeneity of the cell population, reproducibility, and growth rate. The Caco-2 cell line, originating from a colon carcinoma, is a widely used in vitro model for small intestinal epithelium. Cancer cells have an altered metabolism, making it difficult to infer their representativity for the tissue from which they are derived. This study was designed to compare the protein expression pattern of Caco-2 cells with the patterns of intestinal epithelial cells from human small and large intestine. HT-29 intestinal cells, Hep G2 liver cells and TE 671 muscle cells were included too, the latter two as negative controls. Results Two-dimensional gel electrophoresis was performed on each tissue and cell line protein sample. Principal component and cluster analysis revealed that global expression of intestinal epithelial scrapings differed from that of intestinal epithelial cell lines. Since all cultured cell lines clustered together, this finding was ascribed to an adaptation of cells to culture conditions and their tumor origin, and responsible proteins were identified by mass spectrometry. When investigating the profiles of Caco-2 cells and small intestinal cells in detail, a considerable overlap was observed. Conclusion Numerous proteins showed a similar expression in Caco-2 cells, HT-29 cells, and both the intestinal scrapings, of which some appear to be characteristic to human intestinal epithelium in vivo. In addition, several biologically significant proteins are expressed at comparable levels in Caco-2 cells and small intestinal scrapings, indicating the usability of this in vitro model. Caco-2 cells, however, appear to over-express as well as under-express certain proteins, which needs to be considered by scientists using this cell line. Hence, care should be taken to prevent misinterpretation of

  1. Establishment and characterization of two new cell lines derived from human metastatic breast carcinomas.

    Science.gov (United States)

    Zoli, W; Roncuzzi, L; Zini, N; Lenzi, L; Gruppioni, R; Barzanti, F; Sensi, A; Amadori, D; Gasperi-Campani, A

    1997-04-01

    Two human cancer cell lines (MA 2 and MA 3) were established from pleural effusions of infiltrating ductal carcinomas of the breast. The lines were maintained in continuous monolayer culture with doubling times of 70 (MA 2) and 78 (MA 3) hr for more than two years and possessed extensively rearranged abnormal karyo-types with modal chromosome number of 83 (MA 2) and 81 (MA 3) and DNA index values of 1.65 and 1.77, respectively. No amplifications or rearrangements were evident in the c-myc, int-2, c-erb B2, c-Ha-ras, or hst 1 genes in MA 2 and MA 3 cell lines. The clinical histories of the patients from whom the cell lines were derived are reported and compared with the results observed in the cell lines in vitro. The presence of CEA, CA 15-3, and MCA tumor markers observed in the primary tumor tissues was retained by the established cell lines. While the primary tumor tissues were ER+/PgR borderline+ (MA 2) and ER-/PgR+ (MA 3), the MA 2 line was ER+/PgR- and the MA 3 line remained ER-/PgR+. The MDR P-glycoprotein was not expressed either in primary tumor tissues or in the respective cell lines. High expression of cytokeratins 7, 18, and 19 was evident by immunohistochemical analysis in each cell line. whereas cytokeratins 8 and 17 were poorly or not at all expressed. The treatment history of the patients from whom the cell lines were derived involved CMF followed six months later by novantrone and cisplatin plus VP 16 (MA 2) and FEC followed four years later by CMF (MA 3). The chemosensitivity pattern assay of the cell lines indicated that the MA 2 line was sensitive to doxorubicin, cisplatin, and vinblastine, whereas the MA 3 line was sensitive to doxorubicin and cisplatin. The characteristics of these cell lines indicate them to be a good experimental model to investigate breast cancer biology and anticancer drug response.

  2. Hepatitis C virus infection of cholangiocarcinoma cell lines

    NARCIS (Netherlands)

    Fletcher, Nicola F.; Humphreys, Elizabeth; Jennings, Elliott; Osburn, William; Lissauer, Samantha; Wilson, Garrick K.; van Ijzendoorn, Sven C. D.; Baumert, Thomas F.; Balfe, Peter; Afford, Simon; McKeating, Jane A.

    Hepatitis C virus (HCV) infects the liver and hepatocytes are the major cell type supporting viral replication. Hepatocytes and cholangiocytes derive from a common hepatic progenitor cell that proliferates during inflammatory conditions, raising the possibility that cholangiocytes may support HCV

  3. Adhesion of Epstein–Barr virus-positive natural killer cell lines to cultured endothelial cells stimulated with inflammatory cytokines

    Science.gov (United States)

    Kanno, H; Watabe, D; Shimizu, N; Sawai, T

    2008-01-01

    Chronic active Epstein–Barr virus (EBV) infection (CAEBV) is characterized by chronic recurrent infectious mononucleosis-like symptoms. Approximately one-fourth of CAEBV patients develop vascular lesions with infiltration of EBV-positive lymphoid cells. Furthermore, EBV-positive natural killer (NK)/T cell lymphomas often exhibit angiocentric or angiodestructive lesions. These suggest an affinity of EBV-positive NK/T cells to vascular components. In this study, we evaluated the expression of adhesion molecules and cytokines in EBV-positive NK lymphoma cell lines, SNK1 and SNK6, and examined the role of cytokines in the interaction between NK cell lines and endothelial cells. SNKs expressed intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) at much higher levels than those in EBV-negative T cell lines. SNKs produced the larger amount of tumour necrosis factor (TNF)-α, which caused increased expression of ICAM-1 and VCAM-1 in cultured human endothelial cells, than that from EBV-negative T cell lines. Furthermore, SNKs exhibited increased adhesion to cultured endothelial cells stimulated with TNF-α or interleukin (IL)-1β, and the pretreatment of cytokine-stimulated endothelial cells with anti-VCAM-1-antibodies reduced cell adhesion. These indicate that the up-regulated expression of VCAM-1 on cytokine-stimulated endothelial cells would be important for the adhesion of EBV-positive NK cells and might initiate the vascular lesions. PMID:18190605

  4. Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks

    Science.gov (United States)

    Johnen, Georg; Rozynek, Peter; von der Gathen, Yvonne; Bryk, Oleksandr; Zdrenka, Ricarda; Johannes, Christian; Weber, Daniel G.; Igwilo-Okuefuna, O′Brien; Raiko, Irina; Hippler, Jörg; Brüning, Thomas; Dopp, Elke

    2013-01-01

    Background UROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity. Methods UROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR) profiling was applied. Results All tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly. Conclusions It is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments. PMID:23691160

  5. Cross-contamination of a UROtsa stock with T24 cells--molecular comparison of different cell lines and stocks.

    Directory of Open Access Journals (Sweden)

    Georg Johnen

    Full Text Available BACKGROUND: UROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity. METHODS: UROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR profiling was applied. RESULTS: All tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly. CONCLUSIONS: It is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments.

  6. Successful Reconstruction of Tooth Germ with Cell Lines Requires Coordinated Gene Expressions from the Initiation Stage

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    Yasuhiro Tomooka

    2012-10-01

    Full Text Available Tooth morphogenesis is carried out by a series of reciprocal interactions between the epithelium and mesenchyme in embryonic germs. Previously clonal dental epithelial cell (epithelium of molar tooth germ (emtg lines were established from an embryonic germ. They were odontogenic when combined with a dental mesenchymal tissue, although the odontogenesis was quantitatively imperfect. To improve the microenvironment in the germs, freshly isolated dental epithelial cells were mixed with cells of lines, and germs were reconstructed in various combinations. The results demonstrated that successful tooth construction depends on the mixing ratio, the age of dental epithelial cells and the combination with cell lines. Analyses of gene expression in these germs suggest that some signal(s from dental epithelial cells makes emtg cells competent to communicate with mesenchymal cells and the epithelial and mesenchymal compartments are able to progress  odontogenesis from the initiation stage.

  7. Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Mu-Yun [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Shen, Yuh-Chiang [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); National Research Institute of Chinese Medicine, Taipei, Taiwan (China); Lu, Chien-Hsing [Department of Obstetrics and Gynecology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Obstetrics and Gynecology, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Yang, Shu-Yi [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Ho, Tsing-Fen [Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan (China); Peng, Yu-Ta [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Chang, Chia-Che, E-mail: chia_che@dragon.nchu.edu.tw [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China)

    2012-12-15

    Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ER stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosin's tumoricidal effect. Mechanistically, prodigiosin engages the IRE1–JNK and PERK–eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death. Highlights: ► Prodigiosin is a bacterial tripyrrole pigment with potent anticancer effect. ► Prodigiosin is herein identified

  8. SU-C-204-04: Irradiation of Human Cell Lines Using Various Ions

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Y; McMahon, S; Kaminuma, T; Held, K [Harvard Medical School, Boston MA (United States); Tessa, C; Rusek, A [Brookhaven National Laboratory, Upton, NY (United States)

    2016-06-15

    Purpose: The purpose of this study is to investigate and quantify the biological effects of ion radiation using several human cell lines. We aim to answer the question of whether carbon ion the most ideal ion species for heavy ion radiotherapy. Methods: The cells were irradiated at different positions along the pristine Bragg peak of several ions with different atomic number. The biological effectiveness was evaluated using the clonogenic cell survival assay. Irradiation of three human lung cancer cell lines and a fibroblast cell line were undertaken using the charged particle beam at the NASA Space Radiation Laboratory at Brookhaven National Lab. Four mono-energetic ion beams (carbon, oxygen, helium and lithium) were used to irradiate the cells. Water or media-filled T25 flasks were lined up along the beam line so that the cell-containing surfaces of the flasks were placed at a specific depth along the pristine Bragg curve. Four depths along the curve, representing entrance point, rising peak, peak and distal fall off, were selected to determine biological effectiveness. Gaf-chromic films were placed between the flasks to monitor the irradiation as soon as it was finished. Results: For all ion radiations, the maximum cell killing effect occurs at either peak or distal fall off, depending on the cell lines. For instance, for the fibroblast cell line AGO1522, RBEs of 1.4, 1.2, 1.4 and 1.9 were observed at the Bragg peak for Helium, Lithium, Carbon and Oxygen ions. Comparing positions, RBEs of 0.9, 1.2, 1.4 and 1.8 were observed for carbon irradiation of AGO-1522 cells positions corresponding to entrance, rising peak, peak and distal fall off. Conclusion: RBE values differ with position in the Bragg peak, ion species and cell line. Ions other than carbon may prove more effective in certain irradiation conditions and may contribute to optimized heavy ion therapy.

  9. In vitro segregation of different cell lines with neuronal and glial properties from a stem cell line of rat neurotumor RT4.

    Science.gov (United States)

    Tomozawa, Y; Sueoka, N

    1978-01-01

    A clonal stem cell line, RT4-AC, of the rat peripheral neurotumor RT4 differentiates in culture into morphologically distinct cell types RT4-B, RT4-D, and RT4-E (cell type conversion). The multipotential stem cell type RT4-AC and cell type RT4-D produce a glial marker, S100 protein, but RT4-B and RT4-E do not. The stem cells also show a small but significant response to veratridine on voltage-dependent Na+ influx. Cell types RT4-B and RT4-E show a clear response of voltage-dependent Na+ influx to veratridine, typical of neuronal cells, whereas cell type RT4-D is completely negative. These results indicate that (i) the stem cell type RT4-AC shows both neuronal and glial properties, (ii) cell types RT4-B and RT4-E have a neuronal property, and (iii) cell type RT4-D has a glial property. Therefore, cell type conversion of stem cell RT4-AC to RT4-B and RT4-E cells seems to result in differentiation towards neuronal cell types, and cell type conversion of RT4-AC to RT4-D results in differentiation towards a glial type in culture. PMID:282647

  10. Mogoltacin enhances vincristine cytotoxicity in human transitional cell carcinoma (TCC) cell line.

    Science.gov (United States)

    Behnam Rassouli, F; Matin, M M; Iranshahi, M; Bahrami, A R; Neshati, V; Mollazadeh, S; Neshati, Z

    2009-03-01

    Bladder cancer is the second common cancer of the genitourinary system throughout the world and intravesical chemotherapy is usually used to reduce tumour recurrence and progression. Human transitional cell carcinoma (TCC) is an epithelial-like adherent cell line originally established from primary bladder carcinoma. Here we report the effect of mogoltacin, a sesquiterpene coumarin from Ferula badrakema on TCC cells. Mogoltacin was isolated from the fruits of F. badrakema, using silica gel column chromatography and preparative thin layer chromatography. Mogoltacin did not have any significant cytotoxicity effect on neoplastic TCC cells at 16, 32, 64, 128, 200 and 600 microg ml(-1) concentrations. In order to analyse its combination effect, TCC cells were cultured in the presence of various combining concentrations of mogoltacin and vincristine. Cells were then observed for morphological changes (by light microscopy) and cytotoxicity using MTT assay. The effect of mogoltacin on vincristine toxicity was studied after 24, 48 and 72 h of drug administration. The results of MTT assay showed that mogoltacin can significantly enhance the cytotoxicity of vincristine and confirmed the morphological observations. Results revealed that combination of 40 microg ml(-1) vincristine with 16 microg ml(-1) mogoltacin increased the cytotoxicity of vincristine after 48 h by 32.8%.

  11. Identification of a cancer stem cell-like side population in the HeLa human cervical carcinoma cell line.

    Science.gov (United States)

    Wang, Kefang; Zeng, Jianfang; Luo, Lijing; Yang, Jiaxin; Chen, Jie; Li, Bin; Shen, Keng

    2013-12-01

    The present study aimed to identify the stem cell characteristics of side population (SP) cells sorted from the widely-used HeLa human cervical carcinoma cell line. The SP cells were sorted from the HeLa cell line using fluorescence-activating cell sorting (FACS). Stem cell characteristics of the SP cells, including proliferation, self-renewal, differentiation and the ability to form xenografts, were investigated in vitro and in vivo . The SP cells demonstrated strong tumorigenesis following in vivo transplantation into five to six-week-old female Balb/c mice. The SP cells were observed to be more resistant to chemotherapy and radiotherapy compared with non-side population (NSP) cells. A higher expression of CD133 was observed in the SP cells compared with the NSP cells following FACS. The results demonstrated that the SP cells from the HeLa human cervical carcinoma cell line exhibit stem cell characteristics in vitro and also have a strong ability to form tumors in vivo . The cell surface marker CD133 may serve as a potential molecular marker for the identification of cervical cancer stem cells (CSCs).

  12. CD34+ cells cultured in stem cell factor and interleukin-2 generate CD56+ cells with antiproliferative effects on tumor cell lines

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    Hensel Nancy

    2005-04-01

    Full Text Available Abstract In vitro stimulation of CD34+ cells with IL-2 induces NK cell differentiation. In order to define the stages of NK cell development, which influence their generation from CD34 cells, we cultured G-CSF mobilized peripheral blood CD34+ cells in the presence of stem cell factor and IL-2. After three weeks culture we found a diversity of CD56+ subsets which possessed granzyme A, but lacked the cytotoxic apparatus required for classical NK-like cytotoxicity. However, these CD56+ cells had the unusual property of inhibiting proliferation of K562 and P815 cell lines in a cell-contact dependent fashion.

  13. Animal model of naturally occurring bladder cancer: Characterization of four new canine transitional cell carcinoma cell lines

    OpenAIRE

    Rathore, Kusum; Cekanova, Maria

    2014-01-01

    Background Development and further characterization of animal models for human cancers is important for the improvement of cancer detection and therapy. Canine bladder cancer closely resembles human bladder cancer in many aspects. In this study, we isolated and characterized four primary transitional cell carcinoma (K9TCC) cell lines to be used for future in vitro validation of novel therapeutic agents for bladder cancer. Methods Four K9TCC cell lines were established from naturally-occurring...

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

    OpenAIRE

    Damstrup, L; Rude Voldborg, B.; Spang-Thomsen, M.; Br?nner, N; Skovgaard Poulsen, H.

    1998-01-01

    Formation of metastasis is a multistep process involving attachment to the basement membrane, local proteolysis and migration into surrounding tissues, lymph or bloodstream. In the present study, we have analysed the correlation between in vitro invasion and presence of the epidermal growth factor receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labe...

  15. Permissivity of the NCI-60 cancer cell lines to oncolytic Vaccinia Virus GLV-1h68

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    Bedognetti Davide

    2011-10-01

    Full Text Available Abstract Background Oncolytic viral therapy represents an alternative therapeutic strategy for the treatment of cancer. We previously described GLV-1h68, a modified Vaccinia Virus with exclusive tropism for tumor cells, and we observed a cell line-specific relationship between the ability of GLV-1h68 to replicate in vitro and its ability to colonize and eliminate tumor in vivo. Methods In the current study we surveyed the in vitro permissivity to GLV-1h68 replication of the NCI-60 panel of cell lines. Selected cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV strain. In order to identify correlates of permissity to viral infection, we measured transcriptional profiles of the cell lines prior infection. Results We observed highly heterogeneous permissivity to VACV infection amongst the cell lines. The heterogeneity of permissivity was independent of tissue with the exception of B cell derivation. Cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV strain and a significant correlation was found suggesting a common permissive phenotype. While no clear transcriptional pattern could be identified as predictor of permissivity to infection, some associations were observed suggesting multifactorial basis permissivity to viral infection. Conclusions Our findings have implications for the design of oncolytic therapies for cancer and offer insights into the nature of permissivity of tumor cells to viral infection.

  16. Anticancer Effect of Bovine Lactoferrin on Human Esophagus Cancer Cell Line

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    Mohammad Ali Farziyan

    2016-02-01

    Full Text Available Background: Lactoferrin (Lf is a glycoprotein, a member of the transferrin family.From ten known mechanisms of anti-cancer chemoperotecive compounds, Lf alone, has six of these functions and inhibits cancer. In this study, the effect of lactoferrin purified from bovine colostrum was studied as an anti-cancer agent on esophageal cancer cell line. Materials and Methods: Bovine colostrum were collected immediately after giving birth. At first, the fat, casein, and some of the milk proteins were removed. Then, lactoferrin was purified using CM-Sephadex-C50 cation exchange chromatography by FPLC system. Purified lactoferrin with 80 kDa molecular weight and 2mg/ml concentration was obtained. Esophageal cancer cell line KYSE-30 and normal cell line HEK were cultured. After appropriate confluency, different concentrations of Lf were added to KYSE-30 and HEK for 20 h and its anti-cancer effect was evaluated by MTT and flow cytometric methods. The maximum concentration inhibitory effect was studied at different times using MTT method. Results: MTT test determined that 500 µg/ml of lactoferrin reduced cell viability in esophageal cancer cell lines KYSE by 53% and 80% after 20 and 62 hours, respectively, but had no effect on normal cells. Also, flow cytometric analysis determined that lactoferrin was able to induce apoptosis in KYSE-30 cell line. Conclusion: The isolated lactoferrin from bovine milk showed inhibitory effect on esophageal cancer cell line whereas; it did not have any significant effect on normal cells.

  17. 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...

  18. Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines.

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    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.

  19. N-Glycans influence the in vitro adhesive and invasive behaviour of three metastatic cell lines.

    Science.gov (United States)

    Bironaite, D; Nesland, J M; Dalen, H; Risberg, B; Bryne, M

    2000-01-01

    Alterations in cellular glycosylation may play a key role in metastatic behaviour of tumour cells. We studied three metastatic cell lines, LOX (malignant melanoma), FEMX (malignant melanoma) and MA-11 (mammary carcinoma). These cell lines have a very different metastatic behaviour in vivo, and different glycans have been postulated to be partly responsible for these differences. To further investigate the functional role of carbohydrates, these three cell lines have been treated with tunicamycin, an inhibitor of the biosynthesis of N-glycans and benzyl- alpha-N-acetylgalactosamine (benzyl-alpha-GalNAc; BnGalNAc), an inhibitor of mature O-linked glycans. Various in vitro adhesion and invasion assays were undertaken for functional studies. Tunicamycin significantly inhibited adhesion to laminin, but only slightly affected cell adhesion to collagen IV. The same compound significantly decreased cellular invasiveness through a Matrigel invasion chamber. Moreover, tunicamycin reduced homotypic aggregation of cells. BnGalNAc had generally little effect on cell behaviour in in vitro assay. The effects of the inhibitors were, however, to some extent cell line-specific. We conclude that N-glycans, but probably not mature O-glycans have important in vitro functions in cell adhesion to laminin, cell invasion through Matrigel and cellular aggregation in the studied cell lines. These results support the view that carbohydrates are functionally involved in several steps of the metastatic process. Copyright 2000 S. Karger AG, Basel

  20. In Vitro Study of Influence of Au Nanoparticles on HT29 and SPEV Cell Lines

    Science.gov (United States)

    Pavlovich, Elena; Volkova, Nataliia; Yakymchuk, Elena; Perepelitsyna, Olena; Sydorenko, Michail; Goltsev, Anatoliy

    2017-08-01

    Cell culture models are excellent tools for potential toxicity of nanoparticles and fundamental investigations in cancer research. Thus, information about AuNP potential toxicity and effects on human health is necessary for the use of nanomaterials in clinical settings. The aim of our research is to examine the effects of AuNPs on the epithelial origin cell lines: continuous and oncogenic. Embryonic porcine kidney epithelial inoculated (SPEV) cell line and colorectal carcinoma cell line (HT29) were used. In the test cultures, the cell proliferation, necrosis/apoptosis, and multicellular spheroids generation were evaluated. We demonstrated that AuNP concentrations of 6-12 μg/ml reduced the proliferation of SPEV and HT29 cells and increased the cell number at early and late stages of apoptosis and necrosis. It was shown that small concentrations of AuNPs (1-3 μg/ml) stimulate multicellular spheroid formation by HT29 and SPEV cells. However, higher AuNP concentrations (6-12 μg/ml) had both cytotoxic and anti-cohesive effects on cell in suspension. The large sensitiveness to the action of AuNPs was shown by the line of HT29 (6 μg/ml) as compared to the SPEV cells (12 μg/ml). This experimental study of the effect of AuNPs on SPEV and HT29 cell lines will justify their further application in AuNP-mediated anticancer treatment.

  1. Nicotine-induced protein expression profiling reveals mutually altered proteins across four human cell lines.

    Science.gov (United States)

    Paulo, Joao A; Gygi, Steven P

    2017-01-01

    Mass spectrometry-based proteomic strategies can profile the expression level of proteins in response to external stimuli. Nicotine affects diverse cellular pathways, however, the nicotine-induced alterations on the global proteome across human cell lines have not been fully elucidated. We measured perturbations in protein levels resulting from nicotine treatment in four cell lines-HEK, HeLa, PaSC, and SH-SY5Y-in a single experiment using tandem mass tags (TMT10-plex) and high-resolution mass spectrometry. We quantified 8590 proteins across all cell lines. Of these, nicotine increased the abundance of 31 proteins 1.5-fold or greater in all cell lines. Likewise, considering proteins with altered levels in at least three of the four cell lines, 64 were up-regulated, while one was down-regulated. Gene ontology analysis revealed that ∼40% of these proteins were membrane bound, and functioned in transmembrane signaling and receptor activity. We highlighted proteins, including APP, APLP2, LAPTM4B, and NCOA4, which were dysregulated by nicotine in all cell lines investigated and may have implications in downstream signaling pathways, particularly autophagy. Using the outlined methodology, studies in additional (including primary) cell lines will provide further evidence that alterations in the levels of these proteins are indeed a general response to nicotine and thereby merit further investigation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Multi-omics of 34 colorectal cancer cell lines - a resource for biomedical studies.

    Science.gov (United States)

    Berg, Kaja C G; Eide, Peter W; Eilertsen, Ina A; Johannessen, Bjarne; Bruun, Jarle; Danielsen, Stine A; Bjørnslett, Merete; Meza-Zepeda, Leonardo A; Eknæs, Mette; Lind, Guro E; Myklebost, Ola; Skotheim, Rolf I; Sveen, Anita; Lothe, Ragnhild A

    2017-07-06

    Colorectal cancer (CRC) cell lines are widely used pre-clinical model systems. Comprehensive insights into their molecular characteristics may improve model selection for biomedical studies. We have performed DNA, RNA and protein profiling of 34 cell lines, including (i) targeted deep sequencing (n = 612 genes) to detect single nucleotide variants and insertions/deletions; (ii) high resolution DNA copy number profiling; (iii) gene expression profiling at exon resolution; (iv) small RNA expression profiling by deep sequencing; and (v) protein expression analysis (n = 297 proteins) by reverse phase protein microarrays. The cell lines were stratified according to the key molecular subtypes of CRC and data were integrated at two or more levels by computational analyses. We confirm that the frequencies and patterns of DNA aberrations are associated with genomic instability phenotypes and that the cell lines recapitulate the genomic profiles of primary carcinomas. Intrinsic expression subgroups are distinct from genomic subtypes, but consistent at the gene-, microRNA- and protein-level and dominated by two distinct clusters; colon-like cell lines characterized by expression of gastro-intestinal differentiation markers and undifferentiated cell lines showing upregulation of epithelial-mesenchymal transition and TGFβ signatures. This sample split was concordant with the gene expression-based consensus molecular subtypes of primary tumors. Approximately ¼ of the genes had consistent regulation at the DNA copy number and gene expression level, while expression of gene-protein pairs in general was strongly correlated. Consistent high-level DNA copy number amplification and outlier gene- and protein- expression was found for several oncogenes in individual cell lines, including MYC and ERBB2. This study expands the view of CRC cell lines as accurate molecular models of primary carcinomas, and we present integrated multi-level molecular data of 34 widely used cell lines

  3. Multidrug resistance in tumour cells: characterisation of the multidrug resistant cell line K562-Lucena 1

    Directory of Open Access Journals (Sweden)

    VIVIAN M. RUMJANEK

    2001-03-01

    Full Text Available Multidrug resistance to chemotherapy is a major obstacle in the treatment of cancer patients. The best characterised mechanism responsible for multidrug resistance involves the expression of the MDR-1 gene product, P-glycoprotein. However, the resistance process is multifactorial. Studies of multidrug resistance mechanisms have relied on the analysis of cancer cell lines that have been selected and present cross-reactivity to a broad range of anticancer agents. This work characterises a multidrug resistant cell line, originally selected for resistance to the Vinca alkaloid vincristine and derived from the human erythroleukaemia cell K562. This cell line, named Lucena 1, overexpresses P-glycoprotein and have its resistance reversed by the chemosensitisers verapamil, trifluoperazine and cyclosporins A, D and G. Furthermore, we demonstrated that methylene blue was capable of partially reversing the resistance in this cell line. On the contrary, the use of 5-fluorouracil increased the resistance of Lucena 1. In addition to chemotherapics, Lucena 1 cells were resistant to ultraviolet A radiation and hydrogen peroxide and failed to mobilise intracellular calcium when thapsigargin was used. Changes in the cytoskeleton of this cell line were also observed.A resistência a múltiplos fármacos é o principal obstáculo no tratamento de pacientes com câncer. O mecanismo responsável pela resistência múltipla mais bem caracterizado envolve a expressão do produto do gene MDR-1, a glicoproteína P. Entretanto, o processo de resistência tem fatores múltiplos. Estudos de mecanismos de resistência m��ltipla a fármacos têm dependido da análise de linhagens celulares tumorais que foram selecionadas e apresentam reatividade cruzada a uma ampla faixa de agentes anti-tumorais. Este trabalho caracteriza uma linhagem celular com múltipla resistência a fármacos, selecionada originalmente pela resistência ao alcalóide de Vinca vincristina e derivado

  4. A cell line derived from the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae).

    Science.gov (United States)

    Goodman, Cynthia L; Stanley, David; Ringbauer, Joseph A; Beeman, Richard W; Silver, Kristopher; Park, Yoonseong

    2012-08-01

    The red flour beetle, Tribolium castaneum, is a model organism for agricultural and medical research and its complete genome is sequenced. We established a continuously replicating T. castaneum cell line to complement existing physiological, genetic, and genomic research tools. We set up trial cell cultures from egg, pupa, and adult stages as tissue sources and incubated them in six separate cell culture media to determine the optimal combination of tissue source and medium for cell replication. Our most promising culture was generated by co-culturing adult (∼75 %) and pupal tissues in EX-CELL 420 medium containing 9 % FBS. Our new cell culture is designated BCIRL-TcA-CLG1 (TcA) and it has been subcultured more than 90 times. Amplification of genomic DNA with species-specific primers yielded DNA fragments of the expected sizes and with sequences identical to those from the published Tribolium genome. Additionally, we characterized this line using DNA fingerprinting (DAF-PCR) and compared it with three other coleopteran cell lines and its conspecific pupae to confirm identity. Its doubling time is 155.2 hr. Early passages consisted of attached cells and vesicles in suspension, whereas later passages consisted primarily of attached, spherical cells. Similar to other established cell lines, the ploidy of TcA cells was variable, ranging from 20 chromosomes/cell (diploid) to above 30 chromosomes/cell. TcA cells withstood incubation at 40°C for 1 h with no decrease in viability. We recorded increased levels of one heat shock protein (43 kDa) and of the hsp68a transcript following exposure to 40°C. Taken together, this represents the first report of a continuously replicating T. castaneum cell line. We expect the BCIRL-TcA-CLG1 line will become a useful tool in Tribolium research.

  5. An investigation of the RWPE prostate derived family of cell lines using FTIR spectroscopy.

    Science.gov (United States)

    Baker, M J; Clarke, C; Démoulin, D; Nicholson, J M; Lyng, F M; Byrne, H J; Hart, C A; Brown, M D; Clarke, N W; Gardner, P

    2010-05-01

    Interest in developing robust, quicker and easier diagnostic tests for cancer has lead to an increased use of Fourier transform infrared (FTIR) spectroscopy to meet that need. In this study we present the use of different experimental modes of infrared spectroscopy to investigate the RWPE human prostate epithelial cell line family which are derived from the same source but differ in their mode of transformation and their mode of invasive phenotype. Importantly, analysis of the infrared spectra obtained using different experimental modes of infrared spectroscopy produces similar results. The RWPE family of cell lines can be separated into groups based upon the method of cell transformation rather than the resulting invasiveness/aggressiveness of the cell line. The study also demonstrates the possibility of using a genetic algorithm as a possible standardised pre-processing step and raises the important question of the usefulness of cell lines to create a biochemical model of prostate cancer progression.

  6. Antiproliferative Effects of Selected Chemotherapeutics in Human Ovarian Cancer Cell Line A2780

    Directory of Open Access Journals (Sweden)

    Kateřina Caltová

    2012-01-01

    Full Text Available The aim of our study was to determine the effect of selected cytostatics on a human ovarian cancer cell line A2780 as a model system for ovarian cancer treatment. This cell line is considered cisplatin-sensitive. Panel of tested cytostatics included cisplatin, paclitaxel, carboplatin, gemcitabine, topotecan and etoposide. These cytostatics have a different mechanism of action. To evaluate cytotoxic potential of the tested compounds, the methods measuring various toxicological endpoints were employed including morphological studies, MTT assay, dynamic monitoring of cell proliferation with xCELLigence, cell cycle analysis, caspase 3 activity and expression of proteins involved in cell cycle regulation and cell death. The A270 cell line showed different sensitivity towards the selected cytostatics, the highest cytotoxic effect was associated with paclitaxel and topotecan.

  7. Electroporation enhances mitomycin C cytotoxicity on T24 bladder cancer cell line

    DEFF Research Database (Denmark)

    Vasquez, Juan Luis; Gehl, Julie; Hermann, Gregers G

    2012-01-01

    Intravesical mitomycin instillation combined with electric pulses is being used experimentally for the treatment of T1 bladder tumors, in patients unfit for surgery. Electroporation may enhance the uptake of chemotherapeutics by permeabilization of cell membranes. We investigated if electroporation...... improves the cytotoxicity of mitomycin. In two cell lines, T24 (bladder cancer cell line) and DC3F (Chinese hamster fibroblast), exposure to different concentrations of mitomycin (0.01-2000μM) was tested with and without electroporation (6 pulses of 1kV/cm, duration: 99μs, frequency: 1Hz). Cell viability...... was assessed by colorimetric assay (MTT). For both cell lines, mitomycin's IC_50 was approximately 1000μM in both pulsed and unpulsed cells. On T24 cells, electroporation and mitomycin caused (relative reduction) RR of survival of: 25%, 31% and 29%, by concentrations 0μM, 500μM and 1000μM respectively. For DC3...

  8. Defining the diversity of phenotypic respecification using multiple cell lines and reprogramming regimens.

    Science.gov (United States)

    Alicea, Bradly; Murthy, Shashanka; Keaton, Sarah A; Cobbett, Peter; Cibelli, Jose B; Suhr, Steven T

    2013-10-01

    To better understand the basis of variation in cellular reprogramming, we performed experiments with two primary objectives: first, to determine the degree of difference, if any, in reprogramming efficiency among cells lines of a similar type after accounting for technical variables, and second, to compare the efficiency of conversion of multiple similar cell lines to two separate reprogramming regimens-induced neurons and induced skeletal muscle. Using two reprogramming regimens, it could be determined whether converted cells are likely derived from a distinct subpopulation that is generally susceptible to reprogramming or are derived from cells with an independent capacity for respecification to a given phenotype. Our results indicated that when technical components of the reprogramming regimen were accounted for, reprogramming efficiency was reproducible within a given primary fibroblast line but varied dramatically between lines. The disparity in reprogramming efficiency between lines was of sufficient magnitude to account for some discrepancies in published results. We also found that the efficiency of conversion to one phenotype was not predictive of reprogramming to the alternate phenotype, suggesting that the capacity for reprogramming does not arise from a specific subpopulation with a generally "weak grip" on cellular identity. Our findings suggest that parallel testing of multiple cell lines from several sources may be needed to accurately assess the efficiency of direct reprogramming procedures, and that testing a larger number of fibroblast lines--even lines with similar origins--is likely the most direct means of improving reprogramming efficiency.

  9. Molecular and phenotypic characterisation of paediatric glioma cell lines as models for preclinical drug development.

    Directory of Open Access Journals (Sweden)

    Dorine A Bax

    Full Text Available Although paediatric high grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. One important factor hampering the development of new targeted therapies is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumours. We have carried out a detailed molecular and phenotypic characterisation of a series of paediatric high grade glioma cell lines in comparison to routinely used adult lines.All lines proliferate as adherent monolayers and express glial markers. Copy number profiling revealed complex genomes including amplification and deletions of genes known to be pivotal in core glioblastoma signalling pathways. Expression profiling identified 93 differentially expressed genes which were able to distinguish between the adult and paediatric high grade cell lines, including a number of kinases and co-ordinated sets of genes associated with DNA integrity and the immune response.These data demonstrate that glioma cell lines derived from paediatric patients show key molecular differences to those from adults, some of which are well known, whilst others may provide novel targets for evaluation in primary tumours. We thus provide the rationale and demonstrate the practicability of using paediatric glioma cell lines for preclinical and mechanistic studies.

  10. Murine transgenic iPS cell line for monitoring and selection of cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Azra Fatima

    2016-09-01

    Full Text Available We report here a transgenic murine induced pluripotent stem cell (iPSC line expressing puromycin N-acetyltransferase (PAC and enhanced green fluorescent protein (EGFP under the control of α-myosin heavy chain promoter. This transgenic cell line reproducibly differentiates into EGFP-expressing cardiomyocytes (CMs which can be generated at high purity with puromycin treatment and exhibit molecular and functional properties of immature heart muscle cells. This genetically modified iPSC line can be used for assessment of the utility of CMs for myocardial repair, pharmacological and toxicological applications and development of improved cardiac differentiation protocols.

  11. Self-renewing Pten-/- TP53-/- protospheres produce metastatic adenocarcinoma cell lines with multipotent progenitor activity.

    Directory of Open Access Journals (Sweden)

    Wassim Abou-Kheir

    Full Text Available Prostate cancers of luminal adenocarcinoma histology display a range of clinical behaviors. Although most prostate cancers are slow-growing and indolent, a proportion is aggressive, developing metastasis and resistance to androgen deprivation treatment. One hypothesis is that a portion of aggressive cancers initiate from stem-like, androgen-independent tumor-propagating cells. Here we demonstrate the in vitro creation of a mouse cell line, selected for growth as self-renewing stem/progenitor cells, which manifests many in vivo properties of aggressive prostate cancer. Normal mouse prostate epithelium containing floxed Pten and TP53 alleles was subjected to CRE-mediated deletion in vitro followed by serial propagation as protospheres. A polyclonal cell line was established from dissociated protospheres and subsequently a clonal daughter line was derived. Both lines demonstrate a mature luminal phenotype in vitro. The established lines contain a stable minor population of progenitor cells with protosphere-forming ability and multi-lineage differentiation capacity. Both lines formed orthotopic adenocarcinoma tumors with metastatic potential to lung. Intracardiac inoculation resulted in brain and lung metastasis, while intra-tibial injection induced osteoblastic bone formation, recapitulating the bone metastatic phenotype of human prostate cancer. The cells showed androgen receptor dependent growth in vitro. Importantly, in vivo, the deprivation of androgens from established orthotopic tumors resulted in tumor regression and eventually castration-resistant growth. These data suggest that transformed prostate progenitor cells preferentially differentiate toward luminal cells and recapitulate many characteristics of the human disease.

  12. Establishment of B-lymphoid cell lines retaining cytogenetic characteristics of Bloom syndrome.

    Science.gov (United States)

    Shiraishi, Y; Kubonishi, I; Sandberg, A A

    1983-06-01

    The present study describes the establishment of and chromosomal changes in B-lymphoid cell lines from cells of Bloom syndrome (BS) patients using Epstein-Barr virus (EBV). Even though PHA-stimulated BS lymphocytes from all five patients studied showed high levels of sister chromatid exchange (SCE), three EBV-transformed BS-B-lymphoid cell lines had normal levels of SCE and two yielded two types of cell populations, i.e., one with increased SCE and chromosome instability (including breaks and quadriradials) and another with normal levels of SCE and without structural aberrations. The karyotypic abnormalities, as observed in the BS lines have not been seen in the cells of any established normal B-lymphoid lines transformed by EBV and strongly suggest that the chromosome abnormalities in the BS--B-cell lines with abnormal karyotypes originated in vivo and not through an in vitro effect of EBV. Furthermore, in the EBV-transformed B-cell lines, we found quadriradial formation between sister chromosomes during endomitoses instead of between homologous chromosomes, strongly suggesting that quadriradial formation may be closely related to SCE. The coexistence in BS subjects of abnormal and normal populations of cells with respect to the number of SCE awaits explanation.

  13. The control of anaplastic thyroid carcinoma cell lines by oncolytic poxviruses.

    Science.gov (United States)

    Mundi, Neil; Um, Sung; Yoo, John; Rizzo, Giananthony; Black, Morgan; Pinto, Nicole; Palma, David A; Fung, Kevin; MacNeil, Danielle; Mymryk, Joe S; Barrett, John W; Nichols, Anthony C

    2014-09-22

    Anaplastic thyroid cancer (ATC) is rare, but its clinical presentation is often dramatic and aggressive and is nearly uniformly fatal. Oncolytic viral therapy is a potential strategy to improve outcomes for patients suffering with this disease. Seven established ATC cell lines were infected with a panel of poxviruses to identify which virus had the most potential as an oncolytic agent. These included myxoma, vaccinia, and tanapox viruses, all modified to express green fluorescence protein (GFP). Viral proliferation was assessed by fluorescence and viral amplification. The effect on cell line growth was assessed by the Presto Blue metabolic assay and a live-dead assay. A replication assay was performed to assess the production of infectious progeny. An additional five ATC cell lines were tested using the assays described above for susceptibility to vaccinia virus. ATC cell lines were differentially susceptible to each virus. Vaccinia virus was superior to myxoma and tanapox viruses for the control of anaplastic thyroid cancer in vitro. Although subsequent investigation using an expanded panel of cell lines revealed differential susceptibility to vaccinia virus, effective control of cell proliferation was still achieved using higher titers. Vaccinia virus was the most potent of the tested poxviruses and was highly effective in controlling proliferation and inducing cell death in ATC cell lines. The efficacy of these viruses offers hope for improving outcomes for patients suffering with ATC. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. STR DNA typing of human cell lines: detection of intra- and interspecies cross-contamination.

    Science.gov (United States)

    Dirks, Wilhelm G; Drexler, Hans G

    2013-01-01

    Inter- and intraspecies cross-contaminations (CCs) of human and animal cells represent a chronic problem in cell cultures leading to false data. Microsatellite loci in the human genome harboring short tandem repeat (STR) DNA markers allow individualization of cell lines at the DNA level. Thus, fluorescence polymerase chain reaction amplification of STR loci D5S818, D13S317, D7S820, D16S539, vWA, TH01, TPOX, CSF1PO, and Amelogenin for gender determination is the gold standard for authentication of human cell lines and represents an international reference technique. The major cell banks of the USA, Germany, and Japan (ATCC, DSMZ, JCRB, and RIKEN, respectively) have built compatible STR databases to ensure the availability of STR reference profiles. Upon determination of an STR profile of a human cell line, the suspected identity can be proven by online verification of customer-made STR data sets on the homepage of the DSMZ institute. Furthermore, an additional tetraplex PCR has been established to detect mitochondrial DNA sequences of rodent cells within a human cell culture population. Since authentic cell lines are the main prerequisite for rational research and biotechnology, the next sections describe a rapid and reliable method available to students, technicians, and scientists for certifying identity and purity of human cell lines of interest.

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

    Science.gov (United States)

    Talbot, Neil C; Blomberg, Le Ann; Garrett, Wesley M; Caperna, Thomas J

    2010-10-01

    The PICM-19 pig liver stem cell line is a bipotent cell line, i.e., capable of forming either bile ductules or hepatocyte monolayers in vitro, that was derived from the primary culture of pig embryonic stem cells. The cell line has been strictly feeder-dependent in that cell replication, morphology, and function were lost if the cells were cultured without STO feeder cells. A method for the feeder-independent continuous culture of PICM-19 cells (FI-PICM-19) is presented. PICM-19 cells were maintained and grown without feeder cells on collagen I-coated tissue culture plastic for 26 passages (P26) with initial split ratios of 1:3 that diminished to split ratios of less than 1:2 after passage 16. Once plated, the FI-PICM-19 cells were overlaid with a 1:12 to 1:50 dilution of Matrigel or related extracellular matrix product. Growth of the cells was stimulated by daily refeedings with STO feeder-cell conditioned medium. The FI-PICM-19 cells grew to an approximate confluence of 50% prior to each passage at 2-wk intervals. Growth curve analysis showed their average cell number doubling time to be ~96 h. Morphologically, the feeder-independent cells closely resembled PICM-19 cells grown on feeder cells, and biliary canalicui were present at cell-to-cell junctions. However, no spontaneous multicellular ductules formed in the monolayers of FI-PICM-19 cells. Ultrastructural subcellular features of the FI-PICM-19 cells were similar to those of PICM-19 cells cultured on feeder cells. The FI-PICM-19 cells produced a spectrum of serum proteins and expressed many liver/hepatocyte-specific genes. Importantly, cytochrome P450 (EROD) activity, ammonia clearance, and urea production were maintained by the feeder-independent cells. This simple method for the propagation of the PICM-19 cell line without feeder cells should simplify the generation and selection of functional mutants within the population and enhances the cell line's potential for use in toxicological

  16. Metabolic Response to NAD Depletion across Cell Lines Is Highly Variable.

    Directory of Open Access Journals (Sweden)

    Yang Xiao

    Full Text Available Nicotinamide adenine dinucleotide (NAD is a cofactor involved in a wide range of cellular metabolic processes and is a key metabolite required for tumor growth. NAMPT, nicotinamide phosphoribosyltransferase, which converts nicotinamide (NAM to nicotinamide mononucleotide (NMN, the immediate precursor of NAD, is an attractive therapeutic target as inhibition of NAMPT reduces cellular NAD levels and inhibits tumor growth in vivo. However, there is limited understanding of the metabolic response to NAD depletion across cancer cell lines and whether all cell lines respond in a uniform manner. To explore this we selected two non-small cell lung carcinoma cell lines that are sensitive to the NAMPT inhibitor GNE-617 (A549, NCI-H1334, one that shows intermediate sensitivity (NCI-H441, and one that is insensitive (LC-KJ. Even though NAD was reduced in all cell lines there was surprising heterogeneity in their metabolic response. Both sensitive cell lines reduced glycolysis and levels of di- and tri-nucleotides and modestly increased oxidative phosphorylation, but they differed in their ability to combat oxidative stress. H1334 cells activated the stress kinase AMPK, whereas A549 cells were unable to activate AMPK as they contain a mutation in LKB1, which prevents activation of AMPK. However, A549 cells increased utilization of the Pentose Phosphate pathway (PPP and had lower reactive oxygen species (ROS levels than H1334 cells, indicating that A549 cells are better able to modulate an increase in oxidative stress. Inherent resistance of LC-KJ cells is associated with higher baseline levels of NADPH and a delayed reduction of NAD upon NAMPT inhibition. Our data reveals that cell lines show heterogeneous response to NAD depletion and that the underlying molecular and genetic framework in cells can influence the metabolic response to NAMPT inhibition.

  17. Isolation and characterization of side population cells from the human ovarian cancer cell line SK-OV-3.

    Science.gov (United States)

    Ruan, Zhengyi; Liu, Jianhua; Kuang, Yanping

    2015-12-01

    Ovarian cancer (OC) is the most malignant type of gynecological tumor due to its high recurrence rate following initial treatment. Previous studies have indicated that cancer stem cells (CSCs) may be a potential cause underlying the high proportion of recurrence. Side population (SP) cells isolated from cancer cell lines have been shown to exhibit characteristics associated with CSCs, but studies on SP cells in human ovarian SK-OV-3 cell line are limited. In the present study, the SP cell fraction (4.83% of the total cell population) was isolated using flow cytometry, and analyzed by immunocytochemical analysis and reverse transcription-quantitative polymerase chain reaction. The results showed that SP cells exhibited a high mean fluorescence intensity for CD44, a CSC marker, in addition to elevated expression of the CSCs-associated genes, ATP-binding cassette sub-family G member 2 and Nestin. These findings indicated the stem cell-like features of the SP cells. Furthermore, a colony formation test showed that the isolated SP cells possessed a marked capacity for self-regeneration and proliferation. In addition, a cell cycle assay involving cisplatin indicated that the SP cells were strongly resistant to chemotherapy. In conclusion, the present results suggested that SP cells isolated from the SK-OV-3 cell line exhibited properties typically associated with CSCs. Therefore, the isolated SP cells may be used to provide novel insight into potential therapies against OC.

  18. A biocompatible micro cell culture chamber for culturing and on-line monitoring of Eukaryotic cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael

    2006-01-01

    at holde celler i live over længere tidsperioder I det foreliggende arbejde præsenteres et nyt perfusions baseret mikro celle dyrknings kultur kammer med integreret termisk overvågning og regulering. Kammeret opretholdt både dyrkning og on-line overvågning af både kræft celler såvel som stam celler over...... at dyrknings betingelserne i kammeret var sammenlignelige med dem i konventionelle celle kultur dyrknings flaske, hvis lys intensiteten på mikroskopet og omgivelserne blev minimeret mest muligt. Overflade modificeringer af den strukturelle fotoresist SU-8, der ofte bliver brugt til fabrikation af mikro kanaler...

  19. Evaluations of thyme extract effects in human normal bronchial and tracheal epithelial cell lines and in human lung cancer cell line.

    Science.gov (United States)

    Oliviero, Marinelli; Romilde, Iannarelli; Beatrice, Morelli Maria; Matteo, Valisi; Giovanna, Nicotra; Consuelo, Amantini; Claudio, Cardinali; Giorgio, Santoni; Filippo, Maggi; Massimo, Nabissi

    2016-08-25

    Thyme (Thymus vulgaris) is used traditionally to prepare herbal remedies possessing expectorant, mucolytic, antitussive and antispasmodic properties. The aim of the present study was to investigate the effects of a standardized hydroalcoholic extract of thyme on primary human airway (bronchial/tracheal) epithelial cell lines in a model of lung inflammation induced by LPS. In addition, the effects of thyme extract on human lung cancer cell line (H460) were analysed. Thyme extract showed significant anti-inflammatory properties by reducing the NF-κB p65 and NF-κB p52 transcription factors protein levels followed by the decrease of pro-inflammatory cytokines (IL-1 beta and IL-8), and Muc5ac secretion in human normal bronchial and tracheal epithelial cells. Moreover, the extract showed cytotoxic effects on H460 cancer cells, modulated the release of IL-1 beta, IL-8 and down-regulated NF-κB p65 and NF-κB p52 proteins. Taken together, these results substantiated the traditional uses of thyme in the treatment of respiratory diseases. Thyme extract might be an effective treatment of chronic diseases based on inflammatory processes when hypersecretion of mucus overwhelms the ciliary clearance and obstructs airways, causing morbidity and mortality. Moreover thyme extract, evaluated in H460 lung cancer cell line, demonstrated to induce cell cytotoxicity in addition to reduce inflammatory cell signals. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. The anticancer agent PB-100, selectively active on malignant cells, inhibits multiplication of sixteen malignant cell lines, even multidrug resistant

    Directory of Open Access Journals (Sweden)

    Beljanski Mirko

    2000-01-01

    Full Text Available The plant-derived anticancer agent PB-100 selectively destroys cancer cells, even when multidrug resistant; yet, it does not inhibit normal (non-malignant cell multiplication. Testing of PB-100 on sixteen malignant cell lines, several multidrug resistant, as well as on five normal cell lines, confirmed our previous results. Flavopereirine and dihydroflavopereirine, the active principles of PB-100, were chemically synthesized and displayed the same selectivity for tumor cells as the purified plant extract, being active at even lower concentrations.

  1. Protein phosphatase magnesium dependent 1A (PPM1A) plays a role in the differentiation and survival processes of nerve cells.

    Science.gov (United States)

    Shohat, Meytal; Ben-Meir, Daniella; Lavi, Sara

    2012-01-01

    The serine/threonine phosphatase type 2C (PPM1A) has a broad range of substrates, and its role in regulating stress response is well established. We have investigated the involvement of PPM1A in the survival and differentiation processes of PC6-3 cells, a subclone of the PC12 cell line. This cell line can differentiate into neuron like cells upon exposure to nerve growth factor (NGF). Overexpression of PPM1A in naive PC6-3 cells caused cell cycle arrest at the G2/M phase followed by apoptosis. Interestingly, PPM1A overexpression did not affect fully differentiated cells. Using PPM1A overexpressing cells and PPM1A knockdown cells, we show that this phosphatase affects NGF signaling in PC6-3 cells and is engaged in neurite outgrowth. In addition, the ablation of PPM1A interferes with NGF-induced growth arrest during differentiation of PC6-3 cells.

  2. cell lines to oxidized multi-walled carbon nanotubes

    African Journals Online (AJOL)

    EB

    of neurotrophin pathways with unknown underlying mechanism. Earlier reports by Xu et al 14 showed that carboxyl-terminated MWCNTs can antagonize the 3 types of potassium channels on undifferentiated. PC12 cells. However the potassium channels cell alteration had no significant effect in the generation of reactive ...

  3. (Ezh2) Enhancement in Human Esophageal Cell Lines

    African Journals Online (AJOL)

    Purpose: To investigate the inhibitory effect of berberine treatment on enhancement of zeste of homolog 2 (Ezh2) expressions in KYSE450 human esophageal cancer cells. Methods: Transwell motility chambers were used to analyze cell migration and invasion. Bio-Rad protein assay was used for the determination of ...

  4. Establishment and characterization of porcine cytolytic cell lines and clones

    NARCIS (Netherlands)

    Bruin, de M.C.M.; Rooij, van E.M.A.; Voermans, J.L.M.; Visser, de Y.E.; Bianchi, A.T.J.; Kimman, T.G.

    1997-01-01

    Although non-major-histocompatibility-complex-restricted cytolytic cells appear to significantly influence antiviral immunity in pigs, the phenotype and functional characteristics of these cells are not well defined. To allow a detailed analysis of these subsets, we established and characterized

  5. 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

    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...

  6. Modulatory effects of quercetin on proliferation and differentiation of the human colorectal cell line Caco-2

    NARCIS (Netherlands)

    Dihal, A.A.; Woutersen, R.A.; Ommen, B.v.; Rietjens, I.M.C.M.; Stierum, R.H.

    2006-01-01

    The effect of the dietary flavonoid quercetin was investigated on proliferation and differentiation of the human colon cancer cell line Caco-2. Confluent Caco-2 monolayers exposed to quercetin showed a biphasic effect on cell proliferation and a decrease in cell differentiation (0.001

  7. Cell cloning-on-the-spot by using an attachable silicone cylinder.

    Science.gov (United States)

    Park, Hong Bum; Son, Wonseok; Chae, Dong Han; Lee, Jisu; Kim, Il-Woung; Yang, Woomi; Sung, Jae Kyu; Lim, Kyu; Lee, Jun Hee; Kim, Kyung-Hee; Park, Jong-Il

    2016-06-10

    Cell cloning is a laboratory routine to isolate and keep particular properties of cultured cells. Transfected or other genetically modified cells can be selected by the traditional microbiological cloning. In addition, common laboratory cell lines are prone to genotypic drift during their continual culture, so that supplementary cloning steps are often required to maintain correct lineage phenotypes. Here, we designed a silicone-made attachable cloning cylinder, which facilitated an easy and bona fide cloning of interested cells. This silicone cylinder was easy to make, showed competent stickiness to laboratory plastics including culture dishes, and hence enabled secure isolation and culture for days of selected single cells, especially, on the spots of preceding cell-plating dishes under microscopic examination of visible cellular phenotypes. We tested the silicone cylinder in the monoclonal subcloning from a heterogeneous population of a breast cancer cell line, MDA-MB-231, and readily established independent MDA-MB-231 subclones showing different sublineage phenotypes. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Bovine annulus fibrosus cell lines isolated from intervertebral discs

    Directory of Open Access Journals (Sweden)

    Petra Kraus

    2016-12-01

    Full Text Available The adult bovine (Bos taurus intervertebral disc is primarily comprised of two major tissue types: The outer annulus fibrosus (AF and the central nucleus pulposus (NP. We isolated several primary cell lineages of passage (P 0 cells from the AF tissue omitting typically used enzymatic tissue digestion protocols. The cells grow past p10 without signs of senescence in DMEM + 10% FCS on 0.1% gelatin coated/uncoated surfaces of standard cell culture plates and survive freeze-thawing. Preliminary analysis of the AF derived cells for expression of the two structural genes Col1a1 and Col2a1 was performed by PISH recapitulating the expression observed in vivo.

  9. Pemetrexed plus dendritic cells as third-line therapy for metastatic esophageal squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Zhang B

    2016-06-01

    Full Text Available Bin Zhang,1,* Rui Li,2,3,* Chun-Xiao Chang,2,3 Yong Han,2,3 Sheng-Bin Shi,2,3 Jing Tian2,3 1Department of Medical Oncology, Shandong Ji Ning First People’s Hospital, 2Department of Medical Oncology, Shandong Cancer Hospital, Shandong University, Shandong 3Department of Medical Oncology, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, Jinan, People’s Republic of China*These authors contributed equally to this workAbstract: This study was conducted to evaluate the toxicity and efficacy of pemetrexed plus dendritic cells (DCs when administered as third-line treatment for metastatic esophageal squamous cell carcinoma (ESCC. All patients in the study group had previously failed first-line treatment with 5-fluorouracil and cisplatin-based regimens, as well as second-line treatment with taxane-based regimens. A total of 31 patients were treated with pemetrexed (500 mg/m2 plus DCs on day 1, every 3 weeks. DCs were given for one cycle of 21 days. Thirty patients were evaluated for their response. No patient had a complete response, three patients (10.0% had a partial response, ten patients (33.3% had stable disease, and 17 patients (56.7% had progressive disease. The overall response rate was 10.0%. The median progression-free survival (PFS time was 2.9 months (95% CI, 2.7–3.2, and the median overall survival (OS time was 7.1 months (95% CI, 6.4–7.9. The median PFS and OS times among patients with high and low levels of miR-143 expression in their blood serum were significantly different: median PFS times =3.2 months (95% CI, 2.9–3.4 and 2.7 months (95% CI, 2.4–3.0, respectively (P=0.017, and median OS times =7.8 months (95% CI, 6.8–8.9 and 6.3 months (95% CI, 5.3–7.3, respectively (P=0.036. No patient experienced Grade 4 toxicity. Combined third-line treatment with pemetrexed and DCs was marginally effective and well tolerated in patients with advanced ESCC. Serum miR-143 levels are a potential

  10. Effect of the coffee ingredient cafestol on head and neck squamous cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Kotowski, Ulana; Heiduschka, Gregor; Eckl-Dorna, Julia; Kranebitter, Veronika; Stanisz, Isabella; Brunner, Markus; Lill, Claudia; Thurnher, Dietmar [Medical University of Vienna, Department of Otorhinolaryngology, Head and Neck Surgery, Vienna (Austria); Seemann,