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Sample records for lymphoblastoid tk6 cells

  1. A combination of in vitro comet assay and micronucleus test using human lymphoblastoid TK6 cells.

    PubMed

    Kimura, Aoi; Miyata, Atsuro; Honma, Masamitsu

    2013-09-01

    The comet assay has been widely used as a genotoxicity test for detecting primary DNA damage in individual cells. The micronucleus (MN) test is also a well-established assay for detecting clastogenicity and aneugenicity. A combination of the comet assay (COM) and MN test is capable of detecting a variety of genotoxic potentials as an in vitro screening system. Although the in vitro MN test has a robust protocol and Organisation for Economic Co-operation and Development (OECD) test guideline, the in vitro COM does not. To establish a robust protocol for the COM and to compare its sensitivity with that of the MN, we conducted COM and MN concurrently for five genotoxic agents (ethyl methanesulfonate, methyl methanesulfonate, hydrogen peroxide, gamma-rays and mitomycin C) and one non-genotoxic agent (triton X-100), using human lymphoblastoid TK6 cells. Relative cell count (RCC), relative population doubling (RPD), relative increase in cell count (RICC) and relative cell viability determined by trypan blue dye-exclusion assay (TBDE) were employed as cytotoxic measurements. However, the relative cell viability determined by TBDE just after the treatment was not an appropriate parameter of cytotoxicity for the genotoxic agents because it remained constant even at the highest doses, which showed severe cytotoxicity by RCC, RPD and RICC. The results of the COM showed qualitative agreement (positive or negative) with those of the MN except for mitomycin C, which is an interstrand cross-linker. The COM always required higher doses than the MN to detect the genotoxic potential of the genotoxic agents under the test conditions applied here. The doses that induced a comet tail always yielded <50% RICC, and do not accord to the OECD test guideline for MN because of their high cytotoxicity. These results are helpful for interpreting the results of the COM and MN in in vitro genotoxic hazard assessments. Further investigation is required to standardise the COM.

  2. Noninvolvement of the X chromosome in radiation-induced chromosome translocations in the human lymphoblastoid cell line TK6

    SciTech Connect

    Jordan, R.; Schwartz, J.L. )

    1994-03-01

    Fluorescence in situ hybridization procedures were used to examine the influence of chromosome locus on the frequency and type of chromosome aberrations induced by [sup 60]Co [gamma] rays in the human lymphoblastoid cell line TK6. Aberrations involving the X chromosome were compared to those involving the similarly sized autosome chromosome 7. When corrected for DNA content, acentric fragments were induced with equal frequency in the X and 7 chromosomes. Dose-dependent increases in chromosomal interchanges involving chromosome 7 were noted, and the frequencies of balanced translocations and dicentrics produced were approximately equal. Chromosome interchanges involving the X chromosome were rare and showed no apparent dose dependence. Thus, while chromosomes 7 and X are equally sensitive to the induction of chromosome breaks, the X chromosome is much less likely to interact with autosomes than chromosome 7. The noninvolvement of the X chromosome in translocations with autosomes may reflect a more peripheral and separate location for the X chromosome in the mammalian nucleus. 20 refs., 2 figs., 1 tab.

  3. Poly(ADP-ribosyl)ation enhances H-RAS protein stability and causes abnormal cell cycle progression in human TK6 lymphoblastoid cells treated with hydroquinone.

    PubMed

    Liu, Linhua; Ling, Xiaoxuan; Tang, Huanwen; Chen, Jialong; Wen, Qiaosheng; Zou, Fei

    2015-08-05

    Hydroquinone (HQ), one of the most important benzene-derived metabolites, can induce aberrant cell cycle progression; however, the mechanism of this induction remains unclear. Poly(ADP-ribosyl)ation (PARylation), which is catalysed primarily by poly(ADP-ribose) polymerase-1 (PARP-1), participates in various biological processes, including cell cycle control. The results of the present study show an accumulation in G1 phase versus S phase of TK6 human lymphoblast cells treated with HQ for 48h compared with PBS-treated cells; after 72h of HQ treatment, the cells transitioned from G1 arrest to S phase arrest. We examined the expression of six genes related to the cell cycle or leukaemia to further explore the reason for this phenomenon. Among these genes, H-RAS was found to be associated with this phenomenon because its mRNA and protein expression decreased at 48h and increased at 72h. Experiments for PARP activity induction and inhibition revealed that the observed PARylation was positively associated with H-RAS expression. Moreover, in cells treated with HQ in conjunction with PARP-1 knockdown, expression of the H-RAS protein decreased and the number of cells in G1 phase increased. The degree of poly(ADP-ribosyl) modification of the H-RAS protein increased in cells treated with HQ for 72h, further supporting that changes in PARylation contributed to the rapid alteration of H-RAS protein expression, followed by abnormal progression of the cell cycle. Co-immunoprecipitation (co-IP) assays were employed to determine whether protein complexes were formed by PARP-1 and H-RAS proteins, and the direct interaction between these proteins indicated that PARylation regulated H-RAS expression. As detected by confocal microscopy, the H-RAS protein was found in the nucleus and cytoplasm. To our knowledge, this study is the first to reveal that H-RAS protein can be modified by PARylation.

  4. Cadmium chloride, benzo[a]pyrene and cyclophosphamide tested in the in vitro mammalian cell micronucleus test (MNvit) in the human lymphoblastoid cell line TK6 at Covance laboratories, Harrogate UK in support of OECD draft Test Guideline 487.

    PubMed

    Fowler, Paul; Whitwell, James; Jeffrey, Laura; Young, Jamie; Smith, Katie; Kirkland, David

    2010-10-29

    The following genotoxic chemicals were tested in the in vitro micronucleus assay, at Covance Laboratories, Harrogate, UK in the human lymphoblastoid cell line TK6. Cadmium chloride (an inorganic carcinogen), benzo[a]pyrene (a polycyclic aromatic hydrocarbon requiring metabolic activation) and cyclophosphamide (an alkylating agent requiring metabolic activation) were treated with and without cytokinesis block (by addition of cytochalasin B). This work formed part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 for the in vitro micronucleus test. The toxicity measures used, capable of detecting both cytostasis and cell death, were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index or cytokinesis blocked proliferation index in the presence of cytokinesis block. All of the chemicals tested gave significant increases in the percentage of micronucleated cells with and without cytokinesis block at concentrations giving approximately 60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used. The outcomes from this series of tests support the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in the in vitro micronucleus assay.

  5. Mitomycin C, 5-fluoruracil, colchicine and etoposide tested in the in vitro mammalian cell micronucleus test (MNvit) in the human lymphoblastoid cell line TK6 at Novartis in support of OECD draft Test Guideline 487.

    PubMed

    Elhajouji, Azeddine

    2010-10-29

    The following reference genotoxic agents were tested in the in vitro micronucleus test, at Novartis, Basel, Switzerland. Mitomycin C, 5-fluoruracil, colchicine and etoposide were tested in the human lymphoblastoid cell line TK6, with and without cytokinesis block (in the presence of cytochalasin B). This was done in support of the toxicity measures recommended in the draft OECD Test Guideline on In Vitro Mammalian Cell Micronucleus Test (MNvit) and was part of an international collaborative work. As toxicity measures, detecting cytostasis and cell death, relative cell counts (RCC), relative increase in cell counts (RICC), and relative population doubling (RPD) were used for treatments in the absence of cytokinesis block, and replication index (RI) or cytokinesis-blocked proliferation in the presence of cytokinesis block. All four reference agents were positive in the assay with and without cytokinesis block at concentrations giving approximately 50% toxicity or less as assessed by all of the toxicity measures used. Accordingly, the results of this work support the use of relative population doubling and relative increase in cell counts, as well as relative cell counts, as appropriate measures of toxicity for the non-cytokinesis-blocked in vitro micronucleus assay.

  6. Effect of caffeine on radiation-induced apoptosis in TK6 cells

    SciTech Connect

    Zhen, W.; Vaughan, A.T.M.

    1995-02-01

    Apoptosis has been measured in cells of the human TK6 lymphoblastoid cell line by recording the release of endonuclease-digested DNA from affected cells using flow cytometry. In asynchronously dividing cells, DNA degradation characteristic of apoptosis was first seen 12 h after irradiation as a defined DNA fluorescent peak of sub-G{sub 1}-phase content, reaching a maximum of 30-50% of the population by 24-72 h. Treating cells with 2 mM caffeine either before or up to 3 h after irradiation eliminated the degradation of DNA entirely. In addition, the percentage of cells in which apoptosis could be detected microscopically decreased from 62.4 {+-} 0.95% to 16.7 {+-} 1.5% 72 h after caffeine treatment. Delaying caffeine treatment for 12 h after irradiation reduced DNA degradation by approximately 50% compared to cells receiving radiation alone. DNA degradation induced by serum deprivation was unaffected by caffeine treatment. These data support the contention that irradiation of TK6 cells produces a long-lived cellular signal which triggers apoptosis. Apoptosis produced by serum deprivation does not operate through the same pathway. 36 refs., 5 figs.

  7. Impact of cell cycle delay on micronucleus frequency in TK6 cells.

    PubMed

    Sobol, Zhanna; Spellman, Richard A; Thiffeault, Catherine; Dobo, Krista L; Schuler, Maik

    2014-01-01

    Previous studies with TK6 cells have shown that extending the recovery period after pulse treatment allows for greater micronucleus expression for some compounds. This study explores the role of cell cycle delay in micronucleus expression after pulse treatment with three model genotoxins [mitomycin C, etoposide (ETOP), vinblastine]. Cells were treated for 4 hr and allowed to recover for 36 hr with samples removed at various time points during the recovery period and analyzed for cell cycle distribution, apoptosis and micronucleus frequency. Our results show that mitomycin C causes cell cycle delay for 20 hr after pulse treatment and cell cycle perturbation is no longer evident after 36 hr of recovery. The micronucleus frequency of cells sampled at 36 hr is doubled when compared with cells sampled at 20 hr after mitomycin C removal. When cells were treated with indirect acting genotoxins (ETOP, vinblastine), cell cycle perturbation was not observed at the 20 hr time point. Micronucleus frequency after treatment with either ETOP or vinblastine did not differ between the 20 hr and the 36 hr time point. All three compounds induced similar levels of apoptosis ranging from 4.5 to 5.6% with maximum induction occurring at the 36-hr time point. We conclude that TK6 cells exhibit extended cell cycle arrest after exposure to MMC and can go on to express micronuclei, after overcoming cell cycle arrest.

  8. Inhibition of autophagy enhances Hydroquinone-induced TK6 cell death.

    PubMed

    Xu, Longmei; Liu, Jiaxian; Chen, Yuting; Yun, Lin; Chen, Shaoyun; Zhou, Kairu; Lai, Bei; Song, Li; Yang, Hui; Liang, Hairong; Tang, Huanwen

    2017-03-02

    Hydroquinone (HQ), one of the metabolic products of benzene, is a carcinogen. It can induce apoptosis in lymphoma cells. However, whether HQ can induce autophagy and what roles autophagy plays in TK6 cells exposured to HQ remains unclear. In this study, we found that HQ could induce autophagy through techniques of qRT-PCR, Western blot, immunofluorescent assay of LC3 and transmission electron microscope. Furthermore, inhibiting autophagy using 3-methyladenine (3-MA) or chloroquine (CQ) significantly enhanced HQ-induced cell apoptosis, suggesting that autophagy may be a survival mechanism. Our study also showed that HQ activated PARP-1. Moreover, knockdown of PARP-1 strongly exhibited decreased autophagy related genes expression. In contrast, the absence of SIRT1 increased that. Altogether, our data provided evidence that HQ induced autophagy in TK6 cells and autophagy protected TK6 from HQ attack-induced injury in vitro, and the autophagy was partially mediated via activation of the PARP-1-SIRT1 signaling pathway.

  9. Both PIGA and PIGL Mutations Cause GPI-a Deficient Isolates in the TK6 Cell Line

    PubMed Central

    Nicklas, Janice A.; Carter, Elizabeth W.; Albertini, Richard J.

    2015-01-01

    Molecular analysis of proaerolysin selected glycosylphosphatidylinositol anchor (GPI-a) deficient isolates in the TK6 cell line was performed. Initial studies found that the expected X-linked PIGA mutations were rare among the spontaneous isolates but did increase modestly after ethyl methane sulfate (EMS) treatment (but to only 50% of isolates). To determine the molecular bases of the remaining GPI-a deficient isolates, real-time analysis for all the 25 autosomal GPI-a pathway genes was performed on the isolates without PIGA mutations, determining that PIGL mRNA was absent for many. Further analysis determined these isolates had several different homozygous deletions of the 5’ region of PIGL (17p12-p22) extending 5’ (telomeric) through NCOR1 and some into the TTC19 gene (total deletion >250,000bp). It was determined that the TK6 parent had a hemizygous deletion in 17p12-p22 (275,712bp) extending from PIGL intron 2 into TTC19 intron 7. Second hit deletions in the other allele in the GPI-a deficient isolates led to the detected homozygous deletions. Several of the deletion breakpoints including the original first hit deletion were sequenced. As strong support for TK6 having a deletion, a number of the isolates without PIGA mutations nor homozygous PIGL deletions had point mutations in the PIGL gene. These studies show that the GPI-a mutation studies using TK6 cell line could be a valuable assay detecting point and deletion mutations in two genes simultaneously. PMID:25970100

  10. Integration of metabolic activation with a predictive toxicogenomics signature to classify genotoxic versus nongenotoxic chemicals in human TK6 cells

    PubMed Central

    Buick, Julie K.; Moffat, Ivy; Williams, Andrew; Swartz, Carol D.; Recio, Leslie; Hyduke, Daniel R.; Li, Heng‐Hong; Fornace, Albert J.; Aubrecht, Jiri

    2015-01-01

    The use of integrated approaches in genetic toxicology, including the incorporation of gene expression data to determine the molecular pathways involved in the response, is becoming more common. In a companion article, a genomic biomarker was developed in human TK6 cells to classify chemicals as genotoxic or nongenotoxic. Because TK6 cells are not metabolically competent, we set out to broaden the utility of the biomarker for use with chemicals requiring metabolic activation. Specifically, chemical exposures were conducted in the presence of rat liver S9. The ability of the biomarker to classify genotoxic (benzo[a]pyrene, BaP; aflatoxin B1, AFB1) and nongenotoxic (dexamethasone, DEX; phenobarbital, PB) agents correctly was evaluated. Cells were exposed to increasing chemical concentrations for 4 hr and collected 0 hr, 4 hr, and 20 hr postexposure. Relative survival, apoptosis, and micronucleus frequency were measured at 24 hr. Transcriptome profiles were measured with Agilent microarrays. Statistical modeling and bioinformatics tools were applied to classify each chemical using the genomic biomarker. BaP and AFB1 were correctly classified as genotoxic at the mid‐ and high concentrations at all three time points, whereas DEX was correctly classified as nongenotoxic at all concentrations and time points. The high concentration of PB was misclassified at 24 hr, suggesting that cytotoxicity at later time points may cause misclassification. The data suggest that the use of S9 does not impair the ability of the biomarker to classify genotoxicity in TK6 cells. Finally, we demonstrate that the biomarker is also able to accurately classify genotoxicity using a publicly available dataset derived from human HepaRG cells. Environ. Mol. Mutagen. 56:520–534, 2015. © 2015 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc. PMID:25733247

  11. The TP53 dependence of radiation-induced chromosome instability in human lymphoblastoid cells

    NASA Technical Reports Server (NTRS)

    Schwartz, Jeffrey L.; Jordan, Robert; Evans, Helen H.; Lenarczyk, Marek; Liber, Howard

    2003-01-01

    The dose and TP53 dependence for the induction of chromosome instability were examined in cells of three human lymphoblastoid cell lines derived from WIL2 cells: TK6, a TP53-normal cell line, NH32, a TP53-knockout created from TK6, and WTK1, a WIL2-derived cell line that spontaneously developed a TP53 mutation. Cells of each cell line were exposed to (137)Cs gamma rays, and then surviving clones were isolated and expanded in culture for approximately 35 generations before the frequency and characteristics of the instability were analyzed. The presence of dicentric chromosomes, formed by end-to-end fusions, served as a marker of chromosomal instability. Unexposed TK6 cells had low levels of chromosomal instability (0.002 +/- 0.001 dicentrics/cell). Exposure of TK6 cells to doses as low as 5 cGy gamma rays increased chromosome instability levels nearly 10-fold to 0.019 +/- 0.008 dicentrics/cell. There was no further increase in instability levels beyond 5 cGy. In contrast to TK6 cells, unexposed cultures of WTK1 and NH32 cells had much higher levels of chromosome instability of 0.034 +/- 0.007 and 0.041 +/- 0.009, respectively, but showed little if any effect of radiation on levels of chromosome instability. The results suggest that radiation exposure alters the normal TP53-dependent cell cycle checkpoint controls that recognize alterations in telomere structure and activate apoptosis.

  12. Application of the TGx‐28.65 transcriptomic biomarker to classify genotoxic and non‐genotoxic chemicals in human TK6 cells in the presence of rat liver S9

    PubMed Central

    Buick, Julie K.; Williams, Andrew; Swartz, Carol D.; Recio, Leslie; Li, Heng‐Hong; Fornace, Albert J.; Thomson, Errol M.; Aubrecht, Jiri

    2016-01-01

    In vitro transcriptional signatures that predict toxicities can facilitate chemical screening. We previously developed a transcriptomic biomarker (known as TGx‐28.65) for classifying agents as genotoxic (DNA damaging) and non‐genotoxic in human lymphoblastoid TK6 cells. Because TK6 cells do not express cytochrome P450s, we confirmed accurate classification by the biomarker in cells co‐exposed to 1% 5,6 benzoflavone/phenobarbital‐induced rat liver S9 for metabolic activation. However, chemicals may require different types of S9 for activation. Here we investigated the response of TK6 cells to higher percentages of Aroclor‐, benzoflavone/phenobarbital‐, or ethanol‐induced rat liver S9 to expand TGx‐28.65 biomarker applicability. Transcriptional profiles were derived 3 to 4 hr following a 4 hr co‐exposure of TK6 cells to test chemicals and S9. Preliminary studies established that 10% Aroclor‐ and 5% ethanol‐induced S9 alone did not induce the TGx‐28.65 biomarker genes. Seven genotoxic and two non‐genotoxic chemicals (and concurrent solvent and positive controls) were then tested with one of the S9s (selected based on cell survival and micronucleus induction). Relative survival and micronucleus frequency was assessed by flow cytometry in cells 20 hr post‐exposure. Genotoxic/non‐genotoxic chemicals were accurately classified using the different S9s. One technical replicate of cells co‐treated with dexamethasone and 10% Aroclor‐induced S9 was falsely classified as genotoxic, suggesting caution in using high S9 concentrations. Even low concentrations of genotoxic chemicals (those not causing cytotoxicity) were correctly classified, demonstrating that TGx‐28.65 is a sensitive biomarker of genotoxicity. A meta‐analysis of datasets from 13 chemicals supports that different S9s can be used in TK6 cells, without impairing classification using the TGx‐28.65 biomarker. Environ. Mol. Mutagen. 57:243–260, 2016. © 2016 Her Majesty the

  13. Cytosine arabinoside, vinblastine, diethylstilboestrol and 2-aminoanthracene tested in the in vitro human TK6 cell line micronucleus test (MNvit) at Institut Pasteur de Lille in support of OECD draft test guideline 487.

    PubMed

    Nesslany, Fabrice; Marzin, Daniel

    2010-10-29

    The reference genotoxic agents Cytosine arabinoside, Vinblastine, Diethylstilboestrol and 2-Aminoanthracene were tested in the in vitro micronucleus assay, in human lymphoblastoid TK6 cells, without cytokinesis block, at the laboratories of Institut Pasteur de Lille, France. This was done in support of the toxicity measures recommended in the late 2007 version of the draft OECD Test Guideline 487 for the testing of chemicals. All four reference agents were positive in the assay at concentrations giving approximately 50% toxicity or less as assessed by draft Test Guideline 487 recommended measures, relative population doublings and relative increase in cell counts. Accordingly, this work supports the premise that relative population doublings and relative increase in cell counts are appropriate measures of toxicity for the non-cytokinesis blocked in vitro micronucleus assay.

  14. Genotoxicity testing of three monohaloacetic acids in TK6 cells using the cytokinesis-block micronucleus assay.

    PubMed

    Liviac, Danae; Creus, Amadeu; Marcos, Ricard

    2010-09-01

    Chemical disinfection of water generates harmful chemical compounds, known as disinfection by-products (DBPs). One class of DBPs is constituted by haloacetic acids (HAAs), the second major group in prevalence (after trihalomethanes) detected in finished drinking water. In this article, we report the results obtained in the evaluation of the chromosome damage induced by three monohaloacetic acids, namely iodoacetic acid (IAA), bromoacetic acid (BAA) and chloroacetic acid (CAA). To evaluate the induction of chromosome damage, we used the cytokinesis-block micronucleus test that measures the ability of genotoxic agents to induce both clastogenic and/or aneugenic effects. No previous data exist on the effects of these compounds on human chromosomes. We tested five doses of each HAA, in addition to the negative and positive controls. The highest dose tested for each HAA was that immediately lower than the dose producing total cytotoxicity. Our results show that none of the three HAAs tested was able to increase significantly the frequency of micronucleus in binucleated TK6 cells, the rank order in decreasing cytotoxicity was IAA > BAA > CAA.

  15. Mitochondrial Permeability and Toxicity of Di ethylhexyl and Mono ethylhexyl Phthalates on TK6 Human Lymphoblasts Cells

    PubMed Central

    Rosado-Berrios, Carlos A.; Vélez, Christian; Zayas, Beatriz

    2011-01-01

    Phthalates are ubiquitous compounds used in the manufacturing industry. Some are known endocrine disruptors, acting as xenoestrogens, others induce reproductive toxicity and damage to DNA among other effects. Studies on apoptosis induction and mitochondrial damage capacity of phthalates on the immune system are limited. This study aims to determine cell viability inhibition and apoptosis induction of diethylhexyl phthalate (DEHP) and monoethylhexyl phthalate (MEHP) on the human TK6 lymphoblast cell line at concentrations found in the environment. Key hallmark events, such as mitochondrial membrane permeability, generation of reactive oxygen species (ROS) and activation of caspase 3 and 7 were measured. Concentrations that inhibit viability of 50% (IC50) of the cells were determined at 24, 48 and 72 hours with doses ranging from 10μM to 500μM. Changes in mitochondrial membrane permeability, ROS generation and activation of caspases 3 and 7, were measured as part of the cell death mechanism. The IC50 at 24 hours was approximately 250 μM for both phthalates; at 48 hours were 234μM and 196μM for DEHP and MEHP, respectively and at 72 hours IC50s were 100 μM and 80 μM for DEHP and MEHP respectively. Overall the longer the time of exposure the lower the IC50's for both compounds. Both compounds affected mitochondrial membrane potential, promoted ROS generation and activated caspases 3 and 7. MEHP is more toxic, promotes higher level of ROS production and caspases activation. Our findings suggest that DEHP and MEHP have the capacity to induce apoptosis in cells of the immune system at concentrations found in the environment. PMID:21864672

  16. Hydroquinone-induced malignant transformation of TK6 cells by facilitating SIRT1-mediated p53 degradation and up-regulating KRAS.

    PubMed

    Chen, Yuting; Chen, Jiajia; Yun, Lin; Xu, Longmei; Liu, Jiaxian; Xu, Yongchun; Yang, Hui; Liang, Hairong; Tang, Huanwen

    2016-09-30

    Hydroquinone (HQ), known as one of the metabolic products of benzene, causes a number of hematologic malignancies. The study evaluated the potential mechanism of Sirtuin 1 (SIRT1) in HQ-induced TK6 cell malignant transformation. The data of our study show that short term exposure of TK6 cells to HQ led to a decrease expression of SIRT1. Knockdown of SIRT1 sensitized to the HQ-induced apoptosis in vitro and increased the expression of p53, p21 and γ-H2AX. Furthermore, chronic HQ-treated (20μM once a week for 19 weeks) caused carcinogenic transformation and was confirmed by abnormal cell proliferation, matrix metalloproteinase 9(MMP9) and subcutaneous tumor formation in nude mice. SIRT1 increased KRAS expression, and decreased H3K9 and H3K18 acetylation, inhibited p53 signaling and the level of caspase-3 in HQ-induced transformation cells. Taken together, these data suggest that SIRT1 is involved in HQ-induced malignant transformation associated with suppressing p53 signaling and activation of KRAS.

  17. Perspectives on fast-neutron mutagenesis of human lymphoblastoid cells.

    PubMed

    Kronenberg, A

    1991-10-01

    The effects of low-fluence exposures to (Pu, Be) neutrons (En = 4.2 MeV) have been studied in a sensitive human B-lymphoblastoid cell line, TK6. Mutations were scored for two genetic loci, hypoxanthine phosphoribosyltransferase (hgprt) and thymidine kinase (tk), as a function of dose and dose rate. For exposures limited to less than one cell cycle, the mutation frequency for the hgprt locus was 1.92 X 10(-7)/cGy. When exposures were protracted over multiple cell generations, mutation yields were increased to 6.07 X 10(-7)/cGy. Similar yields were obtained for the induction of tk-deficient mutants with a normal cell generation time (tk-ng) when exposures were carried out at very low dose rates over multiple cell generations. In the series of data presented here, the results obtained for short-duration neutron exposures are compared with data obtained for monoenergetic heavy charged particles of defined linear energy transfer (LET) produced at the BEVALAC accelerator at Lawrence Berkeley Laboratory. TK6 cells have been exposed to beams ranging in atomic number from 20Ne to 40Ar over an energy range from 330 to 670 MeV/amu. Mutation induction was evaluated for both loci for a subset of these beams. The results obtained with 20Ne ions of 425 MeV/amu (LET = 32 keV/microns) and 28Si ions of 670 MeV/amu (LET = 50 keV/microns) closely resemble the mutation yields obtained for brief exposures to (Pu, Be) neutrons. The nature of alterations in DNA structure induced within the tk locus of tk-ng mutants is reviewed for a series of neutron-induced mutants and a series of mutants induced by exposure to 40Ar ions (470 MeV/amu, LET = 95 keV/microns). The mutational spectra for these two types of mutants were similar and were dominated by allele loss mutations. Multilocus deletions inclusive of the c-erbA1 locus were common among tk-deficient mutants induced by these densely ionizing radiations. For the mutants induced by 40Ar ions, it is likely that the mutations were produced by

  18. Method for cloning lymphoblastoid cells

    SciTech Connect

    Hammerling, U.; Kosinski, S.

    1989-02-14

    A method is described for increasing cloning frequency of human lymphocyte or lumphoblastoid cells which have been transformed with Epstein Barr virus comprising growing the transformed cells in a semi-solid agarose medium. A lower and an upper layer of agarose are used, the lower layer comprising fibroblasts suspended in the agarose layer and the upper layer comprising irradiated fibroblasts and the transformed cells suspended in the agarose layer wherein the upper agarose layer is added after the lower layer has gelled.

  19. Nonlinear responses for chromosome and gene level effects induced by vinyl acetate monomer and its metabolite, acetaldehyde in TK6 cells.

    PubMed

    Budinsky, Robert; Gollapudi, Bhaskar; Albertini, Richard J; Valentine, Rudolph; Stavanja, Mari; Teeguarden, Justin; Fensterheim, Robert; Rick, David; Lardie, Thomas; McFadden, Lisa; Green, Amanda; Recio, Leslie

    2013-12-01

    Vinyl acetate monomer (VAM) produced rat nasal tumors at concentrations in the hundreds of parts per million. However, VAM is weakly genotoxic in vitro and shows no genotoxicity in vivo. A European Union Risk Assessment concluded that VAM's hydrolysis to acetaldehyde (AA), via carboxylesterase, is a critical key event in VAM's carcinogenic potential. In the following study, we observed increases in micronuclei (MN) and thymidine kinase (Tk) mutants that were dependent on the ability of TK6 cell culture conditions to rapidly hydrolyze VAM to AA. Heat-inactivated horse serum demonstrated a high capacity to hydrolyze VAM to AA; this activity was highly correlated with a concomitant increase in MN. In contrast, heat-inactivated fetal bovine serum (FBS) did not hydrolyze VAM and no increase in MN was observed. AA's ability to induce MN was not impacted by either serum since it directly forms Schiff bases with DNA and proteins. Increased mutant frequency at the Tk locus was similarly mitigated when AA formation was not sufficiently rapid, such as incubating VAM in the presence of FBS for 4 hr. Interestingly, neither VAM nor AA induced mutations at the HPRT locus. Finally, cytotoxicity paralleled genotoxicity demonstrating that a small degree of cytotoxicity occurred prior to increases in MN. These results established 0.25 mM as a consistent concentration where genotoxicity first occurred for both VAM and AA provided VAM is hydrolyzed to AA. This information further informs significant key events related to the mode of action of VAM-induced nasal mucosal tumors in rats.

  20. Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to X-rays.

    PubMed

    Varès, Guillaume; Wang, Bing; Tanaka, Kaoru; Kakimoto, Ayana; Eguchi-Kasai, Kyomi; Nenoi, Mitsuru

    2011-01-10

    The ability of cells to adapt low-dose or low-dose rate radiation is well known. High-LET radiation has unique characteristics, and the data concerning low doses effects and high-LET radiation remain fragmented. In this study, we assessed in vitro the ability of low doses of X-rays to induce an adaptive response (AR) to a subsequent challenging dose of heavy-ion radiation. Lymphoblastoid cells (TK6, AHH-1, NH32) were exposed to priming 0.02-0.1Gy X-rays, followed 6h later by challenging 1Gy heavy-ion radiation (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm). Pre-exposure of p53-competent cells resulted in decreased mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and different H2AX phosphorylation kinetics, as compared to cells exposed to challenging radiation alone. This phenomenon did not seem to be linked with cell cycle effects or radiation-induced apoptosis. Taken together, our results suggested the existence of an AR to mutagenic effects of heavy-ion radiation in lymphoblastoid cells and the involvement of double-strand break repair mechanisms.

  1. Effects of Simulated Microgravity on the Expression Profile of Microrna in Human Lymphoblastoid Cells

    NASA Astrophysics Data System (ADS)

    Zhang, Ye; Wu, Honglu; Ramesh, Govindarajan; Rohde, Larry; Story, Michael; Mangala, Lingegowda

    2012-07-01

    EFFECTS OF SIMULATED MICROGRAVITY ON THE EXPRESSION PROFILE OF MICRORNA IN HUMAN LYMPHOBLASTOID CELLS Lingegowda S. Mangala1,2, Ye Zhang1,3, Zhenhua He2, Kamal Emami1, Govindarajan T. Ramesh4, Michael Story 5, Larry H. Rohde2, and Honglu Wu1 1 NASA Johnson Space Center, Houston, Texas, USA 2 University of Houston Clear Lake, Houston, Texas, USA 3 Wyle Integrated Science and Engineering Group, Houston, Texas, USA 4 Norfolk State University, Norfolk, VA, USA 5 University of Texas, Southwestern Medical Center, Dallas, Texas, USA This study explores the changes in expression of microRNA (miRNA) and related genes under simulated microgravity conditions. In comparison to static 1g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. miRNA has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. However, very little is known about the effect of altered gravity on miRNA expression. To test the hypothesis that the miRNA expression profile would be altered in zero gravity resulting in altered regulation of gene expression leading to metabolic or functional changes in cells, we cultured TK6 human lymphoblastoid cells in a High Aspect Ratio Vessel (HARV; bioreactor) for 72 h either in the rotating condition to model microgravity in space or in the static condition as a control. Expression of several miRNA was changed significantly in the simulated microgravity condition including miR-150, miR-34a, miR-423-5p, miR-22 and miR-141, miR-618 and miR-222. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA microarray and validated the related genes using q-RT PCR. Network and pathway analysis of gene and miRNA expression profiles indicates that the regulation of cell communication and catalytic activities, as well as pathways involved in immune response_IL-15

  2. Characterization of a TK6-Bcl-xL gly-159-ala Human Lymphoblast Clone

    SciTech Connect

    Chyall, L.: Gauny, S.; Kronenberg, A.

    2006-01-01

    TK6 cells are a well-characterized human B-lymphoblast cell line derived from WIL-2 cells. A derivative of the TK6 cell line that was stably transfected to express a mutated form of the anti-apoptotic protein Bcl-xL (TK6-Bcl-xL gly-159- ala clone #38) is compared with the parent cell line. Four parameters were evaluated for each cell line: growth under normal conditions, plating efficiency, and frequency of spontaneous mutation to 6‑thioguanine resistance (hypoxanthine phosphoribosyl transferase locus) or trifluorothymidine resistance (thymidine kinase locus). We conclude that the mutated Bcl-xL protein did not affect growth under normal conditions, plating efficiency or spontaneous mutation frequencies at the thymidine kinase (TK) locus. Results at the hypoxanthine phosphoribosyl transferase (HPRT) locus were inconclusive. A mutant fraction for TK6‑Bcl-xL gly-159-ala clone #38 cells exposed to 150cGy of 160kVp x-rays was also calculated. Exposure to x-irradiation increased the mutant fraction of TK6‑Bcl-xL gly-159-ala clone #38 cells.

  3. Effects of Modeled Microgravity on Expression Profiles of Micro RNA in Human Lymphoblastoid Cells

    NASA Technical Reports Server (NTRS)

    Mangala, Lingegowda S.; Emami, Kamal; Story, Michael; Ramesh, Govindarajan; Rohde, Larry; Wu, Honglu

    2010-01-01

    Among space radiation and other environmental factors, microgravity or an altered gravity is undoubtedly the most significant stress experienced by living organisms during flight. In comparison to the static 1g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. Micro RNA (miRNA) has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. miRNA represents a class of single-stranded noncoding regulatory RNA molecules ( 22 nt) that control gene expressions by inhibiting the translation of mRNA to proteins. However, very little is known on the effect of altered gravity on miRNA expression. We hypothesized that the miRNA expression profile will be altered in zero gravity resulting in regulation of the gene expression and functional changes of the cells. To test this hypothesis, we cultured TK6 human lymphoblastoid cells in Synthecon s Rotary cell culture system (bioreactors) for 72 h either in the rotating (10 rpm) to model the microgravity in space or in the static condition. The cell viability was determined before and after culturing the cells in the bioreactor using both trypan blue and guava via count. Expressions of a panel of 352 human miRNA were analyzed using the miRNA PCRarray. Out of 352 miRNAs, expressions of 75 were significantly altered by a change of greater than 1.5 folds and seven miRNAs were altered by a fold change greater than 2 under the rotating culture condition. Among these seven, miR-545 and miR-517a were down regulated by 2 folds, whereas miR-150, miR-302a, miR-139-3p, miR-515-3p and miR-564 were up regulated by 2 to 8 folds. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA Illumina Microarray Analysis and validated the related genes using q-RT PCR.

  4. [Production of a dialysable transfer factor of cell mediated immunity by lymphoblastoid cells in continuous proliferation].

    PubMed

    Goust, J M; Viza, D; Moulias, R; Trejdosiewicz, L; Lesourd, B; Marescot, M R; Prévot, A

    1975-01-20

    Four lymphoblastoid cell lines tested in this work contain normally a dialysable moiety having by ultraviolet spectroscopy, column chromatography (Biogel P 10) and chemically the same properties than human dialysable Transfer Factor (TFd), but unable to transfer cell mediated immune response against common antigens. Two of them are able to do so after incubation with minimal amounts of TFd. Production of a molecule identical to human TFd is possible in some lymphoblastoid cell lines after induction with TFd.

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

    SciTech Connect

    Lee, Jae-Eun; Hong, Eun-Jung; Nam, Hye-Young; Hwang, Meeyul; Kim, Ji-Hyun; Han, Bok-Ghee; Jeon, Jae-Pil

    2012-10-19

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

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

    PubMed Central

    Fernández-Araujo, Andrea; Sánchez, Jon A.; Alfonso, Amparo; Vieytes, Mercedes R.; Botana, Luis M.

    2015-01-01

    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 h 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-562 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-562 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. PMID:26136685

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

    PubMed

    Fernández-Araujo, Andrea; Sánchez, Jon A; Alfonso, Amparo; Vieytes, Mercedes R; Botana, Luis M

    2015-01-01

    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 h 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-562 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-562 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.

  8. Radiation quality and mutagenesis in human lymphoblastoid cells.

    PubMed

    Liber, Howard L; Idate, Rupa; Warner, Christy; Bailey, Susan M

    2014-10-01

    An interesting problem associated with studying the effects of low doses of high atomic number and energy (HZE) particles, as found in space, is that not all cells will necessarily be similarly traversed during exposure, a scenario that greatly complicates the measurement of end points that require time to develop, gene-locus mutation being a perfect example. The standard protocol for measuring mutations at the heterozygous thymidine kinase locus in human lymphoblastoid cells involves waiting three days after treatment for newly induced mutants to fully express, at which time cells are then plated in the presence of the selective agent, and mutants are counted three weeks later. This approach is acceptable as long as all cells are uniformly affected, as is the case with low-linear energy transfer (LET) ionizing radiation. However, for HZE particles some fraction of cells may not be traversed or perhaps would receive fewer than the average number of "hits", and they would continue to grow at or closer to the normal rate, thus outpacing cells that received more damage. As a result, at three days post-treatment, more heavily damaged cells will have been "diluted" by the less damaged ones, and thus the measured mutant frequency (MF) will underestimate actual mutant frequency. We therefore developed a modified approach for measuring mutation that eliminates this problem and demonstrates that the mutagenicity of 1 GeV/n Fe ions are underestimated by a factor of two when using the standard MF protocol. Furthermore, we determined the mutagenic effects of a variety of heavy ions, all of which induced mutations in a linear fashion. We found that the maximal yield of mutations (i.e., highest relative biological efficiency) was about 7.5 times higher at an LET of 70 keV/μ (400 MeV/n Si) than for gamma rays. Nontargeted mutagenicity after treatment with ionizing radiation was also investigated. For each particular ion/energy examined and in agreement with many previous studies

  9. Network signatures of cellular immortalization in human lymphoblastoid cell lines

    SciTech Connect

    Shim, Sung-Mi; Jung, So-Young; Nam, Hye-Young; Kim, Hye-Ryun; Lee, Mee-Hee; Kim, Jun-Woo; Han, Bok-Ghee; Jeon, Jae-Pil

    2013-11-15

    Highlights: •We identified network signatures of LCL immortalization from transcriptomic profiles. •More than 41% of DEGs are possibly regulated by miRNAs in LCLs. •MicroRNA target genes in LCLs are involved in apoptosis and immune-related functions. •This approach is useful to find functional miRNA targets in specific cell conditions. -- Abstract: Human lymphoblastoid cell line (LCL) has been used as an in vitro cell model in genetic and pharmacogenomic studies, as well as a good model for studying gene expression regulatory machinery using integrated genomic analyses. In this study, we aimed to identify biological networks of LCL immortalization from transcriptomic profiles of microRNAs and their target genes in LCLs. We first selected differentially expressed genes (DEGs) and microRNAs (DEmiRs) between early passage LCLs (eLCLs) and terminally differentiated late passage LCLs (tLCLs). The in silico and correlation analysis of these DEGs and DEmiRs revealed that 1098 DEG–DEmiR pairs were found to be positively (n = 591 pairs) or negatively (n = 507 pairs) correlated with each other. More than 41% of DEGs are possibly regulated by miRNAs in LCL immortalizations. The target DEGs of DEmiRs were enriched for cellular functions associated with apoptosis, immune response, cell death, JAK–STAT cascade and lymphocyte activation while non-miRNA target DEGs were over-represented for basic cell metabolisms. The target DEGs correlated negatively with miR-548a-3p and miR-219-5p were significantly associated with protein kinase cascade, and the lymphocyte proliferation and apoptosis, respectively. In addition, the miR-106a and miR-424 clusters located in the X chromosome were enriched in DEmiR–mRNA pairs for LCL immortalization. In this study, the integrated transcriptomic analysis of LCLs could identify functional networks of biologically active microRNAs and their target genes involved in LCL immortalization.

  10. Relative contribution of four nucleases, CtIP, Dna2, Exo1 and Mre11, to the initial step of DNA double-strand break repair by homologous recombination in both the chicken DT40 and human TK6 cell lines.

    PubMed

    Hoa, Nguyen Ngoc; Akagawa, Remi; Yamasaki, Tomomi; Hirota, Kouji; Sasa, Kentaro; Natsume, Toyoaki; Kobayashi, Junya; Sakuma, Tetsushi; Yamamoto, Takashi; Komatsu, Kenshi; Kanemaki, Masato T; Pommier, Yves; Takeda, Shunichi; Sasanuma, Hiroyuki

    2015-12-01

    Homologous recombination (HR) is initiated by double-strand break (DSB) resection, during which DSBs are processed by nucleases to generate 3' single-strand DNA. DSB resection is initiated by CtIP and Mre11 followed by long-range resection by Dna2 and Exo1 in Saccharomyces cerevisiae. To analyze the relative contribution of four nucleases, CtIP, Mre11, Dna2 and Exo1, to DSB resection, we disrupted genes encoding these nucleases in chicken DT40 cells. CtIP and Dna2 are required for DSB resection, whereas Exo1 is dispensable even in the absence of Dna2, which observation agrees with no developmental defect in Exo1-deficient mice. Despite the critical role of Mre11 in DSB resection in S. cerevisiae, loss of Mre11 only modestly impairs DSB resection in DT40 cells. To further test the role of CtIP and Mre11 in other species, we conditionally disrupted CtIP and MRE11 genes in the human TK6 B cell line. As with DT40 cells, CtIP contributes to DSB resection considerably more significantly than Mre11 in TK6 cells. Considering the critical role of Mre11 in HR, this study suggests that Mre11 is involved in a mechanism other than DSB resection. In summary, CtIP and Dna2 are sufficient for DSB resection to ensure efficient DSB repair by HR.

  11. Use of lymphoblastoid cell lines to evaluate the hypersensitivity to ultraviolet radiation in Cockayne syndrome

    SciTech Connect

    Otsuka, F.; Tarone, R.E.; Cayeux, S.; Robbins, J.H.

    1984-05-01

    Cockayne syndrome (CS) is a rare autosomal recessive disease characterized by acute sun sensitivity, cachectic dwarfism, and neurologic and skeletal abnormalities. Cultured skin fibroblasts from patients with this disease are known to be hypersensitive to the lethal effects of 254-nm UV radiation. The authors have studied the sensitivity of 254-nm UV radiation of lymphoblastoid lines derived from 3 typical CS patients, 1 atypical CS patient who had a very late age of onset of clinical manifestations, 2 patients who had both xeroderma pigmentosum (XP) and typical CS, and 3 heterozygous parents of these patients. Post-UV survival was determined by the trypan-blue dye-exclusion method. The lymphoblastoid lines from the 3 typical CS patients, the atypical CS patient, and the 2 patients with both CS and XP had decreased post-UV viability in comparison with lines from normal donors. Lines from the heterozygous parents had normal post-UV viability. The post-UV viability of the typical CS lines was similar to that of a XP complementation group C line. The relative post-UV viability of lymphoblastoid lines from the typical CS patients was similar to the relative post-UV survival of their fibroblast lines. The lymphoblastoid line from the atypical CS patient had a post-UV viability similar to that of the typical CS patients. Thus, the relative hypersensitivity of CS patients cells in vitro does not reflect the severity or age of onset of the patients clinical manifestations. The lymphoblastoid lines from the 2 patients who had both CS and XP were significantly more sensitive to the UV radiation than those from patients with only CS. Our studies demonstrate that lymphoblastoid lines from patients with CS are appropriate and useful cell lines for the study of the inherited hypersensitivity to UV radiation.

  12. Utilization of Lymphoblastoid Cell Lines as a System for the Molecular Modeling of Autism

    ERIC Educational Resources Information Center

    Baron, Colin A.; Liu, Stephenie Y.; Hicks, Chindo; Gregg, Jeffrey P.

    2006-01-01

    In order to provide an alternative approach for understanding the biology and genetics of autism, we performed statistical analysis of gene expression profiles of lymphoblastoid cell lines derived from children with autism and their families. The goal was to assess the feasibility of using this model in identifying autism-associated genes.…

  13. Molecular Mechanisms of Radiation-Induced Genomic Instability in Human Cells

    SciTech Connect

    Howard L. Liber; Jeffrey L. Schwartz

    2005-10-31

    There are many different model systems that have been used to study chromosome instability. What is clear from all these studies is that conclusions concerning chromosome instability depend greatly on the model system and instability endpoint that is studied. The model system for our studies was the human B-lymphoblastoid cell line TK6. TK6 was isolated from a spontaneously immortalized lymphoblast culture. Thus there was no outside genetic manipulation used to immortalize them. TK6 is a relatively stable p53-normal immortal cell line (37). It shows low gene and chromosome mutation frequencies (19;28;31). Our general approach to studying instability in TK6 cells has been to isolate individual clones and analyze gene and chromosome mutation frequencies in each. This approach maximizes the possibility of detecting low frequency events that might be selected against in mass cultures.

  14. Distribution of sensitivity to 4-nitroquinoline 1-oxide among Japanese lymphoblastoid cell lines

    SciTech Connect

    Kiyohara, Chikako; Hirohata, Tomio; Nagayama, Junya ); Kuratsune, Masanori Nakamura Junior Coll., Fukuoka )

    1991-01-01

    The processes through which the UV-mimic chemical carcinogen, 4-nitroquinoline 1-oxide (4NQO), leads to the DNA lesions are well characterized in E. coli, where the formation of stable 4NQO-purine adducts is critical. The DNA excision-repair mechanisms similar to those for E. coli occur in normal human cells. Xeroderma pigmentosum (XP) is an example of a rare recessive autosomal skin disorder which is characterized biochemically as a DNA repair-deficient disease. The fluorescein diacetate (FDA) method was recently used to determine the sensitivity of lymphoblastoid cell lines 4NQO. Viable cells take up, non-fluorescent chemical, FDA and convert it to, a fluorescent molecule, fluorescein by intracellular esterases. DNA damage produced by 4NQO could be evaluated on the basis of the cell lethality by this FDA method. In the present study the authors describe the distribution of sensitivity to 4NQO among lymphoblastoid cell lines established from Japanese.

  15. Duck lymphocytes. VIII. T-lymphoblastoid cell lines from reticuloendotheliosis virus-induced tumours.

    PubMed

    Chan, S W; Bando, Y; Warr, G W; Middleton, D L; Higgins, D A

    1999-04-01

    The T strain of reticuloendotheliosis virus (REV-T) obtained, along with the helper chicken syncytia virus (CSV), from the CSO4 cell line was highly oncogenic and rapidly fatal in ducks. Tumours were mainly seen in spleen, but neoplastic cells were observed microscopically in many organs. In vitro REV transformation of duck lymphocytes failed to yield stable cell lines, so cells from organs (blood, bone marrow, spleen, lymph node, bursa of Fabricius) of infected birds were used to establish cell lines. Some of these cell lines have been cloned. The success rates of establishment and cloning were increased if cells were cultured in a range of media containing different supplements; however, medium containing 5% foetal calf serum (FCS) and 5% duck serum was generally most efficacious for initial establishment, while spent medium from the parental line supplemented with a further 20% FCS gave best results for cloning. Cloned cell lines had the morphology of lymphoblastoid cells, with irregular nuclei and diffuse chromatin. Analysis of mRNA extracted from these cell lines showed that the uncloned lines were strongly expressing the β chain of the T cell antigen receptor (TCR) and weakly expressing immunoglobulin (Ig) polypeptides [λ light chain and μ, υ, υ (ΔFc) and α heavy chains in various proportions], suggesting the presence of T and B cells. The cloned cell lines that could be classified were TCR β+ ve T cells. This is the first report of the establishment, cloning and partial characterization of duck lymphoblastoid cell lines.

  16. Characteristics of genomic instability in clones of TK6 human lymphoblasts surviving exposure to 56Fe ions

    NASA Technical Reports Server (NTRS)

    Evans, Helen H.; Horng, Min-Fen; Ricanati, Marlene; Diaz-Insua, Mireya; Jordan, Robert; Schwartz, Jeffrey L.

    2002-01-01

    Genomic instability in the human lymphoblast cell line TK6 was studied in clones surviving 36 generations after exposure to accelerated 56Fe ions. Clones were assayed for 20 characteristics, including chromosome aberrations, plating efficiency, apoptosis, cell cycle distribution, response to a second irradiation, and mutant frequency at two loci. The primary effect of the 56Fe-ion exposure on the surviving clones was a significant increase in the frequency of unstable chromosome aberrations compared to the very low spontaneous frequency, along with an increase in the phenotypic complexity of the unstable clones. The radiation-induced increase in the frequency of unstable chromosome aberrations was much greater than that observed previously in clones of the related cell line, WTK1, which in comparison to the TK6 cell line expresses an increased radiation resistance, a mutant TP53 protein, and an increased frequency of spontaneous unstable chromosome aberrations. The characteristics of the unstable clones of the two cell lines also differed. Most of the TK6 clones surviving exposure to 56Fe ions showed unstable cytogenetic abnormalities, while the phenotype of the WTK1 clones was more diverse. The results underscore the importance of genotype in the characteristics of instability after radiation exposure.

  17. Lymphoblastoid Cell Lines as a Tool to Study Inter-Individual Differences in the Response to Glucose

    PubMed Central

    Grassi, Michael A.; Rao, Vidhya R.; Chen, Siquan; Cao, Dingcai; Gao, Xiaoyu; Cleary, Patricia A.; Huang, R. Stephanie; Paterson, Andrew D.; Natarajan, Rama; Rehman, Jalees; Kern, Timothy S.

    2016-01-01

    Background White blood cells have been shown in animal studies to play a central role in the pathogenesis of diabetic retinopathy. Lymphoblastoid cells are immortalized EBV-transformed primary B-cell leukocytes that have been extensively used as a model for conditions in which white blood cells play a primary role. The purpose of this study was to investigate whether lymphoblastoid cell lines, by retaining many of the key features of primary leukocytes, can be induced with glucose to demonstrate relevant biological responses to those found in diabetic retinopathy. Methods Lymphoblastoid cell lines were obtained from twenty-three human subjects. Differences between high and standard glucose conditions were assessed for expression, endothelial adhesion, and reactive oxygen species. Results Collectively, stimulation of the lymphoblastoid cell lines with high glucose demonstrated corresponding changes on molecular, cellular and functional levels. Lymphoblastoid cell lines up-regulated expression of a panel of genes associated with the leukocyte-mediated inflammation found in diabetic retinopathy that include: a cytokine (IL-1B fold change = 2.11, p-value = 0.02), an enzyme (PKCB fold change = 2.30, p-value = 0.01), transcription factors (NFKB-p50 fold change = 2.05, p-value = 0.01), (NFKB-p65 fold change = 2.82, p-value = 0.003), and an adhesion molecule (CD18 fold change = 2.59, 0.02). Protein expression of CD18 was also increased (p-value = 2.14x10-5). The lymphoblastoid cell lines demonstrated increased adhesiveness to endothelial cells (p = 1.28x10-5). Reactive oxygen species were increased (p = 2.56x10-6). Significant inter-individual variation among the lymphoblastoid cell lines in these responses was evident (F = 18.70, p < 0.0001). Conclusions Exposure of lymphoblastoid cell lines derived from different human subjects to high glucose demonstrated differential and heterogeneous gene expression, adhesion, and cellular effects that recapitulated features found in

  18. Cytotoxic effect of anti-idiotype antibody-chlorambucil conjugates against human lymphoblastoid cells.

    PubMed Central

    Tung, E; Goust, J M; Chen, W Y; Kang, S S; Wang, I Y; Wang, A C

    1983-01-01

    The secreted IgMs of two human lymphoblastoid cell lines, RPMI-6410 and RPMI-8392, were purified. Antisera against these two IgMs were raised in rabbits and made idiotypically specific to the respective antigens through various absorption procedures. By immunofluorescence and radioimmunoassay techniques, the purified anti-idiotype antibodies were found to react also with the membrane Igs of the respective cell lines, but not with those of other cell lines. The purified anti-idiotype antibodies were then coupled with Chlorambucil to form antibody-drug conjugates, whose effectiveness in the in-vitro killing of target cells was evaluated by a chromium-release cytotoxicity assay. The results showed that these anti-idiotype antibody-Chlorambucil conjugates were specifically cytotoxic to lymphoblastoid cells that bore membrane Igs carrying the respective idiotypic determinant(s). Furthermore, the conjugates were far more effective in causing cytolysis to the target cells than either Chlorambucil or the anti-idiotype antibodies alone. PMID:6350169

  19. Cytotoxic effect of anti-idiotype antibody-chlorambucil conjugates against human lymphoblastoid cells.

    PubMed

    Tung, E; Goust, J M; Chen, W Y; Kang, S S; Wang, I Y; Wang, A C

    1983-09-01

    The secreted IgMs of two human lymphoblastoid cell lines, RPMI-6410 and RPMI-8392, were purified. Antisera against these two IgMs were raised in rabbits and made idiotypically specific to the respective antigens through various absorption procedures. By immunofluorescence and radioimmunoassay techniques, the purified anti-idiotype antibodies were found to react also with the membrane Igs of the respective cell lines, but not with those of other cell lines. The purified anti-idiotype antibodies were then coupled with Chlorambucil to form antibody-drug conjugates, whose effectiveness in the in-vitro killing of target cells was evaluated by a chromium-release cytotoxicity assay. The results showed that these anti-idiotype antibody-Chlorambucil conjugates were specifically cytotoxic to lymphoblastoid cells that bore membrane Igs carrying the respective idiotypic determinant(s). Furthermore, the conjugates were far more effective in causing cytolysis to the target cells than either Chlorambucil or the anti-idiotype antibodies alone.

  20. Autophagy is the predominant process induced by arsenite in human lymphoblastoid cell lines

    SciTech Connect

    Bolt, Alicia M.; Byrd, Randi M.; Klimecki, Walter T.

    2010-05-01

    Arsenic is a widespread environmental toxicant with a diverse array of molecular targets and associated diseases, making the identification of the critical mechanisms and pathways of arsenic-induced cytotoxicity a challenge. In a variety of experimental models, over a range of arsenic exposure levels, apoptosis is a commonly identified arsenic-induced cytotoxic pathway. Human lymphoblastoid cell lines (LCL) have been used as a model system in arsenic toxicology for many years, but the exact mechanism of arsenic-induced cytotoxicity in LCL is still unknown. We investigated the cytotoxicity of sodium arsenite in LCL 18564 using a set of complementary markers for cell death pathways. Markers indicative of apoptosis (phosphatidylserine externalization, PARP cleavage, and sensitivity to caspase inhibition) were uniformly negative in arsenite exposed cells. Interestingly, electron microscopy, acidic vesicle fluorescence, and expression of LC3 in LCL 18564 identified autophagy as an arsenite-induced process that was associated with cytotoxicity. Autophagy, a cellular programmed response that is associated with both cellular stress adaptation as well as cell death appears to be the predominant process in LCL cytotoxicity induced by arsenite. It is unclear, however, whether LCL autophagy is an effector mechanism of arsenite cytotoxicity or alternatively a cellular compensatory mechanism. The ability of arsenite to induce autophagy in lymphoblastoid cell lines introduces a potentially novel mechanistic explanation of the well-characterized in vitro and in vivo toxicity of arsenic to lymphoid cells.

  1. Histone markers identify the mode of action for compounds positive in the TK6 micronucleus assay.

    PubMed

    Cheung, Jennifer R; Dickinson, Donna A; Moss, Jocelyn; Schuler, Maik J; Spellman, Richard A; Heard, Pamela L

    2015-01-01

    The in vitro micronucleus assay with TK6 cells is frequently used as part of the genotoxicity testing battery for pharmaceuticals. Consequently, follow-up testing strategies are needed for positive compounds to determine their mode of action, which would then allow for deployment of appropriate in vivo follow-up strategies. We have chosen 3 micronucleus positive compounds, the clastogen etoposide, the aneugen noscapine and the cytotoxicant tunicamycin to evaluate different approaches to determine their aneugenic or clastogenic properties. Each of the three compounds were evaluated following 4 and 24h of continuous treatment by flow cytometry for micronucleus induction, the aneugenicity markers phosphorylated-histone 3 (p-H3) and polyploidy, the clastogenicity marker γH2AX and the apoptosis marker cleaved caspase 3. They were further evaluated by Western blot for mono-ubiquitinated and γH2AX. Results show that the clastogen etoposide produced a dose related increase in γH2AX and mono-ubiquitinated H2AX and a dose related decrease in p-H3 positive mitotic cells. Conversely, the aneugen produced increases in p-H3 and polyploidy with no significant increases seen in mono-ubiquitinated H2AX or γH2AX. Lastly, the cytotoxicant tunicamycin induced neither an increase in p-H3 nor γH2AX. All three compounds produced dose-related increases in cleaved caspase 3. The results from this study provide evidence that adding clastogenicity and aneugenicity markers to the in vitro micronucleus assay in TK6 cells could help to identify the mode of action of positive compounds. The combination of endpoints suggested here needs to be further evaluated by a broader set of test compounds.

  2. Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism

    PubMed Central

    James, S. Jill; Rose, Shannon; Melnyk, Stepan; Jernigan, Stefanie; Blossom, Sarah; Pavliv, Oleksandra; Gaylor, David W.

    2009-01-01

    Research into the metabolic phenotype of autism has been relatively unexplored despite the fact that metabolic abnormalities have been implicated in the pathophysiology of several other neurobehavioral disorders. Plasma biomarkers of oxidative stress have been reported in autistic children; however, intracellular redox status has not yet been evaluated. Lymphoblastoid cells (LCLs) derived from autistic children and unaffected controls were used to assess relative concentrations of reduced glutathione (GSH) and oxidized disulfide glutathione (GSSG) in cell extracts and isolated mitochondria as a measure of intracellular redox capacity. The results indicated that the GSH/GSSG redox ratio was decreased and percentage oxidized glutathione increased in both cytosol and mitochondria in the autism LCLs. Exposure to oxidative stress via the sulfhydryl reagent thimerosal resulted in a greater decrease in the GSH/GSSG ratio and increase in free radical generation in autism compared to control cells. Acute exposure to physiological levels of nitric oxide decreased mitochondrial membrane potential to a greater extent in the autism LCLs, although GSH/GSSG and ATP concentrations were similarly decreased in both cell lines. These results suggest that the autism LCLs exhibit a reduced glutathione reserve capacity in both cytosol and mitochondria that may compromise antioxidant defense and detoxification capacity under prooxidant conditions.—James, S. J., Rose, S., Melnyk, S., Jernigan, S., Blossom, S., Pavliv, O., Gaylor, D. W. Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism. PMID:19307255

  3. Cell culture-induced aberrant methylation of the imprinted IG DMR in human lymphoblastoid cell lines.

    PubMed

    Saferali, Aabida; Grundberg, Elin; Berlivet, Soizik; Beauchemin, Hugues; Morcos, Lisanne; Polychronakos, Constantin; Pastinen, Tomi; Graham, Jinko; McNeney, Brad; Naumova, Anna K

    2010-01-01

    DNA methylation patterns are often poorly conserved through cell culturing. To determine the effect of cell immortalization and culture on DNA methylation profiles, we analyzed methylation in the differentially methylated regions (DMR) of five imprinted domains: the intergenic (IG) DMR on chromosome 14q32; potassium voltage-gated channel, KQT-like subfamily, member 1, (KCNQ1); small nuclear ribonucleoprotein polypeptide N (SNRPN), mesoderm specific transcript homolog (MEST); and H19 in lymphoblastoid cell lines (LCLs). In the IG DMR we found an aberrant methylation pattern that was consistent through all the cell lines tested and significantly different from that of noncultured peripheral blood cells. Using a generalized linear mixed model to compare methylation profiles, we show that recently derived LCLs significantly differ from the CEPH LCLs. This implies a gradual cell-culture related deterioration of DNA methylation in the IG DMR with at least two steps that may be identified: loss of methylation at CG sites 1 and 8; and loss of allelic differences in DNA methylation. The IG DMR methylation profile also confirms the high level of clonality of the CEPH LCLs. We conclude that non-transformed primary cells may be less susceptible to epigenetic anomalies and therefore may provide a more accurate reflection of gene expression in vivo.

  4. Costimulatory signal provided by a B-lymphoblastoid cell line and its Ia-negative variant.

    PubMed Central

    Reiser, H; Benacerraf, B

    1989-01-01

    We have analyzed the requirements of highly purified, resting murine CD4+ T lymphocytes for activation mediated by the lectin Con A and by monoclonal antibodies against the CD3 and Thy-1 molecules. Our results indicate that both the Ia-positive B-lymphoblastoid cell line M12 and its Ia-negative variant M12.C3 can provide the costimulatory activity necessary for these activation pathways. The costimulatory function is preserved upon fixation with paraformaldehyde, indicating that the costimulatory molecule(s) is (are) constitutively expressed on the cell surface. Our experiments also point to interesting differences between the M12 cell line and syngeneic Ia-positive antigen-presenting cells in generating a syngeneic mixed lymphocyte reaction. Finally, we show that the CD4+ T cell-M12.C3 cell interaction can be used to screen for interesting monoclonal antibodies that affect cell function. Images PMID:2532358

  5. Use of RAPD to detect sodium arsenite-induced DNA damage in human lymphoblastoid cells.

    PubMed

    Lee, Yuan-Cho; Yang, Vivian C; Wang, Tsu-Shing

    2007-09-24

    Inorganic arsenic is a known human carcinogen, yet its mechanism of action remains unclear. Our previous study showed that arsenite significantly induces oxidative DNA adducts and DNA-protein cross-links in several mammalian cell lines. In the present study, we used the random amplified polymorphic DNA (RAPD) assay to evaluate the possible target in the genomic DNA of human lymphoblastoid cells that were exposed to sodium arsenite. Treatment with both 10 and 80 microM arsenite for 4h induced significant changes in RAPD profiles compared with the control pattern. Two 10-mer RAPD primers (D11 and F1) produced the most distinguishable banding profiles between arsenite-treated and control genomic DNA. The sequencing of four arsenite-sensitive RAPD bands showed that the RB1CC1 and PACE4 genes might be the DNA targets of sodium arsenite treatment. We propose that arsenite may induce sequence- or gene-specific damage and then change the RAPD profile in human lymphoblastoid cells. The results of our study also show that RAPD combined with other techniques is a good tool for detecting alterations in genomic DNA and for the direct screening of new molecular markers related to arsenite-induced carcinogenesis.

  6. Control of cell respiration by nitric oxide in Ataxia Telangiectasia lymphoblastoid cells.

    PubMed

    Masci, Alessandra; Mastronicola, Daniela; Arese, Marzia; Piane, Maria; De Amicis, Andrea; Blanck, Thomas J J; Chessa, Luciana; Sarti, Paolo

    2008-01-01

    Ataxia Telangiectasia (AT) patients are particularly sensitive to oxidative-nitrosative stress. Nitric oxide (NO) controls mitochondrial respiration via the reversible inhibition of complex IV. The mitochondrial response to NO of AT lymphoblastoid cells was investigated. Cells isolated from three patients and three intrafamilial healthy controls were selected showing within each group a normal diploid karyotype and homogeneous telomere length. Different complex IV NO-inhibition patterns were induced by varying the electron flux through the respiratory chain, using exogenous cell membrane permeable electron donors. Under conditions of high electron flux the mitochondrial NO inhibition of respiration was greater in AT than in control cells (P< or =0.05). This property appears peculiar to AT, and correlates well to the higher concentration of cytochrome c detected in the AT cells. This finding is discussed on the basis of the proposed mechanism of reaction of NO with complex IV. It is suggested that the peculiar response of AT mitochondria to NO stress may be relevant to the mitochondrial metabolism of AT patients.

  7. Genetic instability on chromosome 16 in a Human B lymphoblastoid cell line

    SciTech Connect

    Smith, L.E.; Grosovsky, A.J. )

    1993-11-01

    Mutagenesis at the aprt locus in TK6 human lymphoblasts has been found to occur at an unusually high rate (1.2 [times] 10[sup [minus]9]) for a homozygous diploid locus. Evaluation of linked microsatellite polymorphisms demonstrated that loss of heterozygosity (LOH) accompanies conventional intragenic sequence alterations in each APRT[sup [minus

  8. Study of nuclear proteins in normal and xeroderma pigmentosum lymphoblastoid cells

    SciTech Connect

    Amari, N.M.B.

    1985-01-01

    Nuclear histone and nonhistone (NHP) proteins from normal human and xeroderma pigmentosum, complementation group A (XP-A) lymphoblastoid cells were compared both qualitatively, quantitatively and for binding affinity for DNA. Histones and four NHP fractions (NHP/sub 1-4/) were isolated from purified cell nuclei. Binding affinity to (/sup 3/H) melanoma DNA of histones and each NHP fraction was then determined using gradient dialysis followed by a filter assay. Histones and each NHP fraction were then sub-fractionated by polyacrylamide gel electrophoresis. Densitometric scans of the separation of these proteins on the gels were qualitatively, and quantitatively analyzed and compared between the two cell lines. No qualitative or quantitative differences were observed between histones from XP-A or normal cells.

  9. Human iPSC-derived neurons and lymphoblastoid cells for personalized medicine research in neuropsychiatric disorders.

    PubMed

    Gurwitz, David

    2016-09-01

    The development and clinical implementation of personalized medicine crucially depends on the availability of high-quality human biosamples; animal models, although capable of modeling complex human diseases, cannot reflect the large variation in the human genome, epigenome, transcriptome, proteome, and metabolome. Although the biosamples available from public biobanks that store human tissues and cells may represent the large human diversity for most diseases, these samples are not always sufficient for developing biomarkers for patient-tailored therapies for neuropsychiatric disorders. Postmortem human tissues are available from many biobanks; nevertheless, collections of neuronal human cells from large patient cohorts representing the human diversity remain scarce. Two tools are gaining popularity for personalized medicine research on neuropsychiatric disorders: human induced pluripotent stem cell-derived neurons and human lymphoblastoid cell lines. This review examines and contrasts the advantages and limitations of each tool for personalized medicine research.

  10. Human iPSC-derived neurons and lymphoblastoid cells for personalized medicine research in neuropsychiatric disorders

    PubMed Central

    Gurwitz, David

    2016-01-01

    The development and clinical implementation of personalized medicine crucially depends on the availability of high-quality human biosamples; animal models, although capable of modeling complex human diseases, cannot reflect the large variation in the human genome, epigenome, transcriptome, proteome, and metabolome. Although the biosamples available from public biobanks that store human tissues and cells may represent the large human diversity for most diseases, these samples are not always sufficient for developing biomarkers for patient-tailored therapies for neuropsychiatric disorders. Postmortem human tissues are available from many biobanks; nevertheless, collections of neuronal human cells from large patient cohorts representing the human diversity remain scarce. Two tools are gaining popularity for personalized medicine research on neuropsychiatric disorders: human induced pluripotent stem cell-derived neurons and human lymphoblastoid cell lines. This review examines and contrasts the advantages and limitations of each tool for personalized medicine research. PMID:27757061

  11. Involvement of recombination in x-ray mutagenesis of human cells

    SciTech Connect

    Amundson, S.A. ); Xia, F.; Liber, H.L. )

    1993-01-01

    Closely related human lymphoblastoid cell lines derived from WI-L2 differ greatly in their responses to X-irradiation. Compared with TK6 (ATCC CRL 8015), WI-L2-NS (ATCC CRL 8155) has an enhanced X-ray survival. The induction of mutation by X-rays is also markedly different. The hemizygous hprt locus is slightly more mutable in WI-L2-NS than in TK6, and the dose response fits best to a linear-quadratic curve rather than the linear fit of TK6X-ray induced mutation at the autosomal tk locus in heterozygotes derived from WI-L2-NS is 20-50 fold higher than in heterozygotes derived from TK6. A larger proportion of WI-L2-NS mutants had lost heterozygosity compared with mutants of TK6. , Fluorescence in situ hybridization indicated that loss of heterozygosity was due almost uniformly to deletion of an allele in mutants of TK6, and to recombination or gene conversion in mutants of WI-L2-NS. These results indicate that recombinational repair contributes to both cell survival and mutation following exposure to ionizing radiation.

  12. Involvement of recombination in x-ray mutagenesis of human cells

    SciTech Connect

    Amundson, S.A.; Xia, F.; Liber, H.L.

    1993-06-01

    Closely related human lymphoblastoid cell lines derived from WI-L2 differ greatly in their responses to X-irradiation. Compared with TK6 (ATCC CRL 8015), WI-L2-NS (ATCC CRL 8155) has an enhanced X-ray survival. The induction of mutation by X-rays is also markedly different. The hemizygous hprt locus is slightly more mutable in WI-L2-NS than in TK6, and the dose response fits best to a linear-quadratic curve rather than the linear fit of TK6X-ray induced mutation at the autosomal tk locus in heterozygotes derived from WI-L2-NS is 20-50 fold higher than in heterozygotes derived from TK6. A larger proportion of WI-L2-NS mutants had lost heterozygosity compared with mutants of TK6. , Fluorescence in situ hybridization indicated that loss of heterozygosity was due almost uniformly to deletion of an allele in mutants of TK6, and to recombination or gene conversion in mutants of WI-L2-NS. These results indicate that recombinational repair contributes to both cell survival and mutation following exposure to ionizing radiation.

  13. Upregulation of TFAM and mitochondria copy number in human lymphoblastoid cells.

    PubMed

    Chakrabarty, Sanjiban; D'Souza, Reena Reshma; Kabekkodu, Shama Prasada; Gopinath, Puthiya M; Rossignol, Rodrigue; Satyamoorthy, Kapaettu

    2014-03-01

    Mitochondria are central to several physiological and pathological conditions in humans. In the present study, we performed copy number analysis of nuclear encoded mitochondrial genes, in peripheral blood mononuclear cells (PBMCs) and its representative lymphoblastoid cells (LCLs). We have observed hyper diploid copies of mitochondrial transcription factor A (TFAM) gene in the LCLs along with increased mtDNA copy number, mitochondrial mass, intracellular ROS and mitochondrial membrane potential, suggesting elevated mitochondrial biogenesis in LCLs. Gene expression analysis confirmed TFAM over-expression in LCLs when compared to PBMC. Based on our observation, we suggest that increased copy number of TFAM gene upregulates its expression, increases mtDNA copy numbers and protects it from oxidative stress induced damage in the transformed LCLs.

  14. Fast neutrons-induced apoptosis is Fas-independent in lymphoblastoid cells

    SciTech Connect

    Fischer, Barbara; Benzina, Sami; Jeannequin, Pierre; Dufour, Patrick; Bergerat, Jean-Pierre; Denis, Jean-Marc; Gueulette, John; Bischoff, Pierre L. . E-mail: Pierre.Bischoff@ircad.u-strasbg.fr

    2005-08-26

    We have previously shown that ionizing radiation-induced apoptosis in human lymphoblastoid cells differs according to their p53 status, and that caspase 8-mediated cleavage of BID is involved in the p53-dependent pathway. In the present study, we investigated the role of Fas signaling in caspase 8 activation induced by fast neutrons irradiation in these cells. Fas and FasL expression was assessed by flow cytometry and by immunoblot. We also measured Fas aggregation after irradiation by fluorescence microscopy. We found a decrease of Fas expression after irradiation, but no change in Fas ligand expression. We also showed that, in contrast to the stimulation of Fas by an agonistic antibody, Fas aggregation did not occur after irradiation. Altogether, our data strongly suggest that fast neutrons induced-apoptosis is Fas-independent, even in p53-dependent apoptosis.

  15. Phorbol diesters and transferrin modulate lymphoblastoid cell transferrin receptor expression by two different mechanisms

    SciTech Connect

    Alcantara, O.; Phillips, J.L.; Boldt, D.H.

    1986-12-01

    Expression of transferrin receptors (TfR) by activated lymphocytes is necessary for lymphocyte DNA synthesis and proliferation. Regulation of TfR expression, therefore, is a mechanism by which the lymphocyte's proliferative potential may be directed and controlled. The authors studied mechanisms by which lymphoblastoid cells modulate TfR expression during treatment with phorbol diesters or iron transferrin (FeTf), agents which cause downregulation of cell surface TfR. Phorbol diester-induced TfR downregulation occurred rapidly, being detectable at 2 min and reaching maximal decreases of 50% by 15 min. It was inhibited by cold but not by agents that destabilize cytoskeletal elements. Furthermore, this downregulation was reversed rapidly by washing or by treatment with the membrane interactive agent, chlorpromazine. In contrast, FeTf-induced TfR downregulation occurred slowly. Decreased expression of TfR was detectable only after 15 min and maximal downregulation was achieved after 60 min. Although FeTf-induced downregulation also was inhibited by cold, it was inhibited in addition by a group of microtubule destabilizing agents (colchicine, vinblastine, podophyllotoxin) or cytochalasin B, a microfilament inhibitor. Furthermore, FeTf-induced downregulation was not reversed readily by washing or by treatment with chlorpromazine. Phorbol diesters cause TfR downregulation by a cytoskeleton-independent mechanism. These data indicate that TfR expression is regulated by two independent mechanisms in lymphoblastoid cells, and they provide the possibility that downregulation of TfR by different mechanisms may result in different effects in these cells.

  16. Ciprofloxacin-induced inhibition of topoisomerase II in human lymphoblastoid cells.

    PubMed Central

    Bredberg, A; Brant, M; Jaszyk, M

    1991-01-01

    The antibacterial activities of the fluorinated 4-quinolones (e.g., ciprofloxacin) have been ascribed to a marked inhibition of bacterial DNA gyrase. In contrast, the influence on purified mammalian DNA enzymes, including topoisomerases, has been reported to be several orders of magnitude weaker, occurring at concentrations higher than 100 micrograms of ciprofloxacin per ml. In this study, using a nondenaturing filter elution method, a marked induction of double-strand DNA breaks in human lymphoblastoid cells exposed to 80 micrograms of ciprofloxacin per ml was seen. The proportion of single-strand versus double-strand DNA breaks was similar to that seen with the topoisomerase II inhibitory antitumor agent VP-16. The cellular recovery was more rapid after treatment with ciprofloxacin than after treatment with VP-16, displaying a normal elution profile within 15 min at 37 degrees C (60 min for VP-16). These data indicate that ciprofloxacin has an effect on intracellularly located topoisomerase II in humans. PMID:1645508

  17. The role of mitochondria in the radiation-induced bystander effect in human lymphoblastoid cells.

    PubMed

    Rajendran, Sountharia; Harrison, Scott H; Thomas, Robert A; Tucker, James D

    2011-02-01

    Cells without intact mitochondrial DNA have been shown to lack the bystander effect, which is an energy-dependent process. We hypothesized that cells harboring mutations in mitochondrial genes responsible for ATP synthesis would show a decreased bystander effect compared to normal cells. Radiation-induced bystander effects were analyzed in two normal and four mitochondrial mutant human lymphoblastoid cells. Medium from previously irradiated cells (conditioned medium) was transferred to unirradiated cells from the respective cell lines and evaluated for the bystander effect using the cytokinesis-block micronucleus assay. Unlike normal cells that were used as a control, mitochondrial mutant cells neither generated nor responded to the bystander signals. The bystander effect was inhibited in normal cells by adding the mitochondrial inhibitors rotenone and oligomycin to the culture medium. Time-controlled blocking of the bystander effect by inhibitors was found to occur either for prolonged exposure to the inhibitor prior to irradiation with an immediate and subsequent removal of the inhibitors or immediate post-application of the inhibitor. Adding the inhibitors just prior to irradiation and removing them immediately after irradiation was uneventful. Fully functional mitochondrial metabolic capability may therefore be essential for the bystander effect.

  18. Cytogenetic characterization of low-dose hyper-radiosensitivity in Cobalt-60 irradiated human lymphoblastoid cells.

    PubMed

    Joshi, Gnanada S; Joiner, Michael C; Tucker, James D

    2014-12-01

    The dose-effect relationships of cells exposed to ionizing radiation are frequently described by linear quadratic (LQ) models over an extended dose range. However, many mammalian cell lines, when acutely irradiated in G2 at doses ≤0.3Gy, show hyper-radiosensitivity (HRS) as measured by reduced clonogenic cell survival, thereby indicating greater cell lethality than is predicted by extrapolation from high-dose responses. We therefore hypothesized that the cytogenetic response in G2 cells to low doses would also be steeper than predicted by LQ extrapolation from high doses. We tested our hypothesis by exposing four normal human lymphoblastoid cell lines to 0-400cGy of Cobalt-60 gamma radiation. The cytokinesis block micronucleus assay was used to determine the frequencies of micronuclei and nucleoplasmic bridges. To characterize the dependence of the cytogenetic damage on dose, univariate and multivariate regression analyses were used to compare the responses in the low- (HRS) and high-dose response regions. Our data indicate that the slope of the response for all four cell lines at ≤20cGy during G2 is greater than predicted by an LQ extrapolation from the high-dose responses for both micronuclei and bridges. These results suggest that the biological consequences of low-dose exposures could be underestimated and may not provide accurate risk assessments following such exposures.

  19. Characterization of an antigen associated with the Marek's disease lymphoblastoid cell line MSB-1.

    PubMed

    Ross, L J

    1982-06-01

    A Marek's disease lymphoblastoid cell line (MSB-1) has been analysed by immunoprecipitation for expression of tumour-associated antigen, Marek's disease virus (MDV)-specific antigens and antigens specific to avian leukosis-sarcoma viruses. Rabbit antisera raised against two independently derived cell lines after extensive absorption with normal chick cells reacted with a polypeptide of mol. wt. 40 000 (40K) in extracts of MSB-1 cells. The 40K polypeptide was not present in myeloblasts or in chick embryo fibroblasts (CEF) infected with MDV and did not react with antiserum raised against normal chicken thymus antigens. The possibility that the 40K polypeptide is a tumour-associated antigen is discussed. Seven MDV-specific antigens were noted in infected CEF (mol. wt. 110K, 100K, 80K, 70K, 50K, 35K and 32K) but none of these was detected in MSB-1 cells. The avian leukosis-sarcoma group-specific antigen P27gag and its precursor Pr76gag were not found in MSB-1 cells, confirming that expression of mature gag protein is not required for transformation by MDV. However, two polypeptides of unknown origin and function (mol. wt. 180K and 110K) were precipitated from MSB-1 cells with a rabbit anti-Rous sarcoma (Schmidt-Rupin, subgroup D) antiserum.

  20. P53 alters the cytotoxicity and genotoxicity for oxidized graphene in human B-lymphoblastoid cells

    NASA Astrophysics Data System (ADS)

    Petibone, Dayton Matthew

    Widespread use of oxidized graphene nanomaterials in industry, medicine, and consumer products raises concern about potential adverse impacts on human health. The p53 tumor suppressor protein is crucial to maintaining cellular and genetic stability to prevent carcinogenesis. Here, we show that oxygen functionalized graphene (f-G) absorption and p53 functional status correlate with cytotoxicity and genotoxicity in human B-lymphoblastoid cells. Trends in f-G absorption by were dose-dependent. Cells with functional p53 exposed to f-G arrested in G0/G1 phase of the cell cycle, suppressed f-G induced reactive oxygen species (ROS), and had elevated apoptosis. While compared to p53 competent cells, the p53 deficient cells exposed to f-G accumulated in S-phase of the cell cycle, had elevated ROS levels, and evaded apoptosis. The f-G genotoxicity was evident as increased loss-of-heterozygosity mutants independent of p53 status, and structural chromosome damage in p53 deficient cells. These findings have broad implications for the safety and efficacy of oxidized graphene nanomaterials in industrial, consumer products and biomedical applications.

  1. Proliferation-dependent positioning of individual centromeres in the interphase nucleus of human lymphoblastoid cell lines.

    PubMed

    Ollion, Jean; Loll, François; Cochennec, Julien; Boudier, Thomas; Escudé, Christophe

    2015-07-01

    The cell nucleus is a highly organized structure and plays an important role in gene regulation. Understanding the mechanisms that sustain this organization is therefore essential for understanding genome function. Centromeric regions (CRs) of chromosomes have been known for years to adopt specific nuclear positioning patterns, but the significance of this observation is not yet completely understood. Here, using a combination of fluorescence in situ hybridization and immunochemistry on fixed human cells and high-throughput imaging, we directly and quantitatively investigated the nuclear positioning of specific human CRs. We observe differential attraction of individual CRs toward both the nuclear border and the nucleoli, the former being enhanced in nonproliferating cells and the latter being enhanced in proliferating cells. Similar positioning patterns are observed in two different lymphoblastoid cell lines. Moreover, the positioning of CRs differs from that of noncentromeric regions, and CRs display specific orientations within chromosome territories. These results suggest the existence of not-yet-characterized mechanisms that drive the nuclear positioning of CRs and therefore pave the way toward a better understanding of how CRs affect nuclear organization.

  2. The impact of FANCD2 deficiency on formaldehyde-induced toxicity in human lymphoblastoid cell lines.

    PubMed

    Ren, Xuefeng; Ji, Zhiying; McHale, Cliona M; Yuh, Jessica; Bersonda, Jessica; Tang, Maycky; Smith, Martyn T; Zhang, Luoping

    2013-01-01

    Formaldehyde (FA), a major industrial chemical and ubiquitous environmental pollutant, has recently been classified by the International Agency for Research on Cancer as a human leukemogen. The major mode of action of FA is thought to be the formation of DNA-protein cross-links (DPCs). Repair of DPCs may be mediated by the Fanconi anemia pathway; however, data supporting the involvement of this pathway are limited, particularly in human hematopoietic cells. Therefore, we assessed the role of FANCD2, a critical component of the Fanconi anemia pathway, in FA-induced toxicity in human lymphoblast cell models of FANCD2 deficiency (PD20 cells) and FANCD2 sufficiency (PD20-D2 cells). After treatment of the cells with 0-150 μM FA for 24 h, DPCs were increased in a dose-dependent manner in both cell lines, with greater increases in FANCD2-deficient PD20 cells. FA also induced cytotoxicity, micronuclei, chromosome aberrations, and apoptosis in a dose-dependent manner in both cell lines, with greater increases in cytotoxicity and apoptosis in PD20 cells. Increased levels of γ-ATR and γ-H2AX in both cell lines suggested the recognition of FA-induced DNA damage; however, the induction of BRCA2 was compromised in FANCD2-deficient PD20 cells, potentially reducing the capacity to repair DPCs. Together, these findings suggest that FANCD2 protein and the Fanconi anemia pathway are essential to protect human lymphoblastoid cells against FA toxicity. Future studies are needed to delineate the role of this pathway in mitigating FA-induced toxicity, particularly in hematopoietic stem cells, the target cells in leukemia.

  3. Suppression of Epstein-Barr virus reactivation in lymphoblastoid cells cultured in simulated microgravity.

    PubMed

    Long, J P; Pierson, S; Hughes, J H

    1999-01-01

    Rotating-wall vessels allow for the growth of cells in simulated microgravity. Lymphoblastoid cells cultured in rotating-wall vessels exhibited significant differences in the expression of both early and late Epstein-Barr Virus (EBV) antigens. Viral protein expression (as measured by indirect immunofluorescence) was significantly suppressed in cells cultured in simulated microgravity. A significantly greater percentage of P3HR-1 cells and Daudi cells were positive for the expression of BamH1-Z-DNA fragment of Epstein-Barr replication activator (ZEBRA), early antigen restricted (EA-R), and viral capsid antigen (VCA) in cells cultured in static tissue culture flasks as compared to cells cultured in rotating-wall vessels. We observed a 7, 11, and 25-fold reduction, respectively, for EA-R, VCA, and ZEBRA protein in P3HR-1 cells cultured in simulated microgravity. Additionally, suspension cultures of P3HR-1 cells exhibited significantly greater ZEBRA antigen expression than cells cultured in rotating-wall vessels. As an independent confirmation of the reduction in ZEBRA-protein production in simulated microgravity in P3HR-1 cells, ZEBRA-mRNA was quantitated by reverse transcription polymerase chain reaction. We observed between a 4 to 10-fold reduction in ZEBRA-mRNA in cells cultured in simulated microgravity as compared to cells cultured at 1 x g in tissue culture flasks. Rotating-wall vessels, by virtue of providing a simple culture environment triggering marked differences in viral activation, provide a model whereby both host and viral factors involved in regulating the maintenance of EBV latency can be examined.

  4. Expression of genes and proteins in human cultured lymphoblastoid cells during spaceflight

    NASA Astrophysics Data System (ADS)

    Takahashi, Akihisa; Suzuki, Hiromi; Omori, Katsunori; Seki, Masaya; Hashizume, Toko; Shimazu, Toru; Ishioka, Noriaki; Ohnishi, Takeo

    2012-07-01

    The space environment contains two major biologically significant influences: space radiations and microgravity. The aim of this study was to clarify the biological effects of space radiations, microgravity and a space environment on the gene and protein expression. Space experiments were performed with human cultured lymphoblastoid cell lines at the first life science experiment to be conducted on the Japanese Experimental Module "Kibo" of the International Space Station (ISS). Under one gravity or microgravity condition, the cells were grown in the cell biology experimental facility (CBEF) of the ISS for 8 days without experiencing the stress during launching and landing because the cells were frozen during these periods. Ground control samples also were cultured for 8 days in the CBEF on the ground during the spaceflight. Gene and protein expression was analyzed by using DNA chip (a 44k whole human genome microarray, Agilent Technologies Inc.) and protein chip (Panorama ^{TM} Ab MicroArray, Sigma-Aldrich Co.), respectively. We already reported the behavior of p53-dependent regulated genes and proteins after exposure to space radiations, microgravity, and the space environment during spaceflight. Next stage, we will profile the expression except for the p53 gene status and discuss the biological meaning during spaceflight

  5. Proteomic analysis of lymphoblastoid cells derived from monozygotic twins discordant for bipolar disorder: a preliminary study.

    PubMed

    Kazuno, An-a; Ohtawa, Kenji; Otsuki, Kaori; Usui, Masaya; Sugawara, Hiroko; Okazaki, Yuji; Kato, Tadafumi

    2013-01-01

    Bipolar disorder is a severe mental illness characterized by recurrent manic and depressive episodes. In bipolar disorder, family and twin studies suggest contributions from genetic and environmental factors; however, the detailed molecular pathogenesis is yet unknown. Thus, identification of biomarkers may contribute to the clinical diagnosis of bipolar disorder. Monozygotic twins discordant for bipolar disorder are relatively rare but have been reported. Here we performed a comparative proteomic analysis of whole cell lysate derived from lymphoblastoid cells of monozygotic twins discordant for bipolar disorder by using two-dimensional differential in-gel electrophoresis (2D-DIGE). We found approximately 200 protein spots to be significantly differentially expressed between the patient and the co-twin (t test, p<0.05). Some of the proteins were subsequently identified by liquid chromatography tandem mass spectrometry and included proteins involved in cell death and glycolysis. To examine whether these proteins could serve as biomarkers of bipolar disorder, we performed Western blot analysis using case-control samples. Expression of phosphoglycerate mutase 1 (PGAM1), which is involved in glycolysis, was significantly up-regulated in patients with bipolar disorder (t test, p<0.05). Although PGAM1 cannot be regarded as a qualified biomarker of bipolar disorder from this preliminary finding, it could be one of the candidates for further study to identify biomarkers of bipolar disorder.

  6. Porcine circovirus type 2 morphogenesis in a clone derived from the l35 lymphoblastoid cell line.

    PubMed

    Rodríguez-Cariño, C; Duffy, C; Sánchez-Chardi, A; McNeilly, F; Allan, G M; Segalés, J

    2011-01-01

    Porcine circovirus type 2 (PCV2) is the essential infectious agent of post-weaning multisystemic wasting syndrome (PMWS), one of the most important diseases of swine. Although several studies have described different biological properties of the virus, some aspects of its replication cycle, including ultrastructural alterations, remain unknown. The aim of the present study was to describe for the first time a complete morphogenesis study of PCV2 in a clone of the lymphoblastoid L35 cell line at the ultrastructural level using electron microscopy techniques. Cells were infected with PCV2 at a multiplicity of infection of 10 and examined at 0, 6, 12, 24, 48, 60 and 72h post-infection. PCV2 was internalized by endocytosis, after which the virus aggregated in intracytoplasmic inclusion bodies (ICIs). Subsequently, PCV2 was closely associated with mitochondria, completing a first cytoplasmic phase. The virus entered the nucleus for replication and virus assembly and encapsidation occurred with the participation of the nuclear membrane. Immature virions left the nucleus and formed ICIs in a second cytoplasmic phase. The results suggest that at the end of the replication cycle (between 24 and 48h), PCV2 was released either by budding of mature virion clusters or by lysis of apoptotic or dead cells. In conclusion, the L35-derived clone represents a suitable in-vitro model for PCV2 morphogenesis studies and characterization of the PCV2 replication cycle.

  7. Diarylheptanoids from Alpinia officinarum Cause Distinct but Overlapping Effects on the Translatome of B Lymphoblastoid Cells

    PubMed Central

    Kakegawa, Tomohito; Takase, Saeko; Masubuchi, Eri; Yasukawa, Ken

    2014-01-01

    Diarylheptanoids (AO-0001, AO-0002, and AO-0003) isolated from Alpinia officinarum inhibit proinflammatory mediators and exhibit cytotoxic and antiviral activity. However, the precise mechanisms of action of these diarylheptanoids are unknown as are their effects on expression of specific genes. Here, we used a translatome analysis to investigate the mechanisms and modes of action of these three diarylheptanoids. Polysome-associated messenger RNAs (mRNAs) were prepared from diarylheptanoids-treated and control cells from a human B lymphoblastoid cell line; these mRNA samples were then used for microarray analysis. Microarray Data Analysis Tool version 3.2 was used to analyze the microarray data analysis; this software uses pathway information of the WikiPathways for gene ontology analysis. Each of the diarylheptanoids caused upregulation or downregulation of the same 37 and 286 genes, respectively. Among the 37 upregulated genes, 16 were related to mRNA processing based on the WikiPathways analysis. Our findings provided new insights into the mode of action of diarylheptanoids from A. officinarum. PMID:25254051

  8. Arsenite exposure in human lymphoblastoid cell lines induces autophagy and coordinated induction of lysosomal genes.

    PubMed

    Bolt, Alicia M; Douglas, Randi M; Klimecki, Walter T

    2010-11-30

    Chronic exposure to inorganic arsenic is associated with diverse, complex diseases, making the identification of the mechanism underlying arsenic-induced toxicity a challenge. An increasing body of literature from epidemiological and in vitro studies has demonstrated that arsenic is an immunotoxicant, but the mechanism driving arsenic-induced immunotoxicity is not well established. We have previously demonstrated that in human lymphoblastoid cell lines (LCLs), arsenic-induced cell death is strongly associated with the induction of autophagy. In this study we utilized genome-wide gene expression analysis and functional assays to characterize arsenic-induced effects in seven LCLs that were exposed to an environmentally relevant, minimally cytotoxic, concentration of arsenite (0.75 μM) over an eight-day time course. Arsenic exposure resulted in inhibition of cellular growth and induction of autophagy (measured by expansion of acidic vesicles) over the eight-day exposure duration. Gene expression analysis revealed that arsenic exposure increased global lysosomal gene expression, which was associated with increased functional activity of the lysosome protease, cathepsin D. The arsenic-induced expansion of the lysosomal compartment in LCL represents a novel target that may offer insight into the immunotoxic effects of arsenic.

  9. Integration sites of Epstein-Barr virus genome on chromosomes of human lymphoblastoid cell lines

    SciTech Connect

    Wuu, K.D.; Chen, Y.J.; Wang-Wuu, S.

    1994-09-01

    Epstein-Barr virus (EBV) is the pathogen of infectious mononucleosis. The viral genome is present in more than 95% of the African cases of Burkitt lymphoma and it is usually maintained in episomal form in the tumor cells. Viral integration has been described only for Nanalwa which is a Burkitt lymphoma cell line lacking episomes. In order to examine the role of EBV in the immortalization of human Blymphocytes, we investigated whether the EBV integration into the human genome is essential. If the integration does occur, we would like to know whether the integration is randomly distributed or whether the viral DNA integrates preferentially at certain sites. Fourteen in vitro immortalized human lymphoblastoid cell lines (LCLs) were examined by fluorescence in situ hybridization (FISH) with a biotinylated EBV BamHI w DNA fragment as probe. The episomal form of EBV DNA was found in all cells of these cell lines, while only about 65% of the cells have the integrated viral DNA. This might suggest that integration is not a pre-requisite for cell immortalization. Although all chromosomes, except Y, have been found with integrated viral genome, chromsomes 1 and 5 are the most frequent EBV DNA carrier (p<0.05). Nine chromosome bands, namely, 1p31, 1q31, 2q32, 3q13, 3q26, 5q14, 6q24, 7q31 and 12q21, are preferential targets for EBV integration (p<0.001). Eighty percent of the total 938 EBV hybridization signals were found to be at G-band-positive area. This suggests that the mechanism of EBV integration might be different from that of the retroviruses, which specifically integrate to G-band-negative areas. Thus, we conclude that the integration of EBV to host genome is non-random and it may have something to do with the structure of chromosome and DNA sequences.

  10. Genome-wide survey of interindividual differences of RNA stability in human lymphoblastoid cell lines

    PubMed Central

    Duan, Jubao; Shi, Jianxin; Ge, Xijin; Dölken, Lars; Moy, Winton; He, Deli; Shi, Sandra; Sanders, Alan R.; Ross, Jeff; Gejman, Pablo V.

    2013-01-01

    The extent to which RNA stability differs between individuals and its contribution to the interindividual expression variation remain unknown. We conducted a genome-wide analysis of RNA stability in seven human HapMap lymphoblastoid cell lines (LCLs) and analyzed the effect of DNA sequence variation on RNA half-life differences. Twenty-six percent of the expressed genes exhibited RNA half-life differences between LCLs at a false discovery rate (FDR) < 0.05, which accounted for ~ 37% of the gene expression differences between individuals. Nonsense polymorphisms were associated with reduced RNA half-lives. In genes presenting interindividual RNA half-life differences, higher coding GC3 contents (G and C percentages at the third-codon positions) were correlated with increased RNA half-life. Consistently, G and C alleles of single nucleotide polymorphisms (SNPs) in protein coding sequences were associated with enhanced RNA stability. These results suggest widespread interindividual differences in RNA stability related to DNA sequence and composition variation. PMID:23422947

  11. Epstein-Barr virus genetic variation in lymphoblastoid cell lines derived from Kenyan pediatric population.

    PubMed

    Simbiri, Kenneth O; Smith, Nicholas A; Otieno, Richard; Wohlford, Eric E M; Daud, Ibrahim I; Odada, Sumba P; Middleton, Frank; Rochford, Rosemary

    2015-01-01

    Epstein-Barr virus (EBV) is associated with Burkitt's lymphoma (BL), and in regions of sub-Saharan Africa where endemic BL is common, both the EBV Type 1 (EBV-1) and EBV Type 2 strains (EBV-2) are found. Little is known about genetic variation of EBV strains in areas of sub-Saharan Africa. In the present study, spontaneous lymphoblastoid cell lines (LCLs) were generated from samples obtained from Kenya. Polymerase chain reaction (PCR) amplification of the EBV genome was done using multiple primers and sequenced by next-generation sequencing (NGS). Phylogenetic analyses against the published EBV-1 and EBV-2 strains indicated that one sample, LCL10 was closely related to EBV-2, while the remaining 3 LCL samples were more closely related to EBV-1. Moreover, single nucleotide polymorphism (SNP) analyses showed clustering of LCL variants. We further show by analysis of EBNA-1, BLLF1, BPLF1, and BRRF2 that latent genes are less conserved than lytic genes in these LCLs from a single geographic region. In this study we have shown that NGS is highly useful for deciphering detailed inter and intra-variations in EBV genomes and that within a geographic region different EBV genetic variations can co-exist, the implications of which warrant further investigation. The findings will enhance our understanding of potential pathogenic variants critical to the development and maintenance of EBV-associated malignancies.

  12. Arsenite-induced autophagy is associated with proteotoxicity in human lymphoblastoid cells.

    PubMed

    Bolt, Alicia M; Zhao, Fei; Pacheco, Samantha; Klimecki, Walter T

    2012-10-15

    Epidemiological studies of arsenic-exposed populations have provided evidence that arsenic exposure in humans is associated with immunosuppression. Previously, we have reported that arsenite-induced toxicity is associated with the induction of autophagy in human lymphoblastoid cell lines (LCL). Autophagy is a cellular process that functions in the degradation of damaged cellular components, including protein aggregates formed by misfolded or damaged proteins. Accumulation of misfolded or damaged proteins in the endoplasmic reticulum (ER) lumen causes ER stress and activates the unfolded protein response (UPR). In an effort to investigate the mechanism of autophagy induction by arsenite in the LCL model, we examined the potential contribution of ER stress and activation of the UPR. LCL exposed to sodium arsenite for 8-days induced expression of UPR-activated genes, including CHOP and GRP78, at the RNA and the protein level. Evidence for activation of the three arms of the UPR was observed. The arsenite-induced activation of the UPR was associated with an accumulation of protein aggregates containing p62 and LC3, proteins with established roles in the sequestration and autophagic clearance of protein aggregates. Taken together, these data provide evidence that arsenite-induced autophagy is associated with the generation of ER stress, activation of the UPR, and formation of protein aggregates that may be targeted to the lysosome for degradation.

  13. Cadmium-induced apoptosis in lymphoblastoid cell line: involvement of caspase-dependent and -independent pathways.

    PubMed

    Coutant, A; Lebeau, J; Bidon-Wagner, N; Levalois, C; Lectard, B; Chevillard, S

    2006-11-01

    Cadmium is a widely used heavy metal that causes severe damage to many organs including liver, kidney and lung. Cadmium toxicity has been described as in vitro and in vivo apoptosis but its molecular mechanisms are not fully understood. In this study, we used the human lymphoblastoid cell line Boleth to characterise cadmium-induced apoptosis further, using sub-lethal (10 microM) and lethal (IC50: 350 microM) doses. At lethal concentration, we observed features of apoptosis between 6 and 8 h after treatment: maturation of caspases 3 and 8, poly(ADP-ribose)polymerase (PARP) cleavage and DNA fragmentation. In order to determine the role of the MAPKs in this process, we investigated p38, ERK1/2 and c-Jun NH2-terminal kinases (JNK) phosphorylation: at lethal concentration, all these pathways were rapidly activated, but no decrease in the apoptotic rate was seen on inhibition of these kinases with drugs. Chemical inhibitors of caspases 3 and 8 blocked cleavage of PARP but not cell death, suggesting the existence of a caspase-independent death. We found that cadmium depolarised membrane potential in less than 1 h, as determined with DiOC6 dye. Interestingly, mitochondrial alteration led to the translocation of apoptosis-inducing factor (AIF) to the nucleus, where we observed chromatin condensation and possibly DNA fragmentation. These results suggest that cadmium-induced apoptosis can occur in the Boleth cell line through caspase-dependent and -independent pathways, independently of activation of major MAPKs.

  14. Arsenite-induced autophagy is associated with proteotoxicity in human lymphoblastoid cells

    SciTech Connect

    Bolt, Alicia M.; Zhao, Fei; Pacheco, Samantha; Klimecki, Walter T.

    2012-10-15

    Epidemiological studies of arsenic-exposed populations have provided evidence that arsenic exposure in humans is associated with immunosuppression. Previously, we have reported that arsenite-induced toxicity is associated with the induction of autophagy in human lymphoblastoid cell lines (LCL). Autophagy is a cellular process that functions in the degradation of damaged cellular components, including protein aggregates formed by misfolded or damaged proteins. Accumulation of misfolded or damaged proteins in the endoplasmic reticulum (ER) lumen causes ER stress and activates the unfolded protein response (UPR). In an effort to investigate the mechanism of autophagy induction by arsenite in the LCL model, we examined the potential contribution of ER stress and activation of the UPR. LCL exposed to sodium arsenite for 8-days induced expression of UPR-activated genes, including CHOP and GRP78, at the RNA and the protein level. Evidence for activation of the three arms of the UPR was observed. The arsenite-induced activation of the UPR was associated with an accumulation of protein aggregates containing p62 and LC3, proteins with established roles in the sequestration and autophagic clearance of protein aggregates. Taken together, these data provide evidence that arsenite-induced autophagy is associated with the generation of ER stress, activation of the UPR, and formation of protein aggregates that may be targeted to the lysosome for degradation. -- Highlights: ► Arsenite induces endoplasmic reticulum stress and the unfolded protein response. ► Arsenite induces the formation of protein aggregates that contain p62 and LC3-II. ► Time-course data suggests that arsenite-induced autophagy precedes ER stress.

  15. Changes in the nucleosomal structure of the Marek's disease virus genome in lymphoblastoid cell line MDCC-MSB1 induced by 5-azacytidine.

    PubMed

    Hayashi, M; Io, K; Furuichi, T; Ren, S; Isogai, E; Watanabe, T; Namioka, S

    1995-02-01

    Marek's disease virus (MDV) DNA in latently infected lymphoblastoid cell lines is considerably methylated. Treatment of the MDV-derived lymphoblastoid cell lines MDCC-MSB1 (MSB1) and MDCC-RP1 (RP1) with 5-azacytidine (5-AzC) results in hypomethylation of MDV DNA. An increase in mRNA from certain portions of MDV DNA, including the BamHI-H region, was observed in 5-AzC-treated MSB1 cells, but not in the agent-treated RP1 cells. After the treatment of cells with 5-AzC, a site hypersensitive to digestion with DNaseI appeared in the BamHI-H region of MDV DNA in MSB1 but not in RP1. These results suggested that the enhancement of mRNA synthesis by 5-AzC is associated with changes in the nucleosomal structure of MDV DNA in lymphoblastoid cell line MSB1.

  16. Identification of low-dose responsive metabolites in X-irradiated human B lymphoblastoid cells and fibroblasts

    PubMed Central

    Tsuyama, Naohiro; Mizuno, Hajime; Katafuchi, Atsushi; Abe, Yu; Kurosu, Yumiko; Yoshida, Mitsuaki; Kamiya, Kenji; Sakai, Akira

    2015-01-01

    Ionizing radiation (IR) induces cellular stress responses, such as signal transduction, gene expression, protein modification, and metabolite change that affect cellular behavior. We analyzed X-irradiated human Epstein-Barr virus-transformed B lymphoblastoid cells and normal fibroblasts to search for metabolites that would be suitable IR-responsive markers by Liquid Chromotography–Mass spectrometry (LC–MS). Mass spectra, as analyzed with principal component analysis, showed that the proportion of peaks with IR-induced change was relatively small compared with the influence of culture time. Dozens of peaks that had either been upregulated or downregulated by IR were extracted as candidate IR markers. The IR-changed peaks were identified by comparing mock-treated groups to 100 mGy-irradiated groups that had recovered after 10 h, and the results indicated that the metabolites involved in nucleoside synthesis increased and that some acylcarnitine levels decreased in B lymphoblastoids. Some peaks changed by as much as 20 mGy, indicating the presence of an IR-sensitive signal transduction/metabolism control mechanism in these cells. On the other hand, we could not find common IR-changed peaks in fibroblasts of different origin. These data suggest that cell phenotype-specific pathways exist, even in low-dose responses, and could determine cell behavior. PMID:25227127

  17. Effects of soluble and particulate Cr(VI) on genome-wide DNA methylation in human B lymphoblastoid cells.

    PubMed

    Lou, Jianlin; Wang, Yu; Chen, Junqiang; Ju, Li; Yu, Min; Jiang, Zhaoqiang; Feng, Lingfang; Jin, Lingzhi; Zhang, Xing

    2015-10-01

    Several previous studies highlighted the potential epigenetic effects of Cr(VI), especially DNA methylation. However, few studies have compared the effects of Cr(VI) on DNA methylation profiles between soluble and particulate chromate in vitro. Accordingly, Illumina Infinium Human Methylation 450K BeadChip array was used to analyze DNA methylation profiles of human B lymphoblastoid cells exposed to potassium dichromate or lead chromate, and the cell viability was also studied. Array based DNA methylation analysis showed that the impacts of Cr(VI) on DNA methylation were limited, only about 40 differentially methylated CpG sites, with an overlap of 15CpG sites, were induced by both potassium dichromate and lead chromate. The results of mRNA expression showed that after Cr(VI) treatment, mRNA expression changes of four genes (TBL1Y, FZD5, IKZF2, and KIAA1949) were consistent with their DNA methylation alteration, but DNA methylation changes of other six genes did not correlate with mRNA expression. In conclusion, both of soluble and particulate Cr(VI) could induce a small amount of differentially methylated sites in human B lymphoblastoid cells, and the correlations between DNA methylation changes and mRNA expression varied between different genes.

  18. Effect of cell-derived growth factors and cytokines on the clonal outgrowth of EBV-infected B cells and established lymphoblastoid cell lines.

    PubMed

    Ifversen, P; Zhang, X M; Ohlin, M; Zeuthen, J; Borrebaeck, C A

    1993-07-01

    Epstein-Barr virus (EBV) is a potent inducer of polyclonal B lymphocyte proliferation and is widely used as a tool for the establishment of B cell lines producing human monoclonal antibodies. However, because of low transformability, low clonability, and the inherent instability of EBV-infected B cells, valuable antibody-producing B cells are often lost during this procedure. We have here examined various cell-derived cytokines for their ability to enhance both the cellular outgrowth of newly infected B cells and the clonability of infected B cells and lymphoblastoid cell lines. Our results show that the murine thymoma cell line EL-4 is superior to peripheral blood mononuclear cells in both cellular outgrowth and cloning experiments, whereas monocyte-derived factors and monocyte cell lines were less capable than peripheral blood mononuclear cells in enhancing cellular outgrowth and cloning. Furthermore, the human T cell hybridoma cell line MP6 that secretes a B cell growth and differentiation factor, recently identified as an isoform of thioredoxin, is also capable of stimulating EBV-infected B cells and lymphoblastoid cell lines. Co-cultivation of EBV-infected B cells with MP6 cells significantly enhanced the cloning efficiency at the 1 cell/well level. The present results also suggest that one potential role of the MP6-derived thioredoxin could be the up regulation of IL-6 receptor expression in EBV-infected B cells.

  19. Protein degradation in a LAMP-2-deficient B-lymphoblastoid cell line from a patient with Danon disease.

    PubMed

    Sánchez-Lanzas, Raul; Alvarez-Castelao, Beatriz; Bermejo, Teresa; Ayuso, Teresa; Tuñón, Teresa; Castaño, José G

    2016-08-01

    Danon disease, a condition characterized by cardiomyopathy, myopathy, and intellectual disability, is caused by mutations in the LAMP-2 gene. Lamp-2A protein, generated by alternative splicing from the Lamp-2 pre-mRNA, is reported to be the lysosomal membrane receptor essential for the chaperone-mediated autophagic pathway (CMA) aimed to selective protein targeting and translocation into the lysosomal lumen for degradation. To study the relevance of Lamp-2 in protein degradation, a lymphoblastoid cell line was obtained by EBV transformation of B-cells from a Danon patient. The derived cell line showed no significant expression of Lamp-2 protein. The steady-state mRNA and protein levels of alpha-synuclein, IΚBα, Rcan1, and glyceraldehyde-3-phosphate dehydrogenase, four proteins reported to be selective substrates of the CMA pathway, were similar in control and Lamp-2-deficient cells. Inhibition of protein synthesis showed that the half-life of alpha-synuclein, IΚBα, and Rcan1 was similar in control and Lamp-2-deficient cells, and its degradation prevented by proteasome inhibitors. Both in control and Lamp-2-deficient cells, induction of CMA and macroautophagy by serum and aminoacid starvation of cells for 8h produced a similar decrease in IΚBα and Rcan1 protein levels and was prevented by the addition of lysosome and autophagy inhibitors. In conclusion, the results presented here showed that Lamp-2 deficiency in human lymphoblastoid cells did not modify the steady-state levels or the degradation of several protein substrates reported as selective substrates of the CMA pathway.

  20. Lymphoblastoid Cell lines: a Continuous in Vitro Source of Cells to Study Carcinogen Sensitivity and DNA Repair

    PubMed Central

    Hussain, Tabish; Mulherkar, Rita

    2012-01-01

    Obtaining a continuous source of normal cells or DNA from a single individual has always been a rate limiting step in biomedical research. Availability of Lymphoblastoid cell lines (LCLs) as a surrogate for isolated or cryopreserved peripheral blood lymphocytes has substantially accelerated the process of biological investigations. LCLs can be established by in vitro infection of resting B cells from peripheral blood with Epstein Barr Virus (EBV) resulting in a continuous source, bearing negligible genetic and phenotypic alterations. Being a spontaneous replicating source, LCLs fulfil the requirement of constant supply of starting material for variety of assays, sparing the need of re-sampling. There is a reason to believe that LCLs are in close resemblance with the parent lymphocytes based on the ample supporting observations from a variety of studies showing significant level of correlation at molecular and functional level. LCLs, which carry the complete set of germ line genetic material, have been instrumental in general as a source of biomolecules and a system to carry out various immunological and epidemiological studies. Furthermore, in recent times their utility for analysing the whole human genome has extensively been documented. This proves the usefulness of LCLs in various genetic and functional studies. There are a few contradictory reports that have questioned the employment of LCLs as parent surrogate. Regardless of some inherent limitations LCLs are increasingly being considered as an important resource for genetic and functional research. PMID:24551762

  1. Targeting Epstein-Barr virus–transformed B lymphoblastoid cells using antibodies with T-cell receptor–like specificities

    PubMed Central

    Lai, Junyun; Tan, Wei Jian; Too, Chien Tei; Choo, Joanna Ai Ling; Wong, Lan Hiong; Mustafa, Fatimah Bte; Srinivasan, Nalini; Lim, Angeline Pei Chiew; Zhong, Youjia; Gascoigne, Nicholas R. J.; Hanson, Brendon J.; Chan, Soh Ha; Chen, Jianzhu

    2016-01-01

    Epstein-Barr virus (EBV) is an oncovirus associated with several human malignancies including posttransplant lymphoproliferative disease in immunosuppressed patients. We show here that anti-EBV T-cell receptor–like monoclonal antibodies (TCR-like mAbs) E1, L1, and L2 bound to their respective HLA-A*0201-restricted EBV peptides EBNA1562-570, LMP1125-133, and LMP2A426-434 with high affinities and specificities. These mAbs recognized endogenously presented targets on EBV B lymphoblastoid cell lines (BLCLs), but not peripheral blood mononuclear cells, from which they were derived. Furthermore, these mAbs displayed similar binding activities on several BLCLs, despite inherent heterogeneity between different donor samples. A single weekly administration of the naked mAbs reduced splenomegaly, liver tumor spots, and tumor burden in BLCL-engrafted immunodeficient NOD-SCID/Il2rg−/− mice. In particular, mice that were treated with the E1 mAb displayed a delayed weight loss and significantly prolonged survival. In vitro, these TCR-like mAbs induced early apoptosis of BLCLs, thereby enhancing their Fc-dependent phagocytic uptake by macrophages. These data provide evidence for TCR-like mAbs as potential therapeutic modalities to target EBV-associated diseases. PMID:27338099

  2. [Growth factor production and autocrine mechanism of cell proliferation regulation in the RPMI-6410t lymphoblastoid line].

    PubMed

    Seregina, T M; Mekshenkov, M I

    1988-03-01

    The human lymphoblastoid B-cell line RPMI-6410t was found to synthesize and secrete into the growth medium a factor necessary to maintain the reproduction of these cells. In the condition of low plating density (concentration 1-1000 cells per ml) cell proliferation can be maintained only in the presence of a definite dose of medium conditioned by 6410t cell growth under high concentration. Using such a medium guaranteed almost 100% cloning efficiency of these cells by the method of limiting dilutions. The cloning of 6410t cells in the presence of feeder cells, such as mouse splenocytes and peritoneal cells, failed. The 6410t cells were shown to bind specifically the growth factor secreted by them, thus suggesting the presence of a growth factor acceptor on their surface. With the help of special selective method some clones were derived which did not secrete growth factor but were likely to have growth factor acceptors on their surface. A comparison of growth properties of clones GF- and GF+ supported the idea of autocrine control of proliferation as one of the mechanisms of malignant cell transformation.

  3. An IgM-producing B lymphoblastoid cell line established from lymphomas induced by a non-defective reticuloendotheliosis virus.

    PubMed

    Nazerian, K; Witter, R L; Crittenden, L B; Noori-Dalloii, M R; Kung, H J

    1982-02-01

    Chick syncytial virus (CSV), a strain of avian reticuloendotheliosis virus (REV) causes lymphoid tumours in chickens after a prolonged incubation period. A number of CSV-induced tumours were examined for cell surface antigen and were found to be of the B cell type and to produce immunoglobulin. Attempts were made to grow in vitro cell lines from CSV-induced tumours and a lymphoblastoid cell line was established from a liver tumour of a chicken that was inoculated with CSV via the yolk sac in embryo. The donor chicken was viraemic at the time the tumour was removed. The cell line is designated RECC-RP13, it produces non-defective REV, is a B cell type and it produces IgM. It is free from infection with endogenous and exogenous avian leukosis virus (ALV) and has an increased number of chromosomes. Sequences specific to REV were detected in at least four sites in cellular DNA from RECC-RP13. Sequences specific to ALV DNA, beyond that normally found in 151(5) X 7(1) cells, were not found in DNA from this cell line.

  4. The effect of uranyl acetate on human lymphoblastoid cells (RPMI 6410) and HeLa cells.

    PubMed Central

    Ghadially, F. N.; Yang-Steppuhn, S. E.; Lalonde, J. M.

    1982-01-01

    RPMI 6410 cells and HeLa cells were exposed to uranyl acetate. In RPMI 6410 cell cultures this produced single-membrane-bound presumably lysosomal bodies (called "uraniosomes") containing electron-dense crystals in the cultured cells and crystalline deposits in extracellular locations. Neither uraniosomes nor extracellular uranium deposits were found in HeLa cell cultures. All uraniosomes and extracellular uranium deposits analysed by electron-probed X-ray analysis were found to contain uranium, potassium and phosphorus. Traces of sulphur were detected in some but not all uraniosomes and extracellular uranium deposits. Traces of calcium were found in all extracellular uranium deposits and in some uraniosomes also. Images Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7093141

  5. Host Genetic Variants and Gene Expression Patterns Associated with Epstein-Barr Virus Copy Number in Lymphoblastoid Cell Lines

    PubMed Central

    Houldcroft, Charlotte J.; Petrova, Velislava; Liu, Jimmy Z.; Frampton, Dan; Anderson, Carl A.; Gall, Astrid; Kellam, Paul

    2014-01-01

    Lymphoblastoid cell lines (LCLs) are commonly used in molecular genetics, supplying DNA for the HapMap and 1000 Genomes Projects, used to test chemotherapeutic agents, and informing the basis of a number of population genetics studies of gene expression. The process of transforming human B cells into LCLs requires the presence of Epstein-Barr virus (EBV), a double-stranded DNA virus which through B-cell immortalisation maintains an episomal virus genome in every cell of an LCL at variable copy numbers. Previous studies have reported that EBV alters host-gene expression and EBV copy number may be under host genetic control. We performed a genome-wide association study of EBV genome copy number in LCLs and found the phenotype to be highly heritable, although no individual SNPs achieved a significant association with EBV copy number. The expression of two host genes (CXCL16 and AGL) was positively correlated and expression of ADARB2 was negatively correlated with EBV copy number in a genotype-independent manner. This study shows an association between EBV copy number and the gene expression profile of LCLs, and suggests that EBV copy number should be considered as a covariate in future studies of host gene expression in LCLs. PMID:25290448

  6. Reduced interferon-alpha production by Epstein-Barr virus transformed B-lymphoblastoid cell lines and lectin-stimulated lymphocytes in congenital dyserythropoietic anaemia type I.

    PubMed

    Wickramasinghe, S N; Hasan, R; Smythe, J

    1997-08-01

    The concentrations of interferon-alpha (IFN-alpha) in supernatants from cultures of Epstein-Barr virus (EBV) transformed B-lymphoblastoid cell lines derived from seven patients with congenital dyserythropoietic anaemia (CDA) type I were below the 95% confidence limits for those derived from six healthy subjects. In contrast, the concentrations of IFN-alpha in supernatants from cultures of EBV-transformed lymphoblastoid cell lines derived from four patients with other types of CDA and four patients with hereditary sideroblastic anaemia were normal. Supernatants from cultures of peripheral blood lymphocytes stimulated with phytohaemagglutinin or pokeweed mitogen contained less IFN-alpha when the cells were derived from patients with CDA type I than when derived from healthy subjects. Since patients with CDA type I show a substantial haematological response to treatment with IFN-alpha, the data suggest that impaired IFN-alpha production may be an important pathogenetic mechanism in CDA type I.

  7. Establishment of clival chordoma cell line MUG-CC1 and lymphoblastoid cells as a model for potential new treatment strategies

    PubMed Central

    Gellner, Verena; Tomazic, Peter Valentin; Lohberger, Birgit; Meditz, Katharina; Heitzer, Ellen; Mokry, Michael; Koele, Wolfgang; Leithner, Andreas; Liegl-Atzwanger, Bernadette; Rinner, Beate

    2016-01-01

    Chordomas are rare malignant tumors that develop from embryonic remnants of the notochord and arise only in the midline from the clivus to the sacrum. Surgery followed by radiotherapy is the standard treatment. As chordomas are resistant to standard chemotherapy, further treatment options are urgently needed. We describe the establishment of a clivus chordoma cell line, MUG-CC1. The cell line is characterized according to its morphology, immunohistochemistry, and growth kinetics. During establishment, cell culture supernatants were collected, and the growth factors HGF, SDF-1, FGF2, and PDGF analyzed using xMAP® technology. A spontaneous lymphoblastoid EBV-positive cell line was also developed and characterized. MUG-CC1 is strongly positive for brachyury, cytokeratin, and S100. The cell line showed gains of the entire chromosomes 7, 8, 12, 13, 16, 18, and 20, and high level gains on chromosomes 1q21–1q24 and 17q21–17q25. During cultivation, there was significant expression of HGF and SDF-1 compared to continuous chordoma cell lines. A new, well-characterized clival chordoma cell line, as well as a non-tumorigenic lymphoblastoid cell line should serve as an in vitro model for the development of potential new treatment strategies for patients suffering from this disease. PMID:27072875

  8. Utility of Lymphoblastoid Cell Lines for Induced Pluripotent Stem Cell Generation

    PubMed Central

    Kumar, Satish; Curran, Joanne E.; Glahn, David C.; Blangero, John

    2016-01-01

    A large number of EBV immortalized LCLs have been generated and maintained in genetic/epidemiological studies as a perpetual source of DNA and as a surrogate in vitro cell model. Recent successes in reprograming LCLs into iPSCs have paved the way for generating more relevant in vitro disease models using this existing bioresource. However, the overall reprogramming efficiency and success rate remain poor and very little is known about the mechanistic changes that take place at the transcriptome and cellular functional level during LCL-to-iPSC reprogramming. Here, we report a new optimized LCL-to-iPSC reprogramming protocol using episomal plasmids encoding pluripotency transcription factors and mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate using this optimized protocol. Further, we investigated the transcriptional changes in mRNA and miRNA levels, using FC-abs ≥ 2.0 and FDR ≤ 0.05 cutoffs; 5,228 mRNAs and 77 miRNAs were differentially expressed during LCL-to-iPSC reprogramming. The functional enrichment analysis of the upregulated genes and activation of human pluripotency pathways in the reprogrammed iPSCs showed that the generated iPSCs possess transcriptional and functional profiles very similar to those of human ESCs. PMID:27375745

  9. Characterization of human lymphoblastoid cell lines as a novel in vitro test system to predict the immunotoxicity of xenobiotics.

    PubMed

    Markovič, Tijana; Gobec, Martina; Gurwitz, David; Mlinarič-Raščan, Irena

    2015-02-17

    Evaluating immunomodulatory effects of xenobiotics is an important component of the toxicity studies. Herein we report on the establishment of a novel invitro test system for the immunotoxicity screening of xenobiotics based on human lymphoblastoid cell lines (LCLs). Four immunotoxic compounds; tributyltin chloride, cyclosporine A, benzo(a)pyrene and verapamil hydrochloride, as well as three immune-inert compounds; urethane, furosemide and mannitol were selected for characterization. The treatment of LCLs with immunosuppressive compounds resulted in reduced viability. The IC50 values determined in human LCLs were in agreement with the data obtained for human peripheral mononuclear cells. Since cytokine production reflects lymphocytes responses to external stimuli, we evaluated the functional responses of LCLs by monitoring their pro-inflammatory and immunoregulatory cytokine production. Our findings prove that LCLs allowed for reliable differentiation between immunomodulatory and immune-inert compounds. Hence, pre-treatment with immunomodulatory compounds led to a decrease in the production of pro-inflammatory TNFα, IL-6 and immunoregulatory IL-2, IL-4, IL-10 and IFNγ cytokines, when compared to untreated ionomycin/PMA stimulated cells. Moreover, testing a panel of ten LCLs derived from unrelated healthy individuals reflects inter-individual variability in response to immunomodulatory xenobiotics. In conclusion, LCLs provide a novel alternative method for the testing of the immunotoxic effects of xenobiotics.

  10. Arginine to lysine 108 substitution in recombinant CYP1A2 abolishes methoxyresorufin metabolism in lymphoblastoid cells

    PubMed Central

    Hadjokas, Nicholas E; Dai, Renke; Friedman, Fred K; Spence, Michael J; Cusack, Barry J; Vestal, Robert E; Ma, Yongsheng

    2002-01-01

    Cytochrome P4501A2 (CYP1A2) activates a large number of procarcinogens to carcinogens. Phytochemicals such as flavones can inhibit CYP1A2 activity competitively, and hydroxylated derivatives of flavone (galangin) may be potent, selective inhibitors of CYP1A2 activity relative to CYP1A1 activity. Molecular modelling of the CYP1A2 interaction with hydroxylated derivatives of flavone suggests that a number of hydrophobic residues of the substrate-binding domain engage in hydrogen bonding with such inhibitors.We have tested this model using site-directed mutagenesis of these residues in expression plasmids transfected into the human B-lymphoblastoid cell line, AHH-1 TK+/−.Consistent with the molecular model's predicted placement in the active site, amino acid substitutions at the predicted residues abolished CYP1A2 enzymatic activity.Transfected cell lines contained equal amounts of immunoreactive CYP1A2.Our results support the molecular model's prediction of the critical amino acid residues present in the hydrophobic active site, residues that can hydrogen bond with CYP1A2 inhibitors and modify substrate binding and/or turnover. PMID:12023936

  11. Molecular and cytogenetic analysis of lymphoblastoid and colon cancer cell lines from cotton-top tamarin (Sagiunus oedipus).

    PubMed

    Mao, X; McGuire, S; Hamoudi, R A

    2000-07-01

    The cotton-top tamarin (CTT) (Sagiunus oedipus) has been used as an animal model to investigate the etiology and pathophysiology of several human diseases, including ulcerative colitis and its associated colorectal carcinoma (CRC). Little is known, however, about genetic synteny between CTT and humans, and about chromosome aberrations in CTT CRC. To address these issues, we have analyzed CTT lymphoblastoid and CRC cell lines using cytogenetics, fluorescence in situ hybridization (Zoo-FISH), and direct sequencing. The CTT lymphocytes had pseudodiploid chromosomes of 46. The CTT CRC cells showed near-diploid chromosomes of 45. Several clonal structural aberrations were observed, including der(1), a marker chromosome, and double minutes. Zoo-FISH using human chromosome 2, 3, 5, 6, 9, 11, 13, 15, 16, 17, 19, 22, and X paints identified homologous chromosomes and subchromosomal regions in the CTT genome. Fluorescence in situ hybridization with human telomeric probe also detected a homologous sequence in CTT genome. Direct sequencing of CTT genomic DNA using primers amplifying exons 4 and 15 of the human APC gene identified DNA sequences in CTT genome with 99% and 95% homology, respectively. These results provide a basis for further comparative studies of CTT and human genome.

  12. Complete genome sequence of Hydrogenobacter thermophilus type strain (TK-6T)

    SciTech Connect

    Zeytun, Ahmet; Sikorski, Johannes; Nolan, Matt; Lapidus, Alla L.; Lucas, Susan; Han, James; Tice, Hope; Cheng, Jan-Fang; Tapia, Roxanne; Goodwin, Lynne A.; Pitluck, Sam; Liolios, Konstantinos; Ivanova, N; Mavromatis, K; Mikhailova, Natalia; Ovchinnikova, Galina; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Ngatchou, Olivier Duplex; Land, Miriam L; Hauser, Loren John; Chang, Yun-Juan; Jeffries, Cynthia; Han, Cliff; Detter, J. Chris; Ubler, Susanne; Rohde, Manfred; Tindall, Brian; Wirth, Reinhard; Woyke, Tanja; Bristow, James; Eisen, Jonathan; Markowitz, Victor; Hugenholtz, Philip; Klenk, Hans-Peter; Kyrpides, Nikos C

    2011-01-01

    Hydrogenobacter thermophilus Kawasumi et al. 1984 is the type species of the genus Hydrogenobacter. H. thermophilus was the first obligate autotrophic organism reported among aerobic hydrogen-oxidizing bacteria. Strain TK-6T is of interest because of the unusually efficient hydrogen-oxidizing ability of this strain, which results in a faster generation time compared to other autotrophs. It is also able to grow anaerobically using nitrate as an electron acceptor when molecular hydrogen is used as the energy source, and able to aerobically fix CO2 via the reductive tricarboxylic acid cycle. This is the fifth completed genome sequence in the family Aquificaceae, and the second genome sequence determined from a strain derived from the original isolate. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,742,932 bp long genome with its 1,899 protein-coding and 49 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  13. NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells

    NASA Technical Reports Server (NTRS)

    Zhang, Ying; Lim, Chang U K.; Williams, Eli S.; Zhou, Junqing; Zhang, Qinming; Fox, Michael H.; Bailey, Susan M.; Liber, Howard L.

    2005-01-01

    Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

  14. Gene conversion is strongly induced in human cells by double-strand breaks and is modulated by the expression of BCL-XL

    SciTech Connect

    Wiese, Claudia; Pierce, Andrew J.; Gauny, Stacey S.; Jasin, Maria; Kronenberg, Amy

    2001-09-25

    Homology-directed repair (HDR) of DNA double-strand breaks (DSBs) is a well-established mechanism that contributes to the maintenance of genomic stability in rodent cells, and it has been assumed that HDR is of similar importance in the repair of DSBs in human cells. However, in addition to promoting genomic stability, some outcomes of homologous recombination can be deleterious, suggesting that factors exist to regulate HDR. We previously demonstrated that overexpression of BCL-2 or BCL-xL enhanced the frequency of x-ray-induced mutations involving the TK1 locus, including loss of heterozygosity (LOH) events presumed to arise by mitotic recombination. The present study was designed to test whether HDR is a prominent DSB repair pathway in human cells, and to directly determine whether ectopic expression of BCL-xL affects HDR. We used the B-lymphoblastoid cell line TK6, which expresses wild-type TP53 and resembles normal lymphocytes in undergoing apoptosis following! genotoxic stress. U sing isogenic derivatives of TK6 cells (TK6-neo, TK6-bcl-xL), we find that a DSB in an integrated HDR reporter stimulates gene conversion 40-50-fold in TK6-neo cells, demonstrating that a DSB can be efficiently repaired by gene conversion in human cells. Significantly, DSB-induced gene conversion events are 3- to 4-fold more frequent in BCL-xL overexpressing cells. The results demonstrate that HDR plays an important role in maintaining genomic integrity in human cells and that ectopic expression of BCL-xL enhances HDR of DSBs. To our knowledge, this is the first study to highlight a function for BCL-xL in modulating DSB repair in human cells.

  15. Ye-1, a monoclonal antibody that cross-reacts with HLA-B27 lymphoblastoid cell lines and an arthritis causing bacteria.

    PubMed Central

    Kono, D H; Ogasawara, M; Effros, R B; Park, M S; Waldord, R L; Yu, D T

    1985-01-01

    A monoclonal antibody, Ye-1, was generated by immunizing BALB/c mice with Yersinia enterocolitica. This antibody also reacted with all of 12 B27 positive lymphoblastoid cell lines, but only four of 31 B27 negative ones. Three of the four reactive B27 negative cell lines were B7 positive. A B27 positive cell line which has lost the B27 expression because of experimentally-induced mutation became unreactive with the Ye-1. These findings support the possibility that there is cross-reactivity between HLA-B27 antigens and Y. enterocolitica. PMID:3878239

  16. In vitro evaluation of human hybrid cell lines generated by fusion of B-lymphoblastoid cells and ex vivo tumour cells as candidate vaccines for haematological malignancies.

    PubMed

    Mohamed, Yehia S; Dunnion, Debbie; Teobald, Iryna; Walewska, Renata; Browning, Michael J

    2012-10-12

    Fusions of dendritic cells (DCs) and tumour cells have been shown to induce protective immunity to tumour challenge in animal models, and to represent a promising approach to cancer immunotherapy. The broader clinical application of this approach, however, is potentially constrained by the lack of replicative capacity and limited standardisation of fusion cell preparations. We show here that fusion of ex vivo tumour cells isolated from patients with a range of haematological malignancies with the human B-lymphoblastoid cell line (LCL), HMy2, followed by chemical selection of the hybridomas, generated stable, self-replicating human hybrid cell lines that grew continuously in tissue culture, and survived freeze/thawing cycles. The hybrid cell lines expressed HLA class I and class II molecules, and the major T-cell costimulatory molecules, CD80 and CD86. All but two of 14 hybrid cell lines generated expressed tumour-associated antigens that were not expressed by HMy2 cells, and were therefore derived from the parent tumour cells. The hybrid cell lines stimulated allogeneic T-cell proliferative responses and interferon-gamma release in vitro to a considerably greater degree than their respective parent tumour cells. The enhanced T-cell stimulation was inhibited by CTLA4-Ig fusion protein, and by blocking antibodies to MHC class I and class II molecules. Finally, all of five LCL/tumour hybrid cell lines tested induced tumour antigen-specific cytotoxic T-cell responses in vitro in PBL from healthy, HLA-A2+ individuals, as detected by HLA-A2-peptide pentamer staining and cellular cytotoxicity. These data show that stable hybrid cell lines, with enhanced immunostimulatory properties and potential for therapeutic vaccination, can be generated by in vitro fusion and chemical selection of B-LCL and ex vivo haematological tumour cells.

  17. NKG2A-Expressing Natural Killer Cells Dominate the Response to Autologous Lymphoblastoid Cells Infected with Epstein–Barr Virus

    PubMed Central

    Hatton, Olivia; Strauss-Albee, Dara Marie; Zhao, Nancy Q.; Haggadone, Mikel D.; Pelpola, Judith Shanika; Krams, Sheri M.; Martinez, Olivia M.; Blish, Catherine A.

    2016-01-01

    Epstein–Barr virus (EBV) is a human γ-herpesvirus that establishes latency and lifelong infection in host B cells while achieving a balance with the host immune response. When the immune system is perturbed through immunosuppression or immunodeficiency, however, these latently infected B cells can give rise to aggressive B cell lymphomas. Natural killer (NK) cells are regarded as critical in the early immune response to viral infection, but their role in controlling expansion of infected B cells is not understood. Here, we report that NK cells from healthy human donors display increased killing of autologous B lymphoblastoid cell lines (LCLs) harboring latent EBV compared to primary B cells. Coculture of NK cells with autologous EBV+ LCL identifies an NK cell population that produces IFNγ and mobilizes the cytotoxic granule protein CD107a. Multi-parameter flow cytometry and Boolean analysis reveal that these functional cells are enriched for expression of the NK cell receptor NKG2A. Further, NKG2A+ NK cells more efficiently lyse autologous LCL than do NKG2A− NK cells. More specifically, NKG2A+2B4+CD16−CD57−NKG2C−NKG2D+ cells constitute the predominant NK cell population that responds to latently infected autologous EBV+ B cells. Thus, a subset of NK cells is enhanced for the ability to recognize and eliminate autologous, EBV-infected transformed cells, laying the groundwork for harnessing this subset for therapeutic use in EBV+ malignancies. PMID:28018364

  18. RECEPTOR FOR SOLUBLE C3 AND C3b ON HUMAN LYMPHOBLASTOID (RAJI) CELLS

    PubMed Central

    Theofilopoulos, Argyrios N.; Bokisch, Viktor A.; Dixon, Frank J.

    1974-01-01

    This study describes the presence of a receptor for fluid phase human C3 and C3b on Raji cell membranes. The binding of C3 and C3b was demonstrated indirectly by a fluoresceinated anti-C3 serum and directly by using radioiodinated proteins. No other complement proteins or serum factors were needed to mediate binding of C3 and C3b to the receptor. The possibility of enzymatic cleavage of C3 before or after its attachment on the cell membrane was ruled out by the demonstration of antigenically intact C3 on Raji cells. Inhibition and dissociation of Raji cell-EAC1423 rosettes by C3 and C3b indicated that both of these proteins bind to the same receptor site or closely associated receptor sites on Raji cells. C3b-bearing Raji cells were immune adherence negative, indicating that C3b binding to the receptor is brought about through the immune adherence region of the molecule and not the C3d portion. The C3 receptor on Raji cell membranes is uniformly distributed and can move on the membrane plane. Approximately 4 x 105 molecules of C3 or C3b bind per Raji cell. The receptor had a higher affinity for C3 than C3b, as was shown by uptake experiments and inhibition of Raji cell-EAC1423 rosette formation. Apart from the described receptor for C3 and C3b another specific receptor for C3b inactivator-cleaved C3b (C3d) bound to red cells was shown to be present on Raji cells. Raji cells cultured in medium containing fresh normal human serum and cobra venom factor were lysed. Similar results were obtained when C3b-bearing Raji cells were cultured in medium with fresh normal human serum. The lytic effect could be abolished by inactivating serum C3 proactivator (C3PA) and required C6. It was concluded that C3b bound to the Raji cell membrane activates the complement system through the alternate pathway and results in membrane damage and cytolysis. It is postulated that cell destruction by this mechanism may play an important role in vivo in controlling cell growth. PMID:4591176

  19. Molecular Signatures of Cardiac Defects in Down Syndrome Lymphoblastoid Cell Lines Suggest Altered Ciliome and Hedgehog Pathways

    PubMed Central

    Ripoll, Clémentine; Rivals, Isabelle; Ait Yahya-Graison, Emilie; Dauphinot, Luce; Paly, Evelyne; Mircher, Clothilde; Ravel, Aimé; Grattau, Yann; Bléhaut, Henri; Mégarbane, André; Dembour, Guy; de Fréminville, Bénédicte; Touraine, Renaud; Créau, Nicole; Potier, Marie Claude; Delabar, Jean Maurice

    2012-01-01

    Forty percent of people with Down syndrome exhibit heart defects, most often an atrioventricular septal defect (AVSD) and less frequently a ventricular septal defect (VSD) or atrial septal defect (ASD). Lymphoblastoid cell lines (LCLs) were established from lymphocytes of individuals with trisomy 21, the chromosomal abnormality causing Down syndrome. Gene expression profiles generated from DNA microarrays of LCLs from individuals without heart defects (CHD−; n = 22) were compared with those of LCLs from patients with cardiac malformations (CHD+; n = 21). After quantile normalization, principal component analysis revealed that AVSD carriers could be distinguished from a combined group of ASD or VSD (ASD+VSD) carriers. From 9,758 expressed genes, we identified 889 and 1,016 genes differentially expressed between CHD− and AVSD and CHD− and ASD+VSD, respectively, with only 119 genes in common. A specific chromosomal enrichment was found in each group of affected genes. Among the differentially expressed genes, more than 65% are expressed in human or mouse fetal heart tissues (GEO dataset). Additional LCLs from new groups of AVSD and ASD+VSD patients were analyzed by quantitative PCR; observed expression ratios were similar to microarray results. Analysis of GO categories revealed enrichment of genes from pathways regulating clathrin-mediated endocytosis in patients with AVSD and of genes involved in semaphorin-plexin-driven cardiogenesis and the formation of cytoplasmic microtubules in patients with ASD-VSD. A pathway-oriented search revealed enrichment in the ciliome for both groups and a specific enrichment in Hedgehog and Jak-stat pathways among ASD+VSD patients. These genes or related pathways are therefore potentially involved in normal cardiogenesis as well as in cardiac malformations observed in individuals with trisomy 21. PMID:22912673

  20. Oxidative metabolism of flunarizine and cinnarizine by microsomes from B-lymphoblastoid cell lines expressing human cytochrome P450 enzymes.

    PubMed

    Kariya, S; Isozaki, S; Uchino, K; Suzuki, T; Narimatsu, S

    1996-11-01

    The oxidative metabolism of cinnarizine [(E)-1-(diphenylmethyl)-4-(3-phenyl-2-propyl)piperazine, CZ] and flunarizine [(E)-1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propyl)piperazine, FZ] was examined in microsomes from lymphoblastoid cells that expressed human cytochrome P450 (CYP) enzymes. Among 10 kinds of CYP enzymes examined, only CYP2D6 catalyzed p-hydroxylation of the cinnamyl phenyl ring of CZ (C-2 formation) and FZ (F-2 formation), and only CYP2B6 exhibited activity for p-hydroxylation (C-4 formation) of the diphenylmethyl group of CZ at a substrate concentration of 50 microM. On the other hand, CYP2C9 together with CYP1A1, -1A2 and/or -2A6 mediated N-desalkylation at the 1- and 4-positions of the piperazine ring of the two drugs that formed C-1 and C-3 from CZ and F-1 and F-3 from FZ, respectively, whereas CYP2C8, -2C19, -2E1 or -3A4 did not show detectable activity for these reactions under the conditions used. We then examined kinetics for the oxidative metabolism of CZ and FZ using CYP2B6 and -2D6 that have considerable activities. CYP2D6 with Km values of 2 to 4 microM had intrinsic clearance values (Vmax/Km) of 0.31 and 0.14 ml/min/nmol CYP for C-2 and F-2 formation, respectively, while CYP2B6 with a Km value of 17 microM exhibited the clearance value of 0.10 ml/min/nmol CYP for C-4 formation. These results suggest that CYP2D6 mainly mediates p-hydroxylation of the cinnamyl phenyl rings of CZ and FZ, and CYP2B6 mediates that of the diphenylmethyl group of CZ.

  1. In vitro adverse effects of iron ore dusts on human lymphoblastoid cells in culture.

    PubMed

    Wang, He; Wang, Jing J; Sanderson, Barbara J S

    2013-01-01

    The aim of this study was to investigate the adverse effects produced by four types of iron (Fe) ore dust using cultured human cells. Genotoxicity and cytotoxicity induced by Fe ore dusts were determined by assays including cytokinesis block micronucleus (CBMN), population growth, and methyl tetrazolium (MTT). Four iron ore dusts were tested, namely, 1002 Limonite & Goethite (1002), HG2 hematite (HG2), HG1 Soutlem Pit (HG1), and HG4. WIL2 -NS cells were incubated for 10 h with extracts from a range of concentrations (0, 75, or 150 μg/ml) of Fe ore dust. Significant decreases in percent cell viability were seen at 150 μg/ml HG2 and 1002 as measured by MTT, with viability that decreased to 75 and 73%, respectively, compared to untreated controls. The cell population regrew to a different extent after Fe ore dust was removed, except for HG1, where population remained declined. An approximately twofold significant increase in the frequency of micronucleated binucleated cells (MNBNC) was seen with 1002, HG2, and HG1 at 150 μg/ml. A significant rise in apoptosis induction was observed at 150 μg/ml HG1. Data indicate that Fe ore dusts at 150 μg/ml produced cytotoxicity and genotoxicity.

  2. Modulation of a human lymphoblastoid B cell line by cyclic AMP. Ig secretion and phosphatidylcholine metabolism

    SciTech Connect

    Shearer, W.T.; Patke, C.L.; Gilliam, E.B.; Rosenblatt, H.M.; Barron, K.S.; Orson, F.M.

    1988-09-01

    A transformed human B cell line, LA350, was found to be sensitive to cAMP-elevating agents by responding with rapid (0 to 2 h) severalfold elevations of intracellular cAMP to treatment with cholera toxin, isobutylmethylxanthine (IBMX), forskolin, and dibutyryl cAMP (all p less than 0.001). These cAMP-elevating agents also produced significant inhibitions of subsequent (48 to 72 h) Ig secretion by the same B cells as measured by a reverse hemolytic plaque assay and an enzyme-linked immunoadsorbent assay for IgM (both p less than 0.001). PMA- and IBMX-treated cells were particularly responsive to the effects of cholera toxin, showing a doubling of cAMP content and profound decrease in Ig production (p less than 0.001). Because our previous studies had correlated activation of the metabolic turnover of the phosphatidylcholine (PC) fraction of membrane phospholipids with enhanced Ig secretion, we examined the sensitivity of PC metabolism to cAMP in control and PMA-stimulated cells. Formation of PC was found to be inhibited by forskolin and IBMX (both p less than 0.002) but breakdown of PC was stimulated (p less than 0.001). These findings imply that as the enzymatic products of PC, choline phosphate and diacylglycerol, are depleted due to the combined effects of cAMP upon synthesis and turnover of PC, there is a decrease in Ig secretion. Since diacylglycerol activates protein kinase C, it appears reasonable that Ig secretion is at least partially regulated by cAMP-responsive alterations in PC metabolism produced by protein kinase C-induced phosphorylation. We conclude that the early cAMP-sensitive changes in PC metabolism in this activated B cell line may signal for subsequent alterations in Ig secretion.

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

    PubMed Central

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

    2011-01-01

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

  4. High-potentiality preliminary selection criteria and transformation time-dependent factors analysis for establishing Epstein-Barr virus transformed human lymphoblastoid cell lines.

    PubMed

    Chang, I-C; Wu, J-Y; Lu, H-I; Ko, H-W; Kuo, J-L; Wang, C-Y; Shen, P-S; Hwang, S-M

    2006-12-01

    Infection of freshly isolated and cryopreserved lymphocytes with Epstein-Barr virus (EBV) leads to the establishment of human B lymphoblastoid cell lines (LCLs). Techniques for optimal infection of the lymphocytes are vital for the establishment of a human biobank. The present study found that more than half (58-86%) of such established LCLs had transport times of less than 48 h, cell densities exceeding 10(6) cells/ml and cell viabilities greater than 90%. After EBV infection, 3306 freshly isolated lymphocytes required 30.0 +/- 0.1 days to become LCLs. Conversely, 1210 cryopreserved lymphocytes required 36.2 +/- 0.4 days. Cell density and viability of the culture affected transformation time in freshly isolated lymphocytes. On the other hand, blood transport time, cryopreservation time and initial cell viability were major factors in cryopreserved specimens. These results contribute to general information concerning the establishment of a human biobank for EBV infected cells.

  5. Leukemia patient-derived lymphoblastoid cell lines exhibit increased induction of leukemia-associated transcripts following high-dose irradiation.

    PubMed

    Spencer, A; Granter, N

    1999-09-01

    Improvement in diagnostic cytogenetic techniques has led to the recognition of an increasing number of leukemia-associated chromosomal translocations and inversions. These genetic lesions frequently are associated with the disruption of putative transcription factors and the production of hybrid transcripts that are implicated in leukemogenesis. Epidemiologic evidence suggests that some, but not all, individuals with a history of gamma-irradiation exposure are at increased risk of developing chronic myeloid leukemia (CML). CML is characterized by the Philadelphia chromosome and transcription of the resulting hybrid BCR-ABL gene. Utilizing the leukemia-associated BCR-ABL p210 transcript as a marker, we sought differences in the induction of illegitimate genetic recombination following high-dose gamma-irradiation of karyotypically normal lymphoblastoid cell lines (LCL) derived from individuals with and without a history of myeloid leukemias. Six LCL [4 leukemia patient derived [2 acute myeloid leukemia and 2 CML] and 2 from normal individuals were analyzed with reverse transcriptase polymerase chain reaction for BCR-ABL under stringent conditions following exposure to 0, 50, or 100 Gy of LET gamma-irradiation delivered via a Varian linear accelerator at 4 MV. Transcripts identical to disease-associated b2a2 and b3a2 transcripts were detected both spontaneously (background illegitimate genetic recombination) and following gamma-irradiation. Background BCR-ABL positivity was demonstrable in 4 of the 6 LCL, with no significant difference in detection between leukemic- and nonleukemic-derived LCL. Overall, increasing gamma-irradiation dose resulted in an increased frequency of BCR-ABL transcript detection (0 Gy vs 50 Gy vs 100 Gy,p = 0.0023, Chi-square test). Within the leukemic- but not the nonleukemic-derived LCL there was significantly greater BCR-ABL positivity after gamma-irradiation compared to unirradiated equivalents. Furthermore, the BCR-ABL positivity of both

  6. Genetic Regulation of Charged Particle Mutagenesis in Human Cells

    NASA Technical Reports Server (NTRS)

    Kronenberg, Amy; Gauny, S.; Cherbonnel-Lasserre, C.; Liu, W.; Wiese, C.

    1999-01-01

    Our studies use a series of syngeneic, and where possible, isogenic human B-lymphoblastoid cell lines to assess the genetic factors that modulate susceptibility apoptosis and their impact on the mutagenic risks of low fluence exposures to 1 GeV Fe ions and 55 MeV protons. These ions are representative of the types of charged particle radiation that are of particular significance for human health in the space radiation environment. The model system employs cell lines derived from the male donor WIL-2. These cells have a single X chromosome and they are hemizygous for one mutation marker, hypoxanthine phosphoribosyltransferase (HPRT). TK6 and WTK1 cells were each derived from descendants of WIL-2 and were each selected as heterozygotes for a second mutation marker, the thymidine kinase (TK) gene located on chromosome 17q. The HPRT and TK loci can detect many different types of mutations, from single basepair substitutions up to large scale loss of heterozygosity (LOH). The single expressing copy of TK in the TK6 and WTKI cell lines is found on the same copy of chromosome 17, and this allele can be identified by a restriction fragment length polymorphism (RFLP) identified when high molecular weight DNA is digested by the SacI restriction endonuclease and hybridized against the cDNA probe for TK. A large series of polymorphic linked markers has been identified that span more than 60 cM of DNA (approx. 60 megabasepairs) and distinguish the copy of chromosome 17 bearing the initially active TK allele from the copy of chromosome 17 bearing the silent TK allele in both TK6 and WTKI cells. TK6 cells express normal p53 protein while WTKI cells express homozygous mutant p53. Expression of mutant p53 can increase susceptibility to x-ray-induced mutations. It's been suggested that the increased mutagenesis in p53 mutant cells might be due to reduced apoptosis.

  7. Activation of 3-nitrobenzanthrone and its metabolites to DNA-damaging species in human B lymphoblastoid MCL-5 cells.

    PubMed

    Arlt, Volker M; Cole, Kathleen J; Phillips, David H

    2004-03-01

    3-Nitrobenzanthrone (3-NBA) is one of the most potent mutagens in the Ames Salmonella typhimurium assay and a suspected human carcinogen recently identified in diesel exhaust and in airborne particulate matter. 3-Aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA) and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) have been identified as 3-NBA metabolites. In the present study we investigated the genotoxic effects of 3-NBA and its metabolites in the human B lymphoblastoid cell line MCL-5. DNA strand breaks were measured using the Comet assay, chromosomal damage was assessed using the micronucleus assay and DNA adduct formation was determined by 32P-post-labelling analysis. DNA strand-breaking activity was observed with each compound in a concentration-dependent manner (1-50 microM, 2 h incubation time). At 50 microM median comet tail lengths (CTLs) were 25.0 microm for 3-NBA, 48.0 microm for 3-ABA, 54.5 microm for 3-Ac-ABA and 65.0 microm for N-Ac-N-OH-ABA. Median CTLs in control incubations were in the range 7.7-13.1 micro m. Moreover, the strand-breaking activity of 3-NBA was more pronounced in the presence of a DNA repair inhibitor, hydroxyurea. Depending on the concentration used (1-20 microM, 24 h incubation time), 3-NBA and its metabolites also showed clastogenic activity in the micronucleus assay. 3-NBA and N-Ac-N-OH-ABA were the most active at low concentrations; at 1 microM the total number of micronuclei per 500 binucleate cells was 4.7 +/- 0.6 in both cases, compared with 1.7-3.0 for controls (P < 0.05). Furthermore, multiple DNA adducts were detected with each compound (1 microM, 24 h incubation time), essentially similar to those found recently in vivo in rats treated with 3-NBA or its metabolites. DNA adduct levels ranged from 1.3 to 42.8 and from 2.0 to 39.8 adducts/10(8) nt using the nuclease P1 and butanol enrichment procedures, respectively. DNA binding was highest for N-Ac-N-OH-ABA, followed by 3-NBA, and much lower for 3-ABA

  8. EBV Zta protein induces the expression of interleukin-13, promoting the proliferation of EBV-infected B cells and lymphoblastoid cell lines

    PubMed Central

    Tsai, Shu-Chun; Lin, Sue-Jane; Chen, Po-Wen; Luo, Wen-Yi; Yeh, Te-Huei; Wang, Hsei-Wei; Chen, Chi-Ju

    2009-01-01

    Epstein-Barr virus (EBV) infection can modify the cytokine expression profiles of host cells and determine the fate of those cells. Of note, expression of interleukin-13 (IL-13) may be detected in EBV-associated Hodgkin lymphoma and the natural killer (NK) cells of chronic active EBV-infected patients, but its biologic role and regulatory mechanisms are not understood. Using cytokine antibody arrays, we found that IL-13 production is induced in B cells early during EBV infection. Furthermore, the EBV lytic protein, Zta (also known as the BZLF-1 product), which is a transcriptional activator, was found to induce IL-13 expression following transfection. Mechanistically, induction of IL-13 expression by Zta is mediated directly through its binding to the IL-13 promoter, via a consensus AP-1 binding site. Blockade of IL-13 by antibody neutralization showed that IL-13 is required at an early stage of EBV-induced proliferation and for long-term maintenance of the growth of EBV immortalized lymphoblastoid cell lines (LCLs). Thus, Zta-induced IL-13 production facilitates B-cell proliferation and may contribute to the pathogenesis of EBV-associated lymphoproliferative disorders, such as posttransplantation lymphoproliferative disease (PTLD) and Hodgkin lymphoma. PMID:19417211

  9. Use of lymphoblastoid cells for the estimation of environmental insults to DNA. Comprehensive report of the overall activities of the contract during the past three years. Progress report, August 1, 1978-June 31, 1981

    SciTech Connect

    1981-01-01

    Research progress is reported on a study to detect chronic low-level exposure of individuals to polycyclic aromatic hydrocarbons by analysis of DNA in cells with low turnover rates. The technique used was to measure the level of excision repair activity in lymphoblastoid and lymphoma cell lines. (ACR)

  10. Host cell reactivation of uv- and X-ray-damaged herpes simplex virus by Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines

    SciTech Connect

    Henderson, E.E.; Long, W.K.

    1981-12-01

    The efficacy of using an infected centers assay, employing herpes simplex virus-infected, Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) as components, to study host cell reactivation has been explored. Herpes simplex virus type 1 (HSV-1) was shown through the infected centers assay to have detectable but varying ability to lytically infect LCLs established from chromosomal breakage syndromes or closely related genetic disorders. The rate of HSV inactivation by ultraviolet (uv) irradiation was faster in LCLs established from Cockaynes's syndrome than in normal LCLs, and faster still in LCLs established from xeroderma pigmentosum. These results indicate that Cockayne's syndrome, while having what appears to be quantitatively normal levels of uv-induced DNA repair replication, shows decreased ability to host cell reactivated uv-damaged HSV. In direct contrast, X-irradiated HSV showed identical survival when assayed on normal LCLs or LCLs established from ataxia telangiectasia showing increased sensitivity to X irradiation as measured by colony formation. Through the infected centers assay, it has also been possible to demonstrate low levels of multiplicity reactivation of mutagen-damaged HSV in permanently proliferating LCLs.

  11. The human T-cell leukemia virus type 1 Rex regulatory protein exhibits an impaired functionality in human lymphoblastoid Jurkat T cells.

    PubMed Central

    Hamaia, S; Cassé, H; Gazzolo, L; Duc Dodon, M

    1997-01-01

    The Rex protein of human T-cell leukemia virus type 1 (HTLV-1) intervenes in the posttranscriptional regulation of proviral gene expression. Its binding to the Rex response element (XRE) present in the 3' long terminal repeat ensures the coordinate cytoplasmic accumulation of spliced and unspliced forms of viral messengers. Consequently, synthesis of viral structural and enzymatic proteins is strictly dependent on the Rex posttranscriptional activity. Here we report that synthesis of HTLV-1 envelope glycoproteins by Jurkat T cells could be detected only when they were regulated in a Rex-independent manner. Indeed, Jurkat T cells transfected with a Rex-dependent env expression vector (encompassing both the env and pX open reading frames) do not produce significant levels of envelope glycoproteins despite the production of significant amounts of Rex protein. The analysis of levels and distribution patterns of the unspliced env and of the singly spliced tax/rex transcripts suggests that the failure in envelope glycoprotein synthesis may be ascribed to a deficiency of Rex in mediating the nucleocytoplasmic transport of unspliced env RNAs in these cells. Furthermore, despite the synthesis of regulatory proteins, HTLV-1 structural proteins were not detected in Jurkat T cells transfected with an HTLV-1 infectious provirus. Conversely, and as expected, structural proteins were produced by Jurkat cells transfected by a human immunodeficiency virus type 1 (HIV-1) infectious provirus. This phenotype appeared to be linked to a specific dysfunction of Rex, since the functionally equivalent Rev protein of HIV-1 was shown to be fully efficient in promoting the synthesis of HTLV-1 envelope glycoproteins in Jurkat cells. Therefore, it seems likely that the block to Rex function in these lymphoblastoid T cells is determined by inefficient Rex-XRE interactions. These observations suggest that the acquisition of this Rex-deficient phenotype by in vivo-infected HTLV-1 T cells may

  12. Low Dose Radiation Response Curves, Networks and Pathways in Human Lymphoblastoid Cells Exposed from 1 to 10 cGy of Acute Gamma Radiation

    SciTech Connect

    Wyrobek, A. J.; Manohar, C. F.; Nelson, D. O.; Furtado, M. R.; Bhattacharya, M. S.; Marchetti, F.; Coleman, M.A.

    2011-04-18

    We investigated the low dose dependency of the transcriptional response of human cells to characterize the shape and biological functions associated with the dose response curve and to identify common and conserved functions of low dose expressed genes across cells and tissues. Human lymphoblastoid (HL) cells from two unrelated individuals were exposed to graded doses of radiation spanning the range of 1-10 cGy were analyzed by transcriptome profiling, qPCR and bioinformatics, in comparison to sham irradiated samples. A set of {approx}80 genes showed consistent responses in both cell lines; these genes were associated with homeostasis mechanisms (e.g., membrane signaling, molecule transport), subcellular locations (e.g., Golgi, and endoplasmic reticulum), and involved diverse signal transduction pathways. The majority of radiation-modulated genes had plateau-like responses across 1-10 cGy, some with suggestive evidence that transcription was modulated at doses below 1 cGy. MYC, FOS and TP53 were the major network nodes of the low-dose response in HL cells. Comparison our low dose expression findings in HL cells with those of prior studies in mouse brain after whole body exposure, in human keratinocyte cultures, and in endothelial cells cultures, indicates that certain components of the low dose radiation response are broadly conserved across cell types and tissues, independent of proliferation status.

  13. Analysis of genome-wide RNA-sequencing data suggests age of the CEPH/Utah (CEU) lymphoblastoid cell lines systematically biases gene expression profiles.

    PubMed

    Yuan, Yuan; Tian, Lei; Lu, Dongsheng; Xu, Shuhua

    2015-01-22

    In human, Lymphoblastoid cell lines (LCLs) from the CEPH/CEU (Centre d'Etude du Polymorphisme Humain - Utah) family resource have been extensively used for examining the genetics of gene expression levels. However, we noted that CEU/CEPH cell lines were collected and transformed approximately thirty years ago, much earlier than the other cell lines from the pertaining individuals, which we suspected could potentially affect gene expression, data analysis and results interpretation. In this study, by analyzing RNA sequencing data of CEU and the other three European populations as well as an African population, we systematically examined and evaluated the potential confounding effect of LCL age on gene expression levels and patterns. Our results indicated that gene expression profiles of CEU samples have been biased by the older age of CEU cell lines. Interestingly, most of CEU-specific expressions are associated with functions related to cell proliferation, which are more likely due to older age of cell lines than intrinsic characters of the population. We suggested the results be carefully explained when CEU LCLs are used for transcriptomic data analysis in future studies.

  14. Studying the protein expression in human B lymphoblastoid cells exposed to 1.8-GHz (GSM) radiofrequency radiation (RFR) with protein microarray

    SciTech Connect

    Zhijian, Chen; Xiaoxue, Li; Wei, Zheng; Yezhen, Lu; Jianlin, Lou; Deqiang, Lu; Shijie, Chen; Lifen, Jin; Jiliang, He

    2013-03-29

    Highlights: ► Protein microarray shows the differential expression of 27 proteins induced by RFR. ► RPA32 related to DNA repair is down-regulated in Western blot. ► p73 related to cell genome stability and apoptosis is up-regulated in Western blot. -- Abstract: In the present study, the protein microarray was used to investigate the protein expression in human B-cell lymphoblastoid cells intermittently exposed to 1.8-GHz GSM radiofrequency radiation (RFR) at the specific absorption rate (SAR) of 2.0 W/kg for 24 h. The differential expression of 27 proteins was found, which were related to DNA damage repair, apoptosis, oncogenesis, cell cycle and proliferation (ratio >1.5-fold, P < 0.05). The results validated with Western blot assay indicated that the expression of RPA32 was significantly down-regulated (P < 0.05) while the expression of p73 was significantly up-regulated in RFR exposure group (P < 0.05). Because of the crucial roles of those proteins in DNA repair and cell apoptosis, the results of present investigation may explain the biological effects of RFR on DNA damage/repair and cell apoptosis.

  15. Synergistic Effects of Incubation in Rotating Bioreactors and Cumulative Low Dose 60Co γ-ray Irradiation on Human Immortal Lymphoblastoid Cells

    NASA Astrophysics Data System (ADS)

    Wei, Lijun; Han, Fang; Yue, Lei; Zheng, Hongxia; Yu, Dan; Ma, Xiaohuan; Cheng, Huifang; Li, Yu

    2012-11-01

    The complex space environments can influence cell structure and function. The research results on space biology have shown that the major mutagenic factors in space are microgravity and ionizing radiation. In addition, possible synergistic effects of radiation and microgravity on human cells are not well understood. In this study, human immortal lymphoblastoid cells were established from human peripheral blood lymphocytes and the cells were treated with low dose (0.1, 0.15 and 0.2 Gy) cumulative 60Co γ-irradiation and simulated weightlessness [obtained by culturing cells in the Rotating Cell Culture System (RCCS)]. The commonly used indexes of cell damage such as micronucleus rate, cell cycle and mitotic index were studied. Previous work has proved that Gadd45 (growth arrest and DNA-damage-inducible protein 45) gene increases with a dose-effect relationship, and will possibly be a new biological dosimeter to show irradiation damage. So Gadd45 expression is also detected in this study. The micronucleus rate and the expression of Gadd45α gene increased with irradiation dose and were much higher after incubation in the rotating bioreactor than that in the static irradiation group, while the cell proliferation after incubation in the rotating bioreactor decreased at the same time. These results indicate synergetic effects of simulated weightlessness and low dose irradiation in human cells. The cell damage inflicted by γ-irradiation increased under simulated weightlessness. Our results suggest that during medium- and long-term flight, the human body can be damaged by cumulative low dose radiation, and the damage will even be increased by microgravity in space.

  16. Purification and characterization of 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6.

    PubMed Central

    Yoon, K S; Ishii, M; Igarashi, Y; Kodama, T

    1996-01-01

    2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa). The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H. thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen). NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective. The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80 degrees C, and the time for a 50% loss of activity at 70 degrees C under anaerobic conditions was 22 h. The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8. The apparent Km values for 2-oxoglutarate and coenzyme A at 70 degrees C were 1.42 mM and 80 microM, respectively. PMID:8655524

  17. Efficient and reliable establishment of lymphoblastoid cell lines by Epstein-Barr virus transformation from a limited amount of peripheral blood

    PubMed Central

    Omi, Natsue; Tokuda, Yuichi; Ikeda, Yoko; Ueno, Morio; Mori, Kazuhiko; Sotozono, Chie; Kinoshita, Shigeru; Nakano, Masakazu; Tashiro, Kei

    2017-01-01

    Lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus (EBV) serve as an unlimited resource of human genomic DNA. The protocol that is widely used to establish LCLs involves peripheral blood mononuclear cell isolation by density gradient centrifugation, however, that method requires as much as 5 ml of peripheral blood. In this study, in order to provide a more simple and efficient method for the generation of LCLs, we developed a new protocol using hemolytic reaction to enrich white blood cells for EBV transformation and found that the hemolytic protocol successfully generated LCLs from a small volume (i.e., 0.1 ml) of peripheral blood. To assess the quality of genomic DNA extracted from LCLs established by the hemolytic protocol (LCL-hemolytic), we performed single nucleotide polymorphism (SNP) microarray genotyping using the GeneChip® 100 K Array Set (Affymetrix, Inc.). The concordances of the SNP genotyping resulting from genomic DNA from LCL-hemolytic (99.92%) were found to be as good as the technical replicate (99.90%), and Kappa statistics results confirmed the reliability. The findings of this study reveal that the hemolytic protocol is a simple and reliable method for the generation of LCLs, even from a small volume of peripheral blood. PMID:28272413

  18. Gene expression profiling of lymphoblastoid cell lines from monozygotic twins discordant in severity of autism reveals differential regulation of neurologically relevant genes

    PubMed Central

    Hu, Valerie W; Frank, Bryan C; Heine, Shannon; Lee, Norman H; Quackenbush, John

    2006-01-01

    Background The autism spectrum encompasses a set of complex multigenic developmental disorders that severely impact the development of language, non-verbal communication, and social skills, and are associated with odd, stereotyped, repetitive behavior and restricted interests. To date, diagnosis of these neurologically based disorders relies predominantly upon behavioral observations often prompted by delayed speech or aberrant behavior, and there are no known genes that can serve as definitive biomarkers for the disorders. Results Here we demonstrate, for the first time, that lymphoblastoid cell lines from monozygotic twins discordant with respect to severity of autism and/or language impairment exhibit differential gene expression patterns on DNA microarrays. Furthermore, we show that genes important to the development, structure, and/or function of the nervous system are among the most differentially expressed genes, and that many of these genes map closely in silico to chromosomal regions containing previously reported autism candidate genes or quantitative trait loci. Conclusion Our results provide evidence that novel candidate genes for autism may be differentially expressed in lymphoid cell lines from individuals with autism spectrum disorders. This finding further suggests the possibility of developing a molecular screen for autism based on expressed biomarkers in peripheral blood lymphocytes, an easily accessible tissue. In addition, gene networks are identified that may play a role in the pathophysiology of autism. PMID:16709250

  19. Serological studies of HL-A on continuous lymphoblastoid cell lines (CLC) and the definition of an antigen on CLC determined by a heat-labile antibody*

    PubMed Central

    Dumble, Lynette; Jack, I.; Morris, P. J.

    1974-01-01

    Continuous lymphoblastoid cell lines (CLC) are more reactive with HL-A antisera in a complement-dependent cytotoxic test than are peripheral blood lymphocytes (PBL). This additional reactivity leads to assignment to a given CLC of more than four HL-A antigens, the maximum allowable under the two locus concept of the genetic control of HL-A. However, absorption of antisera by CLC shows that no more than four HL-A antigens exist on any of the CLC used in this laboratory. The additional reactivity of these cells lines can be explained in three ways. Firstly, it may be due to the presence of sublytic amounts of HL-A antibody in operationally monospecific antisera. Secondly, it may be due to cross-reactivity between HL-A antigens. Both these findings can be explained on the basis of the increased quantity of HL-A antigens on CLC compared to PBL. Thirdly, it may be due to the presence of a heat-labile (56° for 30 min) complement-dependent cytotoxic antibody which is present in 90% of normal human sera, and detects an antigen group tentatively labelled `D'. PMID:4466611

  20. Microwave electromagnetic field regulates gene expression in T-lymphoblastoid leukemia CCRF-CEM cell line exposed to 900 MHz.

    PubMed

    Trivino Pardo, Juan Carlos; Grimaldi, Settimio; Taranta, Monia; Naldi, Ilaria; Cinti, Caterina

    2012-03-01

    Electric, magnetic, and electromagnetic fields are ubiquitous in our society, and concerns have been expressed regarding possible adverse effects of these exposures. Research on Extremely Low-Frequency (ELF) magnetic fields has been performed for more than two decades, and the methodology and quality of studies have improved over time. Studies have consistently shown increased risk for childhood leukemia associated with ELF magnetic fields. There are still inadequate data for other outcomes. More recently, focus has shifted toward Radio Frequencies (RF) exposures from mobile telephony. There are no persuasive data suggesting a health risk, but this research field is still immature with regard to the quantity and quality of available data. This technology is constantly changing and there is a need for continued research on this issue. To investigate whether exposure to high-frequency electromagnetic fields (EMF) could induce adverse health effects, we cultured acute T-lymphoblastoid leukemia cells (CCRF-CEM) in the presence of 900 MHz MW-EMF generated by a transverse electromagnetic (TEM) cell at short and long exposure times. We evaluated the effect of high-frequency EMF on gene expression and we identified functional pathways influenced by 900 MHz MW-EMF exposure.

  1. Zinc carnosine protects against hydrogen peroxide-induced DNA damage in WIL2-NS lymphoblastoid cell line independent of poly (ADP-Ribose) polymerase expression.

    PubMed

    Ooi, Theng Choon; Mohammad, Nur Hafiza; Sharif, Razinah

    2014-12-01

    The aim of this study is to investigate the ability of zinc carnosine to protect the human lymphoblastoid (WIL2-NS) cell line from hydrogen peroxide-induced DNA damage. Cells were cultured with medium containing zinc carnosine at the concentrations of 0.4, 4, 16 and 32 μM for 9 days prior to treatment with 30 μM of hydrogen peroxide (30 min). Zinc carnosine at the concentration 16 μM was optimal in protecting cells from hydrogen peroxide-induced cytotoxicity and gave the lowest percentage of apoptotic and necrotic cells. Results showed that zinc carnosine was able to induce glutathione production and protect cells from hydrogen peroxide-induced oxidative stress at all concentration and the highest protection was observed at 32-μM zinc carnosine culture. Cytokinesis-block micronucleus cytome assay showed that cells cultured with 4-32 μM of zinc carnosine showed significant reduction in micronuclei formation, nucleoplasmic bridges and nuclear bud frequencies (p < 0.05), suggesting that these concentrations maybe optimal in protecting cells from hydrogen peroxide-induced DNA damage. However, after being challenged with hydrogen peroxide, no increase in poly(ADP-ribose) polymerase expression was observed. Thus, results from this study demonstrate that zinc carnosines possess antioxidant properties and are able to reduce hydrogen peroxide-induced DNA damage in vitro independent of poly(ADP-ribose) polymerase. Further studies are warranted to understand the mechanism of protection of zinc carnosine against hydrogen peroxide-induced damage.

  2. Exposure to 900 MHz electromagnetic field induces an unbalance between pro-apoptotic and pro-survival signals in T-lymphoblastoid leukemia CCRF-CEM cells.

    PubMed

    Marinelli, F; La Sala, D; Cicciotti, G; Cattini, L; Trimarchi, C; Putti, S; Zamparelli, A; Giuliani, L; Tomassetti, G; Cinti, Caterina

    2004-02-01

    It has been recently established that low-frequency electromagnetic field (EMFs) exposure induces biological changes and could be associated with increased incidence of cancer, while the issue remains unresolved as to whether high-frequency EMFs can have hazardous effect on health. Epidemiological studies on association between childhood cancers, particularly leukemia and brain cancer, and exposure to low- and high-frequency EMF suggested an etiological role of EMFs in inducing adverse health effects. To investigate whether exposure to high-frequency EMFs could affect in vitro cell survival, we cultured acute T-lymphoblastoid leukemia cells (CCRF-CEM) in the presence of unmodulated 900 MHz EMF, generated by a transverse electromagnetic (TEM) cell, at various exposure times. We evaluated the effects of high-frequency EMF on cell growth rate and apoptosis induction, by cell viability (MTT) test, FACS analysis and DNA ladder, and we investigated pro-apoptotic and pro-survival signaling pathways possibly involved as a function of exposure time by Western blot analysis. At short exposure times (2-12 h), unmodulated 900 MHz EMF induced DNA breaks and early activation of both p53-dependent and -independent apoptotic pathways while longer continuous exposure (24-48 h) determined silencing of pro-apoptotic signals and activation of genes involved in both intracellular (Bcl-2) and extracellular (Ras and Akt1) pro-survival signaling. Overall our results indicate that exposure to 900 MHz continuous wave, after inducing an early self-defense response triggered by DNA damage, could confer to the survivor CCRF-CEM cells a further advantage to survive and proliferate.

  3. Instability of the expanded (CTG){sub n} repeats in the myotonin protein kinase gene in cultured lymphoblastoid cell lines from patients with myotonic dystrophy

    SciTech Connect

    Ashizawa, Tetsuo; Patel, B.J.; Monckton, D.G.

    1996-08-15

    The mutation associated with myotonic dystrophy (DM) is the expansion of an unstable trinucleotide repeat, (CTG){sub n}, in the 3{prime}-untranslated region of the myotonin protein kinase gene. Although expanded repeats show both germline and somatic instability, the mechanisms of the instability are poorly understood. To establish a model system in which somatic instability of the DM repeat could be studied in more detail, we established lymphoblastoid cell lines (LBCL) from DM patients. Analysis of the DNA from DM LBCL using Southern blotting showed that the (CTG). repeats were apparently stable up to 29 passages in culture. To study infrequent repeat size mutations that are undetectable due to the size heterogeneity, we established LBCL of single-cell origins by cloning using multiple steps of limiting dilution. After expansion to approximately 10{sup 6} cells (equivalent to approximately 20 cell cycles), the DNAs of these cell lines were analyzed by the small pool PCR technique using primers flanking the (CTG), repeat region. Two types of mutations of the expanded (CTG){sub n} repeat alleles were detected: (1) frequent mutations that show small changes of the (CTG){sub n} repeat size, resulting in alleles in a normal distribution around the progenitor allele, and (2) relatively rare mutations with large changes of the (CTG){sub n} repeat size, with a bias toward contraction. The former may represent the mechanism responsible for the so matic heterogeneity of the (CTG), repeat size observe in blood cells of DM patients. This in vitro experimental system will be useful for further studies on mechanisms involved in the regulation of the somatic stability of the (CTG). repeats in DM. 24 refs., 4 figs.

  4. RNA sequencing of transformed lymphoblastoid cells from siblings discordant for autism spectrum disorders reveals transcriptomic and functional alterations: Evidence for sex-specific effects.

    PubMed

    Tylee, Daniel S; Espinoza, Alfred J; Hess, Jonathan L; Tahir, Muhammad A; McCoy, Sarah Y; Rim, Joshua K; Dhimal, Totadri; Cohen, Ori S; Glatt, Stephen J

    2017-03-01

    Genome-wide expression studies of samples derived from individuals with autism spectrum disorder (ASD) and their unaffected siblings have been widely used to shed light on transcriptomic differences associated with this condition. Females have historically been under-represented in ASD genomic studies. Emerging evidence from studies of structural genetic variants and peripheral biomarkers suggest that sex-differences may exist in the biological correlates of ASD. Relatively few studies have explicitly examined whether sex-differences exist in the transcriptomic signature of ASD. The present study quantified genome-wide expression values by performing RNA sequencing on transformed lymphoblastoid cell lines and identified transcripts differentially expressed between same-sex, proximal-aged sibling pairs. We found that performing separate analyses for each sex improved our ability to detect ASD-related transcriptomic differences; we observed a larger number of dysregulated genes within our smaller set of female samples (n = 12 sibling pairs), as compared with the set of male samples (n = 24 sibling pairs), with small, but statistically significant overlap between the sexes. Permutation-based gene-set analyses and weighted gene co-expression network analyses also supported the idea that the transcriptomic signature of ASD may differ between males and females. We discuss our findings in the context of the relevant literature, underscoring the need for future ASD studies to explicitly account for differences between the sexes. Autism Res 2017, 10: 439-455. © 2016 International Society for Autism Research, Wiley Periodicals, Inc.

  5. Insulin-like Growth Factor 1 Differentially Affects Lithium Sensitivity of Lymphoblastoid Cell Lines from Lithium Responder and Non-responder Bipolar Disorder Patients.

    PubMed

    Milanesi, Elena; Hadar, Adva; Maffioletti, Elisabetta; Werner, Haim; Shomron, Noam; Gennarelli, Massimo; Schulze, Thomas G; Costa, Marta; Del Zompo, Maria; Squassina, Alessio; Gurwitz, David

    2015-07-01

    Bipolar disorder (BD) is a chronic psychiatric illness with an unknown etiology. Lithium is considered the cornerstone in the management of BD, though about 50-60 % of patients do not respond sufficiently to chronic treatment. Insulin-like growth factor 1 (IGF1) has been identified as a candidate gene for BD susceptibility, and its low expression has been suggested as a putative biomarker for lithium unresponsiveness. In this study, we examined the in vitro effects of insulin-like growth factor 1 (IGF-1) on lithium sensitivity in lymphoblastoid cell lines (LCLs) from lithium responder (R) and non-responder (NR) bipolar patients. Moreover, we evaluated levels of microRNA let-7c, a small RNA predicted to target IGF1. We found that exogenous IGF-1 added to serum-free media increased lithium sensitivity selectively in LCLs from NR BD patients. However, no significant differences were observed when comparing let-7c expression in LCLs from R vs. NR BD patients. Our data support a key role for IGF-1 in lithium resistance/response in the treatment of bipolar disorder.

  6. Statin-induced expression change of INSIG1 in lymphoblastoid cell lines correlates with plasma triglyceride statin response in a sex-specific manner

    PubMed Central

    Theusch, Elizabeth; Kim, Kyungpil; Stevens, Kristen; Smith, Joshua D.; Chen, Yii-Der I.; Rotter, Jerome I.; Nickerson, Deborah A.; Medina, Marisa W.

    2016-01-01

    Statins are widely prescribed to lower plasma LDL cholesterol levels. They also modestly reduce plasma triglycerides (TG), an independent cardiovascular disease risk factor, in most people. The mechanism and inter-individual variability of TG statin response is poorly understood. We measured statin-induced gene expression changes in lymphoblastoid cell lines derived from 150 participants of a simvastatin clinical trial and identified 23 genes (FDR=15%) with expression changes correlated to plasma TG response. The correlation of insulin-induced gene 1 (INSIG1) expression changes with TG response (rho=0.32, q=0.11) was driven by men (interaction p=0.0055). rs73161338 was associated with INSIG1 expression changes (p=5.4×10−5) and TG response in two statin clinical trials (p=0.0048), predominantly in men. A combined model including INSIG1 expression level and splicing changes accounted for 29.5% of plasma TG statin response variance in men (p=5.6×10−6). Our results suggest that INSIG1 variation may contribute to statin-induced changes in plasma TG in a sex-specific manner. PMID:26927283

  7. Growth of diploid, Epstein-Barr virus-carrying human lymphoblastoid cell lines heterotransplanted into nude mice under immunologically privileged conditions.

    PubMed

    Giovanella, B; Nilsson, K; Zech, L; Yim, O; Klein, G; Stehlin, J S

    1979-07-15

    Human Epstein-Barr virus-carrying lymphoid cell lines which have been classified on the basis of studies on clonality and morphological, chromosomal and functional parameters as lymphoblastoid cell lines (LCL) of presumed non-neoplastic origin were inoculated intracerebrally into nude mice. All eighteen of them grew, killing the host mice within 7 to 25 days, except for 2 which grew more slowly. At autopsy, the brain of the nudes was found to be invaded by infiltrating lymphomas. Sixteen of these lymphomas, when recultured in vitro, gave rise to cell lines with growth properties and morphology indistinguishable from those of the inoculated LCL. Chromosomal examinations showed that 3/7 cell lines injected, which grew as lymphomas in the brain, were still normal diploid on reexplantation whereas the remaining four had become aneuploid. Four lines derived from intracerebral lymphomas (2 diploid, 1 aneuploid and 1 untested) were inoculated subcutaneously into adult nude mice. None of them grew. When the corresponding four original LCL lines were inoculated subcutaneously into newborn nude mice, they grew rapidly, but failed to do so in newborn normal mice or intracerebrally in adult normal mice. One such line, U-1450, was treated with anti-lymphocyte serum (ALS). Small nodules developed at the site of inoculation. From one nodule a cell line was cultured, 1450 ALSAD. It was morphologically indistinguishable from the line of origin. The lines obtained from nude mice inoculated with polyclonal LCL seem to have a restricted clonal representation, but were not monoclonal, as evidenced by analyses of their pattern of immunoglobulin synthesis.

  8. Molecular characterization of the GM 4672 human lymphoblastoid cell line and analysis of its use as a fusion partner in the generation of human-human hybridoma autoantibodies.

    PubMed

    Rioux, J D; Rauch, J; Zdarsky, E; Newkirk, M M

    1993-07-01

    The GM 4672 lymphoblastoid cell line has been used in cell hybridization experiments with peripheral blood lymphocytes (PBLs) in order to generate human-human hybridomas that secrete immunoglobulins directed against a number of different autoantigens. The GM 4672 cells were fused with PBLs isolated from patients with rheumatoid arthritis or systemic lupus erythematosus, or from normal individuals, and the resulting hybridomas were screened for reactivity to platelets, erythrocytes, DNA, cardiolipin, human IgG-Fc, phosphatidylethanolamine, and for lupus anticoagulant activity. This report analyzes the results from 149 fusion experiments completed over a period of nine years. Fifty to sixty-six percent of the fusion experiments resulted in immunoglobulin-secreting clones, with an average of 15 clones/fusion. The hybridoma antibodies were predominantly of the IgM heavy chain isotype, and 67% expressed kappa light chains. Although most hybridoma antibodies (78%) recognized a single autoantigen, 22% recognized more than one autoantigen and were considered polyreactive. In addition, the light and heavy chain variable regions of the antibody secreted by the GM 4672 cell line were amplified by the polymerase chain reaction technique and sequenced. The GM 4672 light chain was encoded by a VkI gene and used a Jk4 minigene. The GM 4672 heavy chain was derived for the rearrangement of a gene from the VH4 subgroup and used a JH4 minigene. The 8 amino acid long diversity region was generated by the fusion of the DK1 and DLR2 genes. The hybridomas generated in fusion experiments, when examined, did not appear to secrete antibodies using the immunoglobulin variable regions derived from the GM 4672 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Proliferation rates and gene expression profiles in human lymphoblastoid cell lines from patients with depression characterized in response to antidepressant drug therapy

    PubMed Central

    Breitfeld, J; Scholl, C; Steffens, M; Brandenburg, K; Probst-Schendzielorz, K; Efimkina, O; Gurwitz, D; Ising, M; Holsboer, F; Lucae, S; Stingl, J C

    2016-01-01

    The current therapy success of depressive disorders remains in need of improvement due to low response rates and a delay in symptomatic improvement. Reliable functional biomarkers would be necessary to predict the individual treatment outcome. On the basis of the neurotrophic hypothesis of antidepressant's action, effects of antidepressant drugs on proliferation may serve as tentative individual markers for treatment efficacy. We studied individual differences in antidepressant drug effects on cell proliferation and gene expression in lymphoblastoid cell lines (LCLs) derived from patients treated for depression with documented clinical treatment outcome. Cell proliferation was characterized by EdU (5-ethynyl-2'-deoxyuridine) incorporation assays following a 3-week incubation with therapeutic concentrations of fluoxetine. Genome-wide expression profiling was conducted by microarrays, and candidate genes such as betacellulin—a gene involved in neuronal stem cell regeneration—were validated by quantitative real-time PCR. Ex vivo assessment of proliferation revealed large differences in fluoxetine-induced proliferation inhibition between donor LCLs, but no association with clinical response was observed. Genome-wide expression analyses followed by pathway and gene ontology analyses identified genes with different expression before vs after 21-day incubation with fluoxetine. Significant correlations between proliferation and gene expression of WNT2B, FZD7, TCF7L2, SULT4A1 and ABCB1 (all involved in neurogenesis or brain protection) were also found. Basal gene expression of SULT4A1 (P=0.029), and gene expression fold changes of WNT2B by ex vivo fluoxetine (P=0.025) correlated with clinical response and clinical remission, respectively. Thus, we identified potential gene expression biomarkers eventually being useful as baseline predictors or as longitudinal targets in antidepressant therapy. PMID:27845776

  10. Proliferation rates and gene expression profiles in human lymphoblastoid cell lines from patients with depression characterized in response to antidepressant drug therapy.

    PubMed

    Breitfeld, J; Scholl, C; Steffens, M; Brandenburg, K; Probst-Schendzielorz, K; Efimkina, O; Gurwitz, D; Ising, M; Holsboer, F; Lucae, S; Stingl, J C

    2016-11-15

    The current therapy success of depressive disorders remains in need of improvement due to low response rates and a delay in symptomatic improvement. Reliable functional biomarkers would be necessary to predict the individual treatment outcome. On the basis of the neurotrophic hypothesis of antidepressant's action, effects of antidepressant drugs on proliferation may serve as tentative individual markers for treatment efficacy. We studied individual differences in antidepressant drug effects on cell proliferation and gene expression in lymphoblastoid cell lines (LCLs) derived from patients treated for depression with documented clinical treatment outcome. Cell proliferation was characterized by EdU (5-ethynyl-2'-deoxyuridine) incorporation assays following a 3-week incubation with therapeutic concentrations of fluoxetine. Genome-wide expression profiling was conducted by microarrays, and candidate genes such as betacellulin-a gene involved in neuronal stem cell regeneration-were validated by quantitative real-time PCR. Ex vivo assessment of proliferation revealed large differences in fluoxetine-induced proliferation inhibition between donor LCLs, but no association with clinical response was observed. Genome-wide expression analyses followed by pathway and gene ontology analyses identified genes with different expression before vs after 21-day incubation with fluoxetine. Significant correlations between proliferation and gene expression of WNT2B, FZD7, TCF7L2, SULT4A1 and ABCB1 (all involved in neurogenesis or brain protection) were also found. Basal gene expression of SULT4A1 (P=0.029), and gene expression fold changes of WNT2B by ex vivo fluoxetine (P=0.025) correlated with clinical response and clinical remission, respectively. Thus, we identified potential gene expression biomarkers eventually being useful as baseline predictors or as longitudinal targets in antidepressant therapy.

  11. Mutagenicity and clastogenicity of extracts of Helicobacter pylori detected by the Ames test and in the micronucleus test using human lymphoblastoid cells.

    PubMed

    Arimoto-Kobayashi, Sakae; Ohta, Kaori; Yuhara, Yuta; Ayabe, Yuka; Negishi, Tomoe; Okamoto, Keinosuke; Nakajima, Yoshihiro; Ishikawa, Takeshi; Oguma, Keiji; Otsuka, Takanao

    2015-07-01

    Epidemiological studies have demonstrated a close association between infection with Helicobacter pylori (H.pylori) and the development of gastric carcinoma. Chronic H.pylori infection increases the frequency of mutation in gastric epithelial cells. However, the mechanism by which infection of H.pylori leads to mutation in gastric epithelial cells is unclear. We suspected that components in H.pylori may be related to the mutagenic response associated with DNA alkylation, and could be detected with the Ames test using a more sensitive strain for alkylating agents. Our investigation revealed that an extract of H.pylori was mutagenic in the Ames test with Salmonella typhimurium YG7108, which is deficient in the DNA repair of O(6)-methylguanine. The extract of H.pylori may contain methylating or alkylating agents, which might induce O (6)-alkylguanine in DNA. Mutagenicity of the alkylating agents N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N-nitrosoguanidine in the Ames test with S.typhimurium TA1535 was enhanced significantly in the presence of the extract of H.pylori. The tested extracts of H.pylori resulted in a significant induction of micronuclei in human-derived lymphoblastoid cells. Heat instability and dialysis resistance of the extracts of H.pylori suggest that the mutagenic component in the extracts of H.pylori is a heat-unstable large molecule or a heat-labile small molecule strongly attached or adsorbed to a large molecule. Proteins in the extracts of H.pylori were subsequently fractionated using ammonium sulphate precipitation. However, all fractions expressed enhancing effects toward MNU mutagenicity. These results suggest the mutagenic component is a small molecule that is absorbed into proteins in the extract of H.pylori, which resist dialysis. Continuous and chronic exposure of gastric epithelial cells to the alkylative mutagenic component from H.pylori chronically infected in the stomach might be a causal factor in the gastric carcinogenesis

  12. Necrosis is increased in lymphoblastoid cell lines from children with autism compared with their non-autistic siblings under conditions of oxidative and nitrosative stress

    PubMed Central

    Fenech, Michael F.

    2013-01-01

    Autism spectrum disorders are a heterogeneous group of neurodevelopmental conditions characterised by impairments in reciprocal social interaction, communication and stereotyped behaviours. As increased DNA damage events have been observed in a range of other neurological disorders, it was hypothesised that they would be elevated in lymphoblastoid cell lines (LCLs) obtained from children with autism compared with their non-autistic siblings. Six case–sibling pairs of LCLs from children with autistic disorder and their non-autistic siblings were obtained from the Autism Genetic Resource Exchange (AGRE) and cultured in standard RPMI-1640 tissue culture medium. Cells were exposed to medium containing either 0, 25, 50, 100 and 200 µM hydrogen peroxide (an oxidative stressor) or 0, 5, 10, 20 and 40 µM s-nitroprusside (a nitric oxide producer) for 1h. Following exposure, the cells were microscopically scored for DNA damage, cytostasis and cytotoxicity biomarkers as measured using the cytokinesis-block micronucleus cytome assay. Necrosis was significantly increased in cases relative to controls when exposed to oxidative and nitrosative stress (P = 0.001 and 0.01, respectively). Nuclear division index was significantly lower in LCLs from children with autistic disorder than their non-autistic siblings when exposed to hydrogen peroxide (P = 0.016), but there was no difference in apoptosis, micronucleus frequency, nucleoplasmic bridges or nuclear buds. Exposure to s-nitroprusside significantly increased the number of micronuclei in non-autistic siblings compared with cases (P = 0.003); however, other DNA damage biomarkers, apoptosis and nuclear division did not differ significantly between groups. The findings of this study show (i) that LCLs from children with autism are more sensitive to necrosis under conditions of oxidative and nitrosative stress than their non-autistic siblings and (ii) refutes the hypothesis that children with autistic disorder are abnormally

  13. Necrosis is increased in lymphoblastoid cell lines from children with autism compared with their non-autistic siblings under conditions of oxidative and nitrosative stress.

    PubMed

    Main, Penelope A E; Thomas, Philip; Esterman, Adrian; Fenech, Michael F

    2013-07-01

    Autism spectrum disorders are a heterogeneous group of neurodevelopmental conditions characterised by impairments in reciprocal social interaction, communication and stereotyped behaviours. As increased DNA damage events have been observed in a range of other neurological disorders, it was hypothesised that they would be elevated in lymphoblastoid cell lines (LCLs) obtained from children with autism compared with their non-autistic siblings. Six case-sibling pairs of LCLs from children with autistic disorder and their non-autistic siblings were obtained from the Autism Genetic Resource Exchange (AGRE) and cultured in standard RPMI-1640 tissue culture medium. Cells were exposed to medium containing either 0, 25, 50, 100 and 200 µM hydrogen peroxide (an oxidative stressor) or 0, 5, 10, 20 and 40 µM s-nitroprusside (a nitric oxide producer) for 1h. Following exposure, the cells were microscopically scored for DNA damage, cytostasis and cytotoxicity biomarkers as measured using the cytokinesis-block micronucleus cytome assay. Necrosis was significantly increased in cases relative to controls when exposed to oxidative and nitrosative stress (P = 0.001 and 0.01, respectively). Nuclear division index was significantly lower in LCLs from children with autistic disorder than their non-autistic siblings when exposed to hydrogen peroxide (P = 0.016), but there was no difference in apoptosis, micronucleus frequency, nucleoplasmic bridges or nuclear buds. Exposure to s-nitroprusside significantly increased the number of micronuclei in non-autistic siblings compared with cases (P = 0.003); however, other DNA damage biomarkers, apoptosis and nuclear division did not differ significantly between groups. The findings of this study show (i) that LCLs from children with autism are more sensitive to necrosis under conditions of oxidative and nitrosative stress than their non-autistic siblings and (ii) refutes the hypothesis that children with autistic disorder are abnormally

  14. A Study of Alterations in DNA Epigenetic Modifications (5mC and 5hmC) and Gene Expression Influenced by Simulated Microgravity in Human Lymphoblastoid Cells

    PubMed Central

    Wang, Zhiping; Liu, Yunlong; Lossie, Amy C.; Thimmapuram, Jyothi; Irudayaraj, Joseph

    2016-01-01

    Cells alter their gene expression in response to exposure to various environmental changes. Epigenetic mechanisms such as DNA methylation are believed to regulate the alterations in gene expression patterns. In vitro and in vivo studies have documented changes in cellular proliferation, cytoskeletal remodeling, signal transduction, bone mineralization and immune deficiency under the influence of microgravity conditions experienced in space. However microgravity induced changes in the epigenome have not been well characterized. In this study we have used Next-generation Sequencing (NGS) to profile ground-based “simulated” microgravity induced changes on DNA methylation (5-methylcytosine or 5mC), hydroxymethylation (5-hydroxymethylcytosine or 5hmC), and simultaneous gene expression in cultured human lymphoblastoid cells. Our results indicate that simulated microgravity induced alterations in the methylome (~60% of the differentially methylated regions or DMRs are hypomethylated and ~92% of the differentially hydroxymethylated regions or DHMRs are hyperhydroxymethylated). Simulated microgravity also induced differential expression in 370 transcripts that were associated with crucial biological processes such as oxidative stress response, carbohydrate metabolism and regulation of transcription. While we were not able to obtain any global trend correlating the changes of methylation/ hydroxylation with gene expression, we have been able to profile the simulated microgravity induced changes of 5mC over some of the differentially expressed genes that includes five genes undergoing differential methylation over their promoters and twenty five genes undergoing differential methylation over their gene-bodies. To the best of our knowledge, this is the first NGS-based study to profile epigenomic patterns induced by short time exposure of simulated microgravity and we believe that our findings can be a valuable resource for future explorations. PMID:26820575

  15. The use of genome-wide eQTL associations in lymphoblastoid cell lines to identify novel genetic pathways involved in complex traits.

    PubMed

    Min, Josine L; Taylor, Jennifer M; Richards, J Brent; Watts, Tim; Pettersson, Fredrik H; Broxholme, John; Ahmadi, Kourosh R; Surdulescu, Gabriela L; Lowy, Ernesto; Gieger, Christian; Newton-Cheh, Chris; Perola, Markus; Soranzo, Nicole; Surakka, Ida; Lindgren, Cecilia M; Ragoussis, Jiannis; Morris, Andrew P; Cardon, Lon R; Spector, Tim D; Zondervan, Krina T

    2011-01-01

    The integrated analysis of genotypic and expression data for association with complex traits could identify novel genetic pathways involved in complex traits. We profiled 19,573 expression probes in Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) from 299 twins and correlated these with 44 quantitative traits (QTs). For 939 expressed probes correlating with more than one QT, we investigated the presence of eQTL associations in three datasets of 57 CEU HapMap founders and 86 unrelated twins. Genome-wide association analysis of these probes with 2.2 m SNPs revealed 131 potential eQTLs (1,989 eQTL SNPs) overlapping between the HapMap datasets, five of which were in cis (58 eQTL SNPs). We then tested 535 SNPs tagging the eQTL SNPs, for association with the relevant QT in 2,905 twins. We identified nine potential SNP-QT associations (P<0.01) but none significantly replicated in five large consortia of 1,097-16,129 subjects. We also failed to replicate previous reported eQTL associations with body mass index, plasma low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides levels derived from lymphocytes, adipose and liver tissue. Our results and additional power calculations suggest that proponents may have been overoptimistic in the power of LCLs in eQTL approaches to elucidate regulatory genetic effects on complex traits using the small datasets generated to date. Nevertheless, larger tissue-specific expression data sets relevant to specific traits are becoming available, and should enable the adoption of similar integrated analyses in the near future.

  16. Buparvaquone but not cyclosporin A prevents Theileria annulata-infected bovine lymphoblastoid cells from stimulating uninfected lymphocytes.

    PubMed

    Rintelen, M; Schein, E; Ahmed, J S

    1990-06-01

    The influence of Buparvaquone on the morphology, proliferation, and stimulation with T and B cell mitogens of Theileria annulata-infected cells was studied. In addition, the stimulatory capacity of the infected cells before and after treatment with Buparvaquone or cyclosporin A (CsA) was also examined and compared to that of ConA-stimulated bovine peripheral blood cells (PBL). After incubation of the cells for 4 days with Buparvaquone only few schizonts were detectable in the cells. Prolongation of the incubation time to 8, 12, or 14 days eliminated completely the parasites. Despite the elimination of the parasites, the cells were still unable to undergo a proliferative response to Con A or PWM. However, the drug did not interfere with the response of normal PBL to these mitogens. Furthermore, Buparvaquone but not CsA inhibits the generation of mixed lymphocyte reaction (MLR). None of the drugs could prevent ConA-blasts from stimulating autologous PBL. These results suggest that the antigen expressed by the infected cells and recognised by the responder PBL was induced by the schizonts.

  17. Interferon-dependent induction of mRNA for the major histocompatibility antigens in human fibroblasts and lymphoblastoid cells.

    PubMed Central

    Fellous, M; Nir, U; Wallach, D; Merlin, G; Rubinstein, M; Revel, M

    1982-01-01

    In human cells treated with interferons, there is an increase in the amount of HLA-A,B,C and beta 2-microglobulin exposed on the cell surface. We have used a cloned HLA-A,B,C cDNA probe to demonstrate by molecular hybridization that this effect of interferon is preceded by a large increase in the amount of HLA mRNA in the cell. This effect was found in five different human cell lines, with purified leukocyte and fibroblast interferons. The increase in HLA mRNA is comparable in its kinetics and dose-response to the induction of (2'-5') oligo(A) synthetase mRNA by interferons. Therefore, interferons seem to activate at least two cellular genes which have different biochemical functions. Images PMID:6179076

  18. Theileria-mediated constitutive expression of the casein kinase II-alpha subunit in bovine lymphoblastoid cells.

    PubMed

    Shayan, P; Ahmed, J S

    1997-01-01

    Theileria-infected cells are induced to undergo a transformation that is reversible, since their proliferation is inhibited after elimination of the schizonts by the theilericidal drug buparvaquone. The molecular mechanisms of the transformation remain unknown. The experiments described in the present report deal with the role of casein kinase (CK) II, a serine/threonine protein kinase, in the permanent proliferation of the parasitized cells and show that the CK II-alpha subunit is expressed in both T. annulata- and T. parva-infected cells and that its expression is closely related to the presence of the parasites in the host-cell cytoplasm. Thus, elimination of the schizonts by buparvaquone leads to the inhibition of CK II-alpha subunit mRNA expression without affecting the expression of actin. Cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) are inhibited in a dose-dependent manner from under-going DNA synthesis as measured by [3H]-thymidine incorporation and from expressing CK II. Furthermore, a host-cell-specific CK II-alpha antisense inhibits DNA synthesis in a dose-dependent manner. In the present study, 6 microM antisense reduced [3H]-thymidine incorporation by Theileria-infected bovine cells to about 50%. Using a primer derived from T. parva CK II, we detected a parasite-specific CK II mRNA in T. parva-infected cell lines. Interestingly. DRB also inhibited the expression of the parasite-specific CK II. However, to date we have not detected a target sequence for this primer in T. annulata schizonts.

  19. Induction of anti-EBNA-1 protein by 12-O-tetradecanoylphorbol-13-acetate treatment of human lymphoblastoid cells

    SciTech Connect

    Wen, Longthung; Tanaka, Akiko; Nonoyama, Meihan )

    1989-08-01

    Binding of the Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) to BamHI-C DNA was studied by affinity column chromatography followed by immunoblotting with human serum specific for EBNA-1. Two species of EBNA-1 (68 and 70 kilodaltons) were identified in nuclear extracts of the EBV-positive Burkitt's lymphoma cell line Raji and not in nuclear extracts of the EBV-negative Burkitt's lymphoma cell line BJAB. Both EBNA-1s bound specifically to the region required for EBV plasmid DNA maintenance (oriP) located in the BamHI-C fragment. Upon treatment with 12-O-tetradecanoylphorbol-13-acetate, which activates latent EBV genome in Raji cells, the 68-kilodalton EBNA-1 was uncoupled from binding to EBV oriP. Nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated BJAB cells also uncoupled the binding of both EBNA-1s to oriP. DNA-cellulose column chromatography identified two protein species which competed for and uncoupled the binding of EBNA-1 to oriP. The two cellular competitors the authors called anti-EBNA-1 proteins had molecular masses of 60 and 40 kilodaltons, respectively. They were not found in nuclear extracts of BJAB cells not activated by 12-O-tetradecanoylphorbol-13-acetate.

  20. Transfer and expression of three cloned human non-HLA-A,B,C class I major histocompatibility complex genes in mutant lymphoblastoid cells.

    PubMed Central

    Shimizu, Y; Geraghty, D E; Koller, B H; Orr, H T; DeMars, R

    1988-01-01

    The HLA-A, -B, and -C class I human histocompatibility antigens and the genes that encode them have been isolated and characterized. Apparently complete class I non-HLA-A, B, C genes have been identified on HindIII-generated 5.4-kilobase (kb), 6.0-kb, and 6.2-kb DNA fragments derived from lymphoblastoid cell line (LCL) 721. We studied the expressibility of these genes by subcloning them into the nonintegrating pHeBo vector and transferring the chimeric plasmids into mutant LCL 721.221. This mutant was derived from LCL 721 by means of immunoselections following gamma-ray mutagenesis that eliminated expressions of the HLA-A, -B, and -C alpha chains. The HLA-A, B, C-null phenotype of mutant 721.221 made it possible to monitor the expression of class I genes transferred into it by assaying cell surface binding of monoclonal antibodies BBM.1 and W6/32, which recognize beta 2-microglobulin and HLA class I alpha-chain epitopes, respectively. Increased binding of BBM.1 and W6/32 was clearly observed in transferents containing the class I gene of the 6.0-kb DNA fragment but not in transferents containing the class I genes of the 5.4- and 6.2-kb DNA fragments. However, one-dimensional gel electrophoresis of BBM.1 and W6/32 immunoprecipitates made with [35S]methionine-labeled cell lysates showed that transfer of each non-HLA-A, B, C class I gene into 721.221 resulted in the appearance of an alpha chain that coprecipitated with beta 2-microglobulin. The three previously unreported alpha chains differed from each other in size and were smaller than HLA-A, -B, and -C alpha chains. These observations clearly show that these three cloned, nonallelic, non-HLA-A, B, C class I genes encode alpha chains that can be expressed in human cells. Images PMID:3257565

  1. Nitroxide TEMPO: a genotoxic and oxidative stress inducer in cultured cells.

    PubMed

    Guo, Xiaoqing; Mittelstaedt, Roberta A; Guo, Lei; Shaddock, Joseph G; Heflich, Robert H; Bigger, Anita H; Moore, Martha M; Mei, Nan

    2013-08-01

    2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) is a low molecular weight nitroxide and stable free radical. In this study, we investigated the cytotoxicity and genotoxicity of TEMPO in mammalian cells using the mouse lymphoma assay (MLA) and in vitro micronucleus assay. In the absence of metabolic activation (S9), 3mM TEMPO produced significant cytotoxicity and marginal mutagenicity in the MLA; in the presence of S9, treatment of mouse lymphoma cells with 1-2mM TEMPO resulted in dose-dependent decreases of the relative total growth and increases in mutant frequency. Treatment of TK6 human lymphoblastoid cells with 0.9-2.3mM TEMPO increased the frequency of both micronuclei (a marker for clastogenicity) and hypodiploid nuclei (a marker of aneugenicity) in a dose-dependent manner; greater responses were produced in the presence of S9. Within the dose range tested, TEMPO induced reactive oxygen species and decreased glutathione levels in mouse lymphoma cells. In addition, the majority of TEMPO-induced mutants had loss of heterozygosity at the Tk locus, with allele loss of ⩽34Mbp. These results indicate that TEMPO is mutagenic in the MLA and induces micronuclei and hypodiploid nuclei in TK6 cells. Oxidative stress may account for part of the genotoxicity induced by TEMPO in both cell lines.

  2. Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain

    PubMed Central

    Nguyen, AnhThu; Rauch, Tibor A.; Pfeifer, Gerd P.; Hu, Valerie W.

    2010-01-01

    Autism is currently considered a multigene disorder with epigenetic influences. To investigate the contribution of DNA methylation to autism spectrum disorders, we have recently completed large-scale methylation profiling by CpG island microarray analysis of lymphoblastoid cell lines derived from monozygotic twins discordant for diagnosis of autism and their nonautistic siblings. Methylation profiling revealed many candidate genes differentially methylated between discordant MZ twins as well as between both twins and nonautistic siblings. Bioinformatics analysis of the differentially methylated genes demonstrated enrichment for high-level functions including gene transcription, nervous system development, cell death/survival, and other biological processes implicated in autism. The methylation status of 2 of these candidate genes, BCL-2 and retinoic acid-related orphan receptor alpha (RORA), was further confirmed by bisulfite sequencing and methylation-specific PCR, respectively. Immunohistochemical analyses of tissue arrays containing slices of the cerebellum and frontal cortex of autistic and age- and sex-matched control subjects revealed decreased expression of RORA and BCL-2 proteins in the autistic brain. Our data thus confirm the role of epigenetic regulation of gene expression via differential DNA methylation in idiopathic autism, and furthermore link molecular changes in a peripheral cell model with brain pathobiology in autism.—Nguyen, A., Rauch, T. A., Pfeifer, G. P., Hu, V. W. Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain. PMID:20375269

  3. Investigation of post-transcriptional gene regulatory networks associated with autism spectrum disorders by microRNA expression profiling of lymphoblastoid cell lines

    PubMed Central

    2010-01-01

    Background Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by abnormalities in reciprocal social interactions and language development and/or usage, and by restricted interests and repetitive behaviors. Differential gene expression of neurologically relevant genes in lymphoblastoid cell lines from monozygotic twins discordant in diagnosis or severity of autism suggested that epigenetic factors such as DNA methylation or microRNAs (miRNAs) may be involved in ASD. Methods Global miRNA expression profiling using lymphoblasts derived from these autistic twins and unaffected sibling controls was therefore performed using high-throughput miRNA microarray analysis. Selected differentially expressed miRNAs were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis, and the putative target genes of two of the confirmed miRNA were validated by knockdown and overexpression of the respective miRNAs. Results Differentially expressed miRNAs were found to target genes highly involved in neurological functions and disorders in addition to genes involved in gastrointestinal diseases, circadian rhythm signaling, as well as steroid hormone metabolism and receptor signaling. Novel network analyses of the putative target genes that were inversely expressed relative to the relevant miRNA in these same samples further revealed an association with ASD and other co-morbid disorders, including muscle and gastrointestinal diseases, as well as with biological functions implicated in ASD, such as memory and synaptic plasticity. Putative gene targets (ID3 and PLK2) of two RT-PCR-confirmed brain-specific miRNAs (hsa-miR-29b and hsa-miR-219-5p) were validated by miRNA overexpression or knockdown assays, respectively. Comparisons of these mRNA and miRNA expression levels between discordant twins and between case-control sib pairs show an inverse relationship, further suggesting that ID3 and PLK2 are in vivo targets of the

  4. DNA alkylation lesions and their repair in human cells: modification of the comet assay with 3-methyladenine DNA glycosylase (AlkD).

    PubMed

    Hašplová, Katarína; Hudecová, Alexandra; Magdolénová, Zuzana; Bjøras, Magnar; Gálová, Eliška; Miadoková, Eva; Dušinská, Mária

    2012-01-05

    3-methyladenine DNA glycosylase (AlkD) belongs to a new family of DNA glycosylases; it initiates repair of cytotoxic and promutagenic alkylated bases (its main substrates being 3-methyladenine and 7-methylguanine). The modification of the comet assay (single cell gel electrophoresis) using AlkD enzyme thus allows assessment of specific DNA alkylation lesions. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay they appear as DNA strand breaks. The alkylating agent methyl methanesulfonate (MMS) was used to induce alkylation lesions and to optimize conditions for the modified comet assay method with AlkD on human lymphoblastoid (TK6) cells. We also studied cellular and in vitro DNA repair of alkylated bases in DNA in TK6 cells after treatment with MMS. Results from cellular repair indicate that 50% of DNA alkylation is repaired in the first 60 min. The in vitro repair assay shows that while AlkD recognises most alkylation lesions after 60 min, a cell extract from TK6 cells recognises most of the MMS-induced DNA adducts already in the first 15 min of incubation, with maximum detection of lesions after 60 min' incubation. Additionally, we tested the in vitro repair capacity of human lymphocyte extracts from 5 individuals and found them to be able to incise DNA alkylations in the same range as AlkD. The modification of the comet assay with AlkD can be useful for in vitro and in vivo genotoxicity studies to detect alkylation damage and repair and also for human biomonitoring and molecular epidemiology studies.

  5. Endogenous antigen presentation by autoantigen-transfected Epstein-Barr virus-lymphoblastoid cells. I. Generation of human thyroid peroxidase-reactive T cells and their T cell receptor repertoire.

    PubMed Central

    Martin, A; Magnusson, R P; Kendler, D L; Concepcion, E; Ben-Nun, A; Davies, T F

    1993-01-01

    To develop a model for endogenous thyroid autoantigen presentation, we transfected EBV-transformed B lymphoblastoid cell lines (EBV-LCL), established from patients with autoimmune thyroid disease and normal controls, with cDNA for the human thyroid autoantigen thyroid peroxidase (hTPO). hTPO-antigen presentation to patient peripheral blood T cells was demonstrated after stimulation in vitro for 7 d with irradiated hTPO-transfected or untransfected autologous EBV-LCL. Anti-hTPO-reactive T cells were subsequently cloned in the presence of irradiated, autologous hTPO-transfected EBV-LCL and IL-2.10 T cell-cloned lines exhibited specific hTPO-induced proliferation (stimulation indices of 2.1-7.9) towards autologous hTPO-transfected EBV-LCL, and were subjected to human T cell receptor (hTCR) V gene analysis, using the PCR for the detection of V alpha and V beta hTcR gene families. The results indicated a preferential use of hTCR V alpha 1 and/or V alpha 3 in 9 of the 10 lines. In contrast, hTCR V beta gene family use was more variable. These data demonstrate a model for the endogenous presentation of human thyroid peroxidase in the absence of other thyroid specific antigens. The high frequency of antigen-specific T cells obtained from PBMC using this technique will facilitate further studies at both the functional and hTCR V gene level. Images PMID:7682574

  6. Repair of I-SceI induced DSB at a specific site of chromosome in human cells: influence of low-dose, low-dose-rate gamma-rays.

    PubMed

    Yatagai, Fumio; Suzuki, Masao; Ishioka, Noriaki; Ohmori, Hitoshi; Honma, Masamitsu

    2008-11-01

    We investigated the influence of low-dose, low-dose-rate gamma-ray irradiation on DNA double strand break (DSB) repair in human lymphoblastoid TK6 cells. A single DSB was introduced at intron 4 of the TK+ allele (chromosome 17) by transfection with the I-SceI expression vector pCBASce. We assessed for DSB repair due to non-homologous end-joining (NHEJ) by determining the generation of TK-deficient mutants in the TK6 derivative TSCE5 (TK +/-) carrying an I-SceI recognition site. We similarly estimated DSB repair via homologous recombination (HR) at the same site in the derived compound heterozygote (TK-/-) cell line TSCER2 that carries an additional point mutation in exon 5. The NHEJ repair of DSB was barely influenced by pre-irradiation of the cells with 30 mGy gamma-rays at 1.2 mGy h(-1). DSB repair by HR, in contrast, was enhanced by approximately 50% after pre-irradiation of the cells under these conditions. Furthermore, when I-SceI digestion was followed by irradiation at a dose of 8.5 mGy, delivered at a dose rate of only 0.125 mGy h(-1), HR repair efficiency was enhanced by approximately 80%. This experimental approach can be applied to characterize DSB repair in the low-dose region of ionizing radiation.

  7. Comparative mutagenesis of human cells in vivo and in vitro

    SciTech Connect

    Thilly, W.G.

    1992-05-01

    This report discusses measuring methods of point mutations; high density cell cultures for low dose studies; measurement and sequence determination of mutations in DNA; the mutational spectra of styrene oxide and ethlyene oxide in TK-6 cells; mutational spectrum of Cr in human lymphoblast cells; mutational spectra of radon in TK-6 cells; and the mutational spectra of smokeless tobacco. (CBS)

  8. Levels of Epstein-Barr virus DNA in lymphoblastoid cell lines are correlated with frequencies of spontaneous lytic growth but not with levels of expression of EBNA-1, EBNA-2, or latent membrane protein.

    PubMed Central

    Metzenberg, S

    1990-01-01

    The process of Epstein-Barr virus (EBV)-induced transformation of human B lymphocytes results in a cell line that is a mixture of latently and lytically infected cells, with the lytic cells composing roughly 5% to less than 0.0001% of the overall population. A set of nine normal lymphoblastoid cell lines that span a 100- to 200-fold range in average EBV DNA content were studied, and the frequency with which these cells entered a lytic phase of viral growth correlated with their EBV DNA copy number (as a population average). However, neither factor correlated with the levels of expression of transcript for the viral genes EBNA-1, EBNA-2, and latent membrane protein, nor did they correlate with the levels of EBNA-2 protein and latent membrane protein. The rate at which a cell line enters into lytic growth spontaneously is therefore not dependent on the overall steady-state levels of expression of these latent-phase genes. Images PMID:2152830

  9. Induction of genomic instability in TK6 human lymphoblasts exposed to 137Cs gamma radiation: comparison to the induction by exposure to accelerated 56Fe particles

    NASA Technical Reports Server (NTRS)

    Evans, Helen H.; Horng, Min-Fen; Ricanati, Marlene; Diaz-Insua, M.; Jordan, Robert; Schwartz, Jeffrey L.

    2003-01-01

    The induction of genomic instability in TK6 human lymphoblasts by exposure to (137)Cs gamma radiation was investigated by measuring the frequency and characteristics of unstable clones isolated approximately 36 generations after exposure. Clones surviving irradiation and control clones were analyzed for 17 characteristics including chromosomal aberrations, growth defects, alterations in response to a second irradiation, and mutant frequencies at the thymidine kinase and Na(+)/K(+) ATPase loci. Putative unstable clones were defined as those that exhibited a significant alteration in one or more characteristics compared to the controls. The frequency and characteristics of the unstable clones were compared in clones exposed to (137)Cs gamma rays or (56)Fe particles. The majority of the unstable clones isolated after exposure to either gamma rays or (56)Fe particles exhibited chromosomal instability. Alterations in growth characteristics, radiation response and mutant frequencies occurred much less often than cytogenetic alterations in these unstable clones. The frequency and complexity of the unstable clones were greater after exposure to (56)Fe particles than to gamma rays. Unstable clones that survived 36 generations after exposure to gamma rays exhibited increases in the incidence of dicentric chromosomes but not of chromatid breaks, whereas unstable clones that survived 36 generations after exposure to (56)Fe particles exhibited increases in both chromatid and chromosome aberrations.

  10. Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain.

    PubMed

    Nguyen, AnhThu; Rauch, Tibor A; Pfeifer, Gerd P; Hu, Valerie W

    2010-08-01

    Autism is currently considered a multigene disorder with epigenetic influences. To investigate the contribution of DNA methylation to autism spectrum disorders, we have recently completed large-scale methylation profiling by CpG island microarray analysis of lymphoblastoid cell lines derived from monozygotic twins discordant for diagnosis of autism and their nonautistic siblings. Methylation profiling revealed many candidate genes differentially methylated between discordant MZ twins as well as between both twins and nonautistic siblings. Bioinformatics analysis of the differentially methylated genes demonstrated enrichment for high-level functions including gene transcription, nervous system development, cell death/survival, and other biological processes implicated in autism. The methylation status of 2 of these candidate genes, BCL-2 and retinoic acid-related orphan receptor alpha (RORA), was further confirmed by bisulfite sequencing and methylation-specific PCR, respectively. Immunohistochemical analyses of tissue arrays containing slices of the cerebellum and frontal cortex of autistic and age- and sex-matched control subjects revealed decreased expression of RORA and BCL-2 proteins in the autistic brain. Our data thus confirm the role of epigenetic regulation of gene expression via differential DNA methylation in idiopathic autism, and furthermore link molecular changes in a peripheral cell model with brain pathobiology in autism.

  11. Epstein-Barr virus nuclear protein 3C binds to the N-terminal (NTD) and beta trefoil domains (BTD) of RBP/CSL; Only the NTD interaction is essential for lymphoblastoid cell growth

    SciTech Connect

    Calderwood, Michael A.; Lee, Sungwook; Holthaus, Amy M.; Blacklow, Stephen C.; Kieff, Elliott; Johannsen, Eric

    2011-05-25

    Association of EBV nuclear proteins EBNA2, EBNA3A and EBNA3C with RBP/CSL, is essential for lymphoblastoid cell line (LCL) proliferation. Conserved residues in the EBNA3 homology domain, required for RBP/CSL interaction, lack the W{Phi}P motif that mediates EBNA2 and Notch binding to the RBP/CSL beta-trefoil domain (BTD). We map RBP/CSL interacting residues within EBNA3A(aa128-204) and EBNA3C(aa211-233). The EBNA3A results are consistent with an earlier report (aa125-222), but the EBNA3C domain is unexpectedly small and includes a 'WTP' sequence. This EBNA3C WTP motif confers RBP/CSL binding in vitro, in yeast, and in mammalian cells. Further, an EBNA3C WTP {yields} STP(W227S) mutation impaired BTD binding whereas EBNA3 homology domain mutations disrupted RBP/CSL N-terminal domain (NTD) binding. WTP was not essential for EBNA3C repression of EBNA2 in reporter assays or for maintenance of LCL growth. Our results indicate that EBNA3 proteins interact with multiple RBP/CSL domains, but only NTD interactions are required for LCL growth.

  12. DNA damage signaling, impairment of cell cycle progression, and apoptosis triggered by 5-ethynyl-2'-deoxyuridine incorporated into DNA.

    PubMed

    Zhao, Hong; Halicka, H Dorota; Li, Jiangwei; Biela, Ewa; Berniak, Krzysztof; Dobrucki, Jurek; Darzynkiewicz, Zbigniew

    2013-11-01

    The "click chemistry" approach utilizing 5-ethynyl-2'-deoxyuridine (EdU) as a DNA precursor was recently introduced to assess DNA replication and adapted to flow- and imaging-cytometry. In the present study, we observed that EdU, once incorporated into DNA, induces DNA damage signaling (DDS) such as phosphorylation of ATM on Ser1981, of histone H2AX on Ser139, of p53 on Ser15, and of Chk2 on Thr68. It also perturbs progression of cells through the cell cycle and subsequently induces apoptosis. These effects were observed in non-small cell lung adenocarcinoma A549 as well as in B-cell human lymphoblastoid TK6 and WTK1 cells, differing in the status of p53 (wt versus mutated). After 1 h EdU pulse-labeling, the most affected was cells progression through the S phase subsequent to that at which they had incorporated EdU. This indicates that DNA replication using the template containing incorporated EdU is protracted and triggers DDS. Furthermore, progression of cells having DNA pulse-labeled with EdU led to accumulation of cells in G2 , likely by activating G2 checkpoint. Consistent with the latter was activation of p53 and Chk2. Although a correlation was observed in A549 cells between the degree of EdU incorporation and the extent of γH2AX induction, such correlation was weak in TK6 and WTK1 cells. The degree of perturbation of the cell cycle kinetics by the incorporated EdU was different in the wt p53 TK6 cells as compared to their sister WTK1 cell line having mutated p53. The data are thus consistent with the role of p53 in modulating activation of cell cycle checkpoints in response to impaired DNA replication. The confocal microscopy analysis of the 3D images of cells exposed to EdU for 1 h pulse and then grown for 24 or 48 h revealed an increased number of colocalized γH2AX and p53BP1 foci considered to be markers of DNA double-strand breaks and enlarged nuclei.

  13. Cell Type-Dependent Changes in CdSe/ZnS Quantum Dot Uptake and Toxic Endpoints

    PubMed Central

    Soenen, Stefaan J.; Al-Ali, Abdullah; Brown, Andy; Hondow, Nicole; Wills, John; Jenkins, Gareth J. S.; Doak, Shareen H.

    2015-01-01

    Toxicity of nanoparticles (NPs) is often correlated with the physicochemical characteristics of the materials. However, some discrepancies are noted in in-vitro studies on quantum dots (QDs) with similar physicochemical properties. This is partly related to variations in cell type. In this study, we show that epithelial (BEAS-2B), fibroblast (HFF-1), and lymphoblastoid (TK6) cells show different biological responses following exposure to QDs. These cells represented the 3 main portals of NP exposure: bronchial, skin, and circulatory. The uptake and toxicity of negatively and positively charged CdSe:ZnS QDs of the same core size but with different surface chemistries (carboxyl or amine polymer coatings) were investigated in full and reduced serum containing media following 1 and 3 cell cycles. Following thorough physicochemical characterization, cellular uptake, cytotoxicity, and gross chromosomal damage were measured. Cellular damage mechanisms in the form of reactive oxygen species and the expression of inflammatory cytokines IL-8 and TNF-α were assessed. QDs uptake and toxicity significantly varied in the different cell lines. BEAS-2B cells demonstrated the highest level of QDs uptake yet displayed a strong resilience with minimal genotoxicity following exposure to these NPs. In contrast, HFF-1 and TK6 cells were more susceptible to toxicity and genotoxicity, respectively, as a result of exposure to QDs. Thus, this study demonstrates that in addition to nanomaterial physicochemical characterization, a clear understanding of cell type-dependent variation in uptake coupled to the inherently different capacities of the cell types to cope with exposure to these exogenous materials are all required to predict genotoxicity. PMID:25601991

  14. Characterization of a lymphoblastoid line deleted for lambda immunoglobulin genes

    SciTech Connect

    Hough, C.A., White, B.N., Holden, J.A.

    1995-04-01

    While characterizing the cat eye syndrome (CES) supernumerary chromosome for the presence of {lambda} immunoglobulin gene region sequences, a lymphoblastoid cell line from one CES patient was identified in which there was selection of cells deleted from some IGLC and IGLV genes. Two distinct deletions, one on each chromosome 22, were identified, presumably arising from independent somatic recombination events occurring during B-lymphocyte differentiation. The extent of the deleted regions was determined using probes from the various IGLV subgroups and they each covered at least 82 kilobases. The precise definition of the deletions was not possible because of conservation of some restriction sites in the IGLV region. The cell line was used to map putative IGLV genes within the recombinant phage {lambda}V{lambda}135 to the distal part of the IGLV gene region. 35 refs., 4 figs.

  15. Persistent reversal of P-glycoprotein-mediated daunorubicin resistance by tetrandrine in multidrug-resistant human T lymphoblastoid leukemia MOLT-4 cells.

    PubMed

    Liu, Zhen-Li; Hirano, Toshihiko; Tanaka, Sachiko; Onda, Kenji; Oka, Kitaro

    2003-11-01

    Multidrug resistance (MDR) represents a major problem in cancer chemotherapy. P-glycoprotein (P-gp), the drug efflux pump that mediates this resistance, can be inhibited by compounds with a variety of pharmacological functions, thus circumventing the MDR phenotype. The present study was performed to evaluate a unique MDR-reversal feature of a bisbenzylisoquinoline alkaloid tetrandrine (TET) in a P-gp expressing MOLT-4 MDR line (MOLT-4/DNR) established in our laboratory. Cell viability was determined by an MTT assay. P-gp function was characterized by determining the Rh123 accumulation/efflux capacity. P-gp overexpression in resistant MOLT-4/DNR cells was confirmed by flow cytometry analysis after staining with phycoerythrin-conjugated anti-P-gp monoclonal antibody 17F9. Compared to ciclosporin A (CsA), TET exhibited stronger activity to reverse drug resistance to daunorubicin (DNR), vinblastine (VLB) and doxorubicin (DOX) in MOLT-4/DNR cells. TET showed no cytotoxic effects on parental MOLT-4 cells lacking P-gp expression or on the resistant MOLT-4/DNR cells. TET modulated DNR cytotoxicity even after it was washed with the medium for 24 h, while CsA almost completely lost its reversal capability 24 h after washing. TET and CsA similarly increased the accumulation of Rh123 in resistant MOLT-4/DNR cells. However, TET inhibited Rh123 efflux from resistant cells even after washing with the medium, while CsA rapidly lost its ability to inhibit Rh123 efflux after washing. The current study suggests that TET enhances the cytotoxicity of anticancer drugs in the P-gp expressing MDR cell line by modulating P-gp in a different manner to the well-known P-gp inhibitor CsA.

  16. Correction of interleukin-2 receptor function in X-SCID lymphoblastoid cells by retrovirally mediated transfer of the gamma-c gene.

    PubMed

    Taylor, N; Uribe, L; Smith, S; Jahn, T; Kohn, D B; Weinberg, K

    1996-04-15

    X-SCID, the most common form of human SCID, is due to mutations in the common gamma chain gene (gamma-c) that encodes an essential component of the cytokine receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. Activation of the Janus family tyrosine kinases Jak1 and Jak3 is necessary for appropriate signalling through the IL-2 receptor (IL-2R). Neither Jak1 nor Jak3 was phosphorylated after IL-2 stimulation of an Epstein-Barr virus-transformed cell line (LCL) from an X-SCID patient with a gamma-c null mutation. However, we now show that appropriate IL-2R function can be restored in an X-SCID LCL by transduction of a wild-type gamma-c gene. A retroviral vector, G1gamma-cSvNa, was constructed and produced in the PG13 packaging line. Transduced X-SCID LCL expressed the G1gamma-cSvNa transcript. IL-2 stimulation of the transduced cell line resulted in appropriate tyrosine phosphorylation of both Jak1 and Jak3. Thus, retroviral-mediated transduction of normal gamma-c can reconstitute downstream signalling through the IL-2R in X-SCID cell lines, suggesting that gene therapy may be a treatment for this disease.

  17. Induction of heme oxygenase: A general response to oxidant stress in cultured mammalian cells

    SciTech Connect

    Applegate, L.A.; Luscher, P.; Tyrrell, R.M. )

    1991-02-01

    Accumulation of heme oxygenase mRNA is strongly stimulated by treatment of cultured human skin fibroblasts with ultraviolet radiation, hydrogen peroxide, or the sulfhydryl reagent sodium arsenite. Since this will result in a transient reduction in the prooxidant state of cells, the phenomenon may represent an important inducible antioxidant defense mechanism. To examine the generality of the response, we have measured the accumulation of the specific mRNA in a variety of human and mammalian cell types after inducing treatments. Induction by sodium arsenite is observed in all additional human cell types tested. This includes primary epidermal keratinocytes and lung and colon fibroblasts as well as established cell lines such as HeLa, TK6 lymphoblastoid, and transformed fetal keratinocytes. Strong induction of heme oxygenase mRNA is also observed following sodium arsenite treatment of cell lines of rat, hamster, mouse, monkey, and marsupial origin. The agents which lead to induction in cultured human skin fibroblasts fall into two categories: (a) those which are oxidants or can generate active intermediates (ultraviolet A radiation, hydrogen peroxide, menadione, and the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate); (b) agents which are known to interact with or modify cellular glutathione levels (buthionine sulfoximine, sodium arsenite, iodoacetamide, diamide, and cadmium chloride). These observations strongly support the hypothesis that induction of the enzyme is a general response to oxidant stress in mammalian cells and are consistent with the possibility that the cellular redox state plays a key role.

  18. Altered kinetic properties of the branched-chain alpha-keto acid dehydrogenase complex due to mutation of the beta-subunit of the branched-chain alpha-keto acid decarboxylase (E1) component in lymphoblastoid cells derived from patients with maple syrup urine disease.

    PubMed Central

    Indo, Y; Kitano, A; Endo, F; Akaboshi, I; Matsuda, I

    1987-01-01

    Branched-chain alpha-keto acid dehydrogenase (BCKDH) complexes of lymphoblastoid cell lines derived from patients with classical maple syrup urine disease (MSUD) phenotypes were studied in terms of their catalytic functions and analyzed by immunoblotting, using affinity purified anti-bovine BCKDH antibody. Kinetic studies on three cell lines derived from patients with the classical phenotype showed sigmoidal or near sigmoidal kinetics for overall BCKDH activity and a deficiency of the E1 component activity. An immunoblot study revealed a markedly decreased amount of the E1 beta subunit accompanied by weak staining of the E1 alpha subunit. The E2 and E3 component exhibited a cross-reactive peptide. Thus, in at least some patients with MSUD, mutations of the E1 beta subunit might provide an explanation for the altered kinetic properties of the BCKDH complex. Images PMID:3597778

  19. TK{sup {minus}} mutants attributable to localized gene conversion in a human cell line

    SciTech Connect

    Giver, C.R.; Grosovsky, A.J.

    1995-11-01

    The human lymphoblastoid cell line TK6 is heterozygous at the tk gene, carrying an inactivating frameshift near the end of the coding sequence, within exon 7 of the functional allele. Here, we describe our use of sequence analyses at these polymorphic sites to identify 8 x-ray induced mutations, out of 184 examined, which exhibit partial conversion of the tk functional allele to the non-functional sequence. These mutants are characterized by loss of heterozygosity at the exon 4 frameshift polymorphism, and remain heterozgousity at exon 7. No restriction fragment length alterations were observed by Southern blotting, and sequencing of the tk cDNA in these mutants revealed the presence of two full length tk transcripts, both having the sequence of the non-functional allele in exon 4, but representing the two different sequences in exon 7. Therefore, the results cannot be explained by a partial deletion of the functional tk allele. Polymorphic microsatellite markers located both proximally and distally to tk on the q arm of chromosome 17 were found to remain heterozygous, ruling out the possibility of a single homologous exchange event. These mutants may be explained by single strand invasion coupled with mismatch repair of the resultant heteroduplex, or by recombinationally mediated repair of a double strand break or gap. We also present microsatellite mapping data which localizes the human tk gene to a 1cM region between markers D17S802 and D17S937.

  20. Differential gene expression profiles in neurons generated from lymphoblastoid B-cell line-derived iPS cells from monozygotic twin cases with treatment-resistant schizophrenia and discordant responses to clozapine.

    PubMed

    Nakazawa, Takanobu; Kikuchi, Masataka; Ishikawa, Mitsuru; Yamamori, Hidenaga; Nagayasu, Kazuki; Matsumoto, Takuya; Fujimoto, Michiko; Yasuda, Yuka; Fujiwara, Mikiya; Okada, Shota; Matsumura, Kensuke; Kasai, Atsushi; Hayata-Takano, Atsuko; Shintani, Norihito; Numata, Shusuke; Takuma, Kazuhiro; Akamatsu, Wado; Okano, Hideyuki; Nakaya, Akihiro; Hashimoto, Hitoshi; Hashimoto, Ryota

    2017-03-01

    Schizophrenia is a chronic psychiatric disorder with complex genetic and environmental origins. While many antipsychotics have been demonstrated as effective in the treatment of schizophrenia, a substantial number of schizophrenia patients are partially or fully unresponsive to the treatment. Clozapine is the most effective antipsychotic drug for treatment-resistant schizophrenia; however, clozapine has rare but serious side-effects. Furthermore, there is inter-individual variability in the drug response to clozapine treatment. Therefore, the identification of the molecular mechanisms underlying the action of clozapine and drug response predictors is imperative. In the present study, we focused on a pair of monozygotic twin cases with treatment-resistant schizophrenia, in which one twin responded well to clozapine treatment and the other twin did not. Using induced pluripotent stem (iPS) cell-based technology, we generated neurons from iPS cells derived from these patients and subsequently performed RNA-sequencing to compare the transcriptome profiles of the mock or clozapine-treated neurons. Although, these iPS cells similarly differentiated into neurons, several genes encoding homophilic cell adhesion molecules, such as protocadherin genes, showed differential expression patterns between these two patients. These results, which contribute to the current understanding of the molecular mechanisms of clozapine action, establish a new strategy for the use of monozygotic twin studies in schizophrenia research.

  1. Polyphenolic Profile and Targeted Bioactivity of Methanolic Extracts from Mediterranean Ethnomedicinal Plants on Human Cancer Cell Lines.

    PubMed

    Pollio, Antonino; Zarrelli, Armando; Romanucci, Valeria; Di Mauro, Alfredo; Barra, Federica; Pinto, Gabriele; Crescenzi, Elvira; Roscetto, Emanuela; Palumbo, Giuseppe

    2016-03-23

    The methanol extracts of the aerial part of four ethnomedicinal plants of Mediterranean region, two non-seed vascular plants, Equisetum hyemale L. and Phyllitis scolopendrium (L.) Newman, and two Spermatophyta, Juniperus communis L. (J. communis) and Cotinus coggygria Scop. (C. coggygria), were screened against four human cells lines (A549, MCF7, TK6 and U937). Only the extracts of J. communis and C. coggygria showed marked cytotoxic effects, affecting both cell morphology and growth. A dose-dependent effect of these two extracts was also observed on the cell cycle distribution. Incubation of all the cell lines in a medium containing J. communis extract determined a remarkable accumulation of cells in the G2/M phase, whereas the C. coggygria extract induced a significant increase in the percentage of G1 cells. The novelty of our findings stands on the observation that the two extracts, consistently, elicited coherent effects on the cell cycle in four cell lines, independently from their phenotype, as two of them have epithelial origin and grow adherent and two are lymphoblastoid and grow in suspension. Even the expression profiles of several proteins regulating cell cycle progression and cell death were affected by both extracts. LC-MS investigation of methanol extract of C. coggygria led to the identification of twelve flavonoids (compounds 1-11, 19) and eight polyphenols derivatives (12-18, 20), while in J. communis extract, eight flavonoids (21-28), a α-ionone glycoside (29) and a lignin (30) were found. Although many of these compounds have interesting individual biological activities, their natural blends seem to exert specific effects on the proliferation of cell lines either growing adherent or in suspension, suggesting potential use in fighting cancer.

  2. Cell fixation in zinc salt solution is compatible with DNA damage response detection by phospho-specific antibodies.

    PubMed

    Zhao, Hong; Li, Jiangwei; Traganos, Frank; Halicka, H Dorota; Zarebski, Mirosław; Dobrucki, Jurek; Darzynkiewicz, Zbigniew

    2011-06-01

    By virtue of superior preservation of proteins and nucleic acids the zinc salt-based fixatives (ZBF) has been proposed as an alternative to precipitants and cross-linking fixatives in histopathology. It was recently reported that ZBF is compatible with analysis of cell surface immunophenotype and detection of intracellular epitopes by flow cytometry. The aim of this study was to explore whether ZBF is also compatible with the detection of DNA damage response assessed by phospho-specific antibodies (Abs) detecting phosphorylation of the key proteins of that pathway. DNA damage in human pulmonary adenocarcinoma A549 cells was induced by treatment with the DNA topoisomerase I inhibitor camptothecin and phosphorylation of histone H2AX on Ser139 (γH2AX) and of ATM on Ser1981 was detected with phospho-specific Abs; cellular fluorescence was measured by laser scanning cytometry (LSC). The sensitivity and accuracy of detection of H2AX and ATM phosphorylation concurrent with the detection of DNA replication by EdU incorporation and "click chemistry" was found in ZBF fixed cells to be comparable to that of cell fixed in formaldehyde. The accuracy of DNA content measurement as evident from the resolution of DNA content frequency histograms of cells stained with DAPI was somewhat better in ZBF- than in formaldehyde-fixed cells. The pattern of chromatin condensation revealed by the intensity of maximal pixel of DAPI that allows one to identify mitotic and immediately post-mitotic cells by LSC was preserved after ZBF fixation. ZBF fixation was also compatible with the detection of γH2AX foci considered to be the hallmarks of induction of DNA double-strand breaks. Analysis of cells by flow cytometry revealed that ZBF fixation of lymphoblastoid TK6 cells led to about 60 and 33% higher intensity of the side and forward light scatter, respectively, compared to formaldehyde fixed cells.

  3. Rb silencing mediated by the down-regulation of MeCP2 is involved in cell transformation induced by long-term exposure to hydroquinone.

    PubMed

    Liu, Linhua; Ling, Xiaoxuan; Wu, Minhua; Chen, Jialong; Chen, Shaoqiao; Tan, Qiang; Chen, Jiansong; Liu, Jiaxian; Zou, Fei

    2017-02-01

    Hydroquinone (HQ), a metabolite of benzene, is a well-known human carcinogen; however, its molecular mechanisms of action remain unclear. MeCP2 has been traditionally described as a transcriptional repressor, though growing evidence indicates that it also activates gene expression. Here, we investigated whether some epigenetic machinery genes are aberrantly expressed as target tumor suppressor genes in HQ-transformed TK6 lymphoblastoid cells. Our results showed that treatment with 5-Aza-2'-deoxycytidine or trichostatin A enhanced the expression of Rb, resulting in cell arrest in G1-phase, and subsequently, an increase in apoptosis and a decrease in cell growth. Moreover, we hypothesised that Rb was silenced by the down-regulation of MeCP2 in HQ-transformed cells, resulting in the dynamic expression of Rb and epigenetic machinery proteins in HQ-transformed cells at different time points. The expression of Rb and MeCP2 in patients with B-cell non-Hodgkin's lymphoma (B-NHL) showed that positive staining for MeCP2 or Rb was significantly lower in B-NHL tumor tissues, and these changes were significantly and negatively correlated with the grade of B-NHL. The restoration of MeCP2 in HQ-transformed cells enhanced the expression of Rb, promoted cell apoptosis, and inhibited cell growth. The changes in the expression patterns of MeCP2 and Rb were inversely correlated with the degree of DNA methylation. A ChiP assay revealed that MeCP2 proteins were recruited to the Rb promoter with lower 5'-methylcytosine levels. In conclusion, we demonstrated that the down-regulation of MeCP2 silences Rb, a process involved in cell transformation resulting from long-term exposure to HQ. © 2016 Wiley Periodicals, Inc.

  4. Ionizing radiation-induced mutation of human cells with different DNA repair capacities

    NASA Astrophysics Data System (ADS)

    Amundson, S. A.; Chen, D. J.

    We have observed significant differences in the response to ionizing radiation of two closely related human cell lines, and now compare the effects on these lines of both low and intermediate LET radiation. Compared to TK6, WTK1 has an enhanced X-ray survival, and is also more resistant to cell killing by alpha-particles. The hprt locus is more mutable in WTK1 than in TK6 by both X-rays and alpha-particles. WTK1 is also more mutable by alpha-particles than by X-rays at the hprt locus. X-ray-induced mutation at the heterozygous tk locus in WTK1 is about 25 fold higher than in TK6, while alpha-particle-induced mutation is nearly 50 fold higher at this locus. Also, the slowly growing tk- mutants, which comprise the majority of spontaneous and X-ray-inducedtk - mutants of TK6, were not induced significantly by alpha-particles. Previously, we showed that TK6 has a reduced capacity for recombination compared with WTK1, and therefore, these results indicate that recombinational repair may contribute to both cell survival and mutation-induction following exposure to ionizing radiation. Such a mechanism may aid cell survival, but could also result in increased deleterious effects such as the unmasking of recessive mutations in cancer suppresser genes.

  5. Ionizing radiation-induced mutation of human cells with different DNA repair capacities

    SciTech Connect

    Amundson, S.A.; Chen, D.J.

    1994-12-31

    We have observed significant differences in the response to ionizing radiation of two closely related human cell lines, and now compare the effects on these lines of both low and intermediate LET radiation. Compared to TK6, WTK1 has an enhanced X-ray survival, and is also more resistant to cell killing by {alpha}-particles. The hprt locus is more mutable in WTK1 than in TK6 by both X-rays and {alpha}-particles. WTK1 is also more mutable by {alpha}-particles than by X-rays at the hprt locus. X-ray-induced mutation at the heterozygous tk locus in WTK1 is about 25 fold higher than in TK6, while {alpha}-particle-induced mutation is nearly 50 fold higher at this locus. Also, the slowly growing tk- mutants, which comprise the majority of spontaneous and X-ray-induced tk- mutants of TK6, were not induced significantly by {alpha}-particles. Previously, we showed that TK6 has a reduced capacity for recombination compared with WTK1, and therefore, these results indicate that recombinational repair may contribute to both cell survival and mutation-induction following exposure to ionizing radiation. Such a mechanism may aid cell survival, but could also result in increased deleterious effects such as the unmasking of recessive mutations in cancer suppresser genes.

  6. Apoptotic regulation and mutagenesis in human cells exposes to charged particles of importance for spaceflight

    NASA Astrophysics Data System (ADS)

    Kronenberg, A.; Gauny, S.; Hain, J.; Wu, P.; Wiese, C.

    Exposure to ionizing radiation can elicit two modes of cell death - necrosis or apoptosis. In human lymphoid cells, the predominant mechanism of radiation- induced cell death is apoptosis. The most likely exposure of individual human cells to heavy ions (e.g. Fe or Si) during spaceflight will result from single particle traversals. Here we report the fluence-response for apoptosis in human TK6 B- lymp hoblasts and provide evidence that single Fe ion traversals can stimulate an apoptotic response. The apoptotic response to charged particle exposures includes scrambling of the phospholipid bilayer in the cell membrane, activation of caspase signaling cascades and degradation of DNA into oligonucleosomes. We have also explored the importance of apoptotic regulation on the frequency and spectrum of mutations arising after exposure to charged particles. We used isogenic derivatives of TK6 cells stably transfected with pSFFV-neo-bcl-xL (encoding the anti-apoptotic gene BCL-XL and the neomycin resistance gene) or with pSFFV neo (encoding only- the neomycin resistance gene). TK6-bclxL cells were more susceptible to mutations at the TK1 locus than TK6-neo cells following exposure to protons, silicon ions or Fe ions. Molecular analysis demonstrated that most Fe-ion-induced mutations arose by loss of heterozygosity (LOH). In TK6-bclxL cells, more of the LOH occurred via mitotic recombination than in TK6-neo cells where the predominant mode of LOH was via deletion. We are currently mapping the LOH tracts to further define the biological bases for the differential sensitivity to Fe-ion-induced mutagenesis as a function of the genotype of the cell at risk. Supported by NASA grant T-964W to A. Kronenberg

  7. Frozen human cells can record radiation damage accumulated during space flight: mutation induction and radioadaptation.

    PubMed

    Yatagai, Fumio; Honma, Masamitsu; Takahashi, Akihisa; Omori, Katsunori; Suzuki, Hiromi; Shimazu, Toru; Seki, Masaya; Hashizume, Toko; Ukai, Akiko; Sugasawa, Kaoru; Abe, Tomoko; Dohmae, Naoshi; Enomoto, Shuichi; Ohnishi, Takeo; Gordon, Alasdair; Ishioka, Noriaki

    2011-03-01

    To estimate the space-radiation effects separately from other space-environmental effects such as microgravity, frozen human lymphoblastoid TK6 cells were sent to the "Kibo" module of the International Space Station (ISS), preserved under frozen condition during the mission and finally recovered to Earth (after a total of 134 days flight, 72 mSv). Biological assays were performed on the cells recovered to Earth. We observed a tendency of increase (2.3-fold) in thymidine kinase deficient (TK(-)) mutations over the ground control. Loss of heterozygosity (LOH) analysis on the mutants also demonstrated a tendency of increase in proportion of the large deletion (beyond the TK locus) events, 6/41 in the in-flight samples and 1/17 in the ground control. Furthermore, in-flight samples exhibited 48% of the ground-control level in TK(-) mutation frequency upon exposure to a subsequent 2 Gy dose of X-rays, suggesting a tendency of radioadaptation when compared with the ground-control samples. The tendency of radioadaptation was also supported by the post-flight assays on DNA double-strand break repair: a 1.8- and 1.7-fold higher efficiency of in-flight samples compared to ground control via non-homologous end-joining and homologous recombination, respectively. These observations suggest that this system can be used as a biodosimeter, because DNA damage generated by space radiation is considered to be accumulated in the cells preserved frozen during the mission, Furthermore, this system is also suggested to be applicable for evaluating various cellular responses to low-dose space radiation, providing a better understanding of biological space-radiation effects as well as estimation of health influences of future space explores.

  8. Preliminary results of space experiment: Implications for the effects of space radiation and microgravity on survival and mutation induction in human cells

    NASA Astrophysics Data System (ADS)

    Yatagai, F.; Honma, M.; Ukai, A.; Omori, K.; Suzuki, H.; Shimazu, T.; Takahashi, A.; Ohnishi, T.; Dohmae, N.; Ishioka, N.

    2012-02-01

    In view of the concern for the health of astronauts that may one day journey to Mars or the Moon, we investigated the effect that space radiation and microgravity might have on DNA damage and repair. We sent frozen human lymphoblastoid TK6 cells to the International Space Station where they were maintained under frozen conditions during a 134-day mission (14 November 2008 to 28 March 2009) except for an incubation period of 8 days under 1G or μG conditions in a CO2 incubator. The incubation period started after 100 days during which the cells had been exposed to 54 mSv of space radiation. The incubated cells were then refrozen, returned to Earth, and compared to ground control samples for the determination of the influence of microgravity on cell survival and mutation induction. The results for both varied from experiment to experiment, yielding a large SD, but the μG sample results differed significantly from the 1G sample results for each of 2 experiments, with the mean ratio of μG to 1G being 0.55 for the concentration of viable cells and 0.59 for the fraction of thymidine kinase deficient (TK-) mutants. Among the mutants, non-loss of zygosity events (point mutations) were less frequent (31%) after μG incubation than after 1G incubation, which might be explained by the influence of μG on cellular metabolic or physiological function. Additional experiments are needed to clarify the effect of μG interferes on DNA repair.

  9. Radiation Induced Bystander Effects in Human Lymphoblastoid Cells

    DTIC Science & Technology

    2003-12-01

    34 observ6 peut etre caus6 par les interactions cellulaires via les prot~ines s~cr~toires 1ib~r~es par les cellules irradi~es en agissant sur les...l’accident du r~acteur de Chernobyl. Nous avons formulk l’hypoth~se que l’effet "bystander" observ6 pouvait 6tre une consdquence d’interactions cellulaires ...qui seraient indicatifs d’expositions biologiques ou chimiques. 11 est pr~vu que certains de ces marqueurs seront communs aux trois agents stressants

  10. Lymphoblastoid lines and skin fibroblasts from patients with tuberous sclerosis are abnormally sensitive to ionizing radiation and to a radiomimetic chemical

    SciTech Connect

    Scudiero, D.A.; Moshell, A.N.; Scarpinato, R.G.; Meyer, S.A.; Clatterbuck, B.E.; Tarone, R.E.; Robbins, J.H.

    1982-03-01

    Lymphoblastoid lines, derived by transforming peripheral blood lymphocytes with Epstein-Barr virus, and skin fibroblast lines were established from two patients with tuberous sclerosis. The number of viable lymphoblastoid cells was determined by their ability to exclude the vital dye trypan blue after their irradiation with x-rays or 254 nm ultraviolet light. The growth of fibroblasts was determined by their ability to form colonies after treatment with the radiomimetic, DNA-damaging chemical N-methyl-N'-nitro-N-nitrosoguanidine. The tuberous sclerosis lymphoblastoid lines were hypersensitive to x-rays but had normal sensitivity to the ultraviolet radiation. The tuberous sclerosis fibroblast lines were hypersensitive to the N-methyl-N'-nitro-N-nitrosoguanidine. The hypersensitivity of tuberous sclerosis cells to x-rays and to N-methyl-N'-nitro-N-nitrosoguanidine is believed to reflect defective repair of DNA damaged by these agents and may provide the basis for in vitro, including prenatal, diagnostic tests for tuberous sclerosis.

  11. Influence of low-dose and low-dose-rate ionizing radiation on mutation induction in human cells

    NASA Astrophysics Data System (ADS)

    Yatagai, F.; Umebayashi, Y.; Suzuki, M.; Abe, T.; Suzuki, H.; Shimazu, T.; Ishioka, N.; Iwaki, M.; Honma, M.

    This is a review paper to introduce our recent studies on the genetic effects of low-dose and low-dose-rate ionizing radiation (IR). Human lymphoblastoid TK6 cells were exposed to γ-rays at a dose-rate of 1.2 mGy/h (total 30 mGy). The frequency of early mutations (EMs) in the thymidine kinase ( TK) gene locus was determined to be 1.7 × 10 -6, or 1.9-fold higher than the level seen in unirradated controls [Umebayashi, Y., Honma, M., Suzuki, M., Suzuki, H., Shimazu, T., Ishioka, N., Iwaki, M., Yatagai, F., Mutation induction in cultured human cells after low-dose and low-dose-rate γ-ray irradiation: detection by LOH analysis. J. Radiat. Res., 48, 7-11, 2007]. These mutants were then analyzed for loss of heterozygosity (LOH) events. Small interstitial-deletion events were restricted to the TK gene locus and were not observed in EMs in unirradated controls, but they comprised about half of the EMs (8/15) after IR exposure. Because of the low level of exposure to IR, this specific type of event cannot be considered to be the direct result of an IR-induced DNA double strand break (DSB). To better understand the effects of low-level IR exposure, the repair efficiency of site-specific chromosomal DSBs was also examined. The pre γ-irradiation under the same condition did not largely influence the efficiency of DSB repair via end-joining, but enhanced such efficiency via homologous recombination to an about 40% higher level (unpublished data). All these results suggest that DNA repair and mutagenesis can be indirectly influenced by low-dose/dose-rate IR.

  12. Mechanisms of mutagenesis in human cells exposed to 55 MeV protons

    NASA Technical Reports Server (NTRS)

    Gauny, S.; Wiese, C.; Kronenberg, A.

    2001-01-01

    Protons represent the major type of charged particle radiation in spaceflight environments. The purpose of this study was to assess mutations arising in human lymphoid cells exposed to protons. Mutations were quantitated at the thymidine kinase (TK1) locus in cell lines derived from the same donor: TK6 cells (wt TP53) and WTK1 cells (mutant TP53). WTK1 cells were much more susceptible to mutagenesis following proton exposure than TK6 cells. Intragenic deletions were observed among early-arising TK1 mutants in TK6 cells, but not in WTK1 cells where all of the mutants arose by LOH. Deletion was the predominant mode of LOH in TK6 cells, while allelic recombination was the major mode of LOH in WTK1 cells. Deletions were of variable lengths, from <1 cM to 64 cM, while mutations that arose by allelic recombination often extended to the telomere. In summary, proton exposures elicited many types of mutations at an autosomal locus in human cells. Most involved large scale loss of genetic information, either through deletion or by recombination.

  13. THE PRODUCTION OF VESICULAR STOMATITIS VIRUS BY ANTIGEN- OR MITOGEN-STIMULATED LYMPHOCYTES AND CONTINUOUS LYMPHOBLASTOID LINES

    PubMed Central

    Nowakowski, Maja; Feldman, Joseph D.; Kano, Shogo; Bloom, Barry R.

    1973-01-01

    A variety of lymphoid cell populations were examined in terms of their ability to replicate vesicular stomatitis virus (VSV), a lytic, RNA-containing virus maturing at the cell surface. The number of cells capable of producing VSV was estimated in terms of infectious centers by the virus plaque assay (VPA), and morphologically by electron microscopy (EM). The lymphoid cells examined in this study included: (a) lymph node cells from delayed hypersensitive guinea pigs stimulated by specific antigen, (b) mouse spleen cells activated by selective bone marrow-derived (B) cell and thymus derived (T) cell mitogens, and (c) cells of human and murine continuous lymphoblastoid or lymphoma lines. In unstimulated cultures of guinea pig lymph node cells there is a background of approximately 1 in 1,000 cells which produces VSV; in purified protein derivative (PPD)-stimulated cultures the number of cells producing virus was 1.6% in the VPA and 1.9% by EM. These cells were large lymphocytes with some morphological features of transformed lymphocytes but were not typical blast cells. A few macrophages were associated with virus in both stimulated and control cultures. These observations indicate that (a) cells responsive to antigens, as detected by a marker virus, were lymphocytes; (b) cells other than lymphocytes (macrophages) were capable of replicating VSV even without antigenic stimulation; and (c) the correlation of results obtained by VPA and morphologic examination was usually quite good. Of the total number of mouse spleen cells stimulated with concanavalin (Con A), a T cell mitogen, 4.5 (EM)–5.7% (VPA) were associated with VSV. These were characteristic transformed lymphocytes, similar to phytohemagglutinin (PHA)-stimulated human lymphocytes. In contrast Escherichia coli lipopolysaccharide (LPS)-treated mouse spleen cultures contained lower numbers of virus plaque-forming cells. The majority of such cells associated with virus displayed extensive rough endoplasmic

  14. Abnormal segregation of alleles in CEPH pedigree DNAs arising from allele loss in lymphoblastoid DNA

    SciTech Connect

    Royle, N.J.; Armour, J.A.L.; Crosier, M.; Jeffreys, A.J. )

    1993-01-01

    Somatic events that result in the reduction to hemior homozygosity at all loci affected by the event have been identified in lymphoblastoid DNA from mothers of two CEPH families. Using suitably informative probes, the allele deficiencies were detected by the abnormal transmission of alleles from grandparents to grandchildren, with the apparent absence of the alleles from the parent. Undetected somatic deficiencies in family DNAs could result in misscoring of recombination events and consequently introduce errors into linkage analysis. 15 refs., 2 figs.

  15. Reduction of immunoglobulin G secretion in vitro following long term lymphoblastoid interferon (Wellferon) treatment in multiple sclerosis patients.

    PubMed Central

    O'Gorman, M R; Oger, J; Kastrukoff, L F

    1987-01-01

    Pokeweed-mitogen-induced IgG secretion, Con A suppression and T cell surface markers were measured in 30 chronic progressive multiple sclerosis (MS) patients and 21 healthy controls. Mean IgG secretion was higher in the MS patients than in the controls (2392 +/- 270 vs 1499 +/- 243); Con A suppression was lower (4 +/- 5% vs 24 +/- 4%) and the CD4/CD8 ratio was higher (4.1 +/- 0.4 vs 2.9 +/- 0.4). The above assays were used in vitro to monitor the effects of Wellferon (lymphoblastoid interferon) injections on this group of MS patients. Before treatment the INF-group (n = 14) did not differ from the PLA-group (n = 16). After 1 week of daily injections the level of IgG secreted was dramatically reduced in the INF group (629 +/- 96 ng/ml) compared to the PLA-group (1756 +/- 319 ng/ml). There was no change in either Con A suppression or T cell surface markers. IgG secretion remained lower in the INF-group for the 6 month treatment period. Following cessation of the injections and a 6 month washout period, IgG secretion in the INF-group rose and was equivalent to that observed in the PLA-group. A series of lymphocyte subset mixing experiments implicates the B lymphocyte subset as being directly affected by interferon injections in vitro. PMID:2957131

  16. Ciprofloxacin as a potential radio-sensitizer to tumor cells and a radio-protectant for normal cells: differential effects on γ-H2AX formation, p53 phosphorylation, Bcl-2 production, and cell death

    PubMed Central

    Kiang, Juliann G.; Garrison, Bradley R.; Smith, Joan T.; Fukumoto, Risaku

    2014-01-01

    Ionizing radiation increases cell mortality in a dose-dependent manner. Increases in DNA double strand breaks, γ-H2AX, p53 phophorylation, and protein levels of p53 and Bax also occur. We investigated the ability of ciprofloxacin (CIP), a widely prescribed antibiotic, to inhibit DNA damage induced by ionizing radiation. Human tumor TK6, NH32 (p53−/− of TK6) cells, and human normal peripheral blood mononuclear cells (PBMCs) were exposed to 2–8 Gy 60Co-γ-photon radiation. γ-H2AX (an indicator of DNA strand breaks), phosphorylated p53 (responsible for cell-cycle arrest), Bcl-2, apoptotic proteins, and cell death were measured. Ionizing irradiation increased γ-H2AX amounts in TK6 cells (p53+/+) within 1 hr in a radiation dose-dependent manner. CIP pretreatment and post-treatment effectively inhibited the increase in γ-H2AX. CIP pretreatment reduced Bcl-2 production but promoted p53 phosphorylation, caspase-3 activation and cell death. In NH32 cells, CIP failed to significantly inhibit the radiation-induced γ-H2AX increase, suggesting that CIP inhibition involves in p53-dependent mechanisms. In normal healthy human PBMCs, CIP failed to block the radiation-induced γ-H2AX increase but effectively increased Bcl-2 production but blocked the phospho-p53 increase and subsequent cell death. CIP increased Gadd45α, and enhanced p21 protein 24 hr postirradiation. Results suggest that CIP exerts its effect in TK6 cells by promoting p53 phosphorylation and inhibiting Bcl-2 production and in PBMCs by inhibiting p53 phosphorylation and increasing Bcl-2 production. Our data are the first to support the view that CIP may be effective to protect normal tissue cells from radiation injury, while enhancing cancer cell death in radiation therapy. PMID:24802382

  17. (Comparative) mutagenesis of human cells in vivo and in vitro

    SciTech Connect

    Thilly, W.G.

    1989-01-01

    We have combined Fischer and Lerman's denaturing gradient gel electrophoresis, high fidelity DNA amplification, and quantitative mutational measurements in a 104 bp sequence within the third exon of the hprt gene in cultured human TK6 cells in order to observe predominant point mutations. We have now characterized the mutations arising spontaneously or after treatment with ICR-191, MNNG, benzo({alpha})pyrene diol epoxide, ultraviolet light, hyperbaric oxygen, and hydrogen peroxide.

  18. Randomised controlled trial of lymphoblastoid interferon for chronic active hepatitis B.

    PubMed Central

    Anderson, M G; Harrison, T J; Alexander, G; Zuckerman, A J; Murray-Lyon, I M

    1987-01-01

    Thirty male patients (27 homosexual) with biopsy proven chronic active hepatitis B were randomised to receive lymphoblastoid interferon (Wellferon) or no treatment. All patients were HBeAg positive and had continuing viral replication. Patients receiving treatment were given a single daily intramuscular injection of interferon for 28 days at a starting dose of 2.5 MU/m2 increasing to a maximum of 7.5 MU/m2/day. Transient side effects of malaise and influenza like symptoms occurred in all patients and resolved rapidly after treatment. Hepatitis B viral replication was suppressed during interferon treatment in all patients but the effect was limited to the period of therapy. After one year there was no appreciable difference in viral markers between the two groups of patients and this treatment schedule appears less effective than the thrice weekly, three month regimes recently reported from other centres. PMID:3297940

  19. [Comparative] mutagenesis of human cells in vivo and in vitro. Progress report, 1989

    SciTech Connect

    Thilly, W.G.

    1989-12-31

    We have combined Fischer and Lerman`s denaturing gradient gel electrophoresis, high fidelity DNA amplification, and quantitative mutational measurements in a 104 bp sequence within the third exon of the hprt gene in cultured human TK6 cells in order to observe predominant point mutations. We have now characterized the mutations arising spontaneously or after treatment with ICR-191, MNNG, benzo({alpha})pyrene diol epoxide, ultraviolet light, hyperbaric oxygen, and hydrogen peroxide.

  20. Small nucleolar RNA host genes and long non-coding RNA responses in directly irradiated and bystander cells.

    PubMed

    Chaudhry, M Ahmad

    2014-04-01

    The irradiated cells communicate with unirradiated cells and induce changes in them through a phenomenon known as the bystander effect. The nature of the bystander signal and how it impacts unirradiated cells remains to be discovered. Examination of molecular changes could lead to the identification of pathways underlying the bystander effect. Apart from microRNAs, little is known about the regulation of other non-coding RNAs (ncRNA) in irradiated or bystander cells. In this study we monitored the transcriptional changes of several small nucleolar RNAs (snoRNAs) host genes and long non-coding RNAs (lncRNAs) that are known to participate in a variety of cellular functions, in irradiated and bystander cells to gain insight into the molecular pathways affected in these cells. We used human lymphoblasts TK6 cells in a medium exchanged bystander effect model system to examine ncRNA expression alterations. The snoRNA host genes SNHG1 and SNHG4 were upregulated in irradiated TK6 cells but were repressed in bystander cells. The SNHG5 and SNHG11 were downregulated in irradiated and bystander cells and the expression levels of these ncRNA were significantly lower in bystander cells. The lncRNA MALAT1, MATR3, SRA1, and SOX2OT were induced in irradiated TK6 cells and their expression levels were repressed in bystander cells. The lncRNA RMST was induced in both irradiated and bystander cells. Taken together, these results indicate that expression levels of ncRNA are modulated in irradiated and bystander cells and these transcriptional changes could be associated with the bystander effect.

  1. Different mechanisms of radiation-induced loss of heterozygosity in two human lymphoid cell lines from a single donor

    NASA Technical Reports Server (NTRS)

    Wiese, C.; Gauny, S. S.; Liu, W. C.; Cherbonnel-Lasserre, C. L.; Kronenberg, A.

    2001-01-01

    Allelic loss is an important mutational mechanism in human carcinogenesis. Loss of heterozygosity (LOH) at an autosomal locus is one outcome of the repair of DNA double-strand breaks (DSBs) and can occur by deletion or by mitotic recombination. We report that mitotic recombination between homologous chromosomes occurred in human lymphoid cells exposed to densely ionizing radiation. We used cells derived from the same donor that express either normal TP53 (TK6 cells) or homozygous mutant TP53 (WTK1 cells) to assess the influence of TP53 on radiation-induced mutagenesis. Expression of mutant TP53 (Met 237 Ile) was associated with a small increase in mutation frequencies at the hemizygous HPRT (hypoxanthine phosphoribosyl transferase) locus, but the mutation spectra were unaffected at this locus. In contrast, WTK1 cells (mutant TP53) were 30-fold more susceptible than TK6 cells (wild-type TP53) to radiation-induced mutagenesis at the TK1 (thymidine kinase) locus. Gene dosage analysis combined with microsatellite marker analysis showed that the increase in TK1 mutagenesis in WTK1 cells could be attributed, in part, to mitotic recombination. The microsatellite marker analysis over a 64-cM region on chromosome 17q indicated that the recombinational events could initiate at different positions between the TK1 locus and the centromere. Virtually all of the recombinational LOH events extended beyond the TK1 locus to the most telomeric marker. In general, longer LOH tracts were observed in mutants from WTK1 cells than in mutants from TK6 cells. Taken together, the results demonstrate that the incidence of radi-ation-induced mutations is dependent on the genetic background of the cell at risk, on the locus examined, and on the mechanisms for mutation available at the locus of interest.

  2. A comparison of the genotoxicity of benzo[a]pyrene in four cell lines with differing metabolic capacity.

    PubMed

    Shah, Ume-Kulsoom; Seager, Anna L; Fowler, Paul; Doak, Shareen H; Johnson, George E; Scott, Sharon J; Scott, Andrew D; Jenkins, Gareth J S

    2016-09-15

    Benzo[a]pyrene (B[a]P) is a known genotoxin and carcinogen, yet its genotoxic response at low level exposure has not been determined. This study was conducted to examine the interplay of dose and metabolic capacity on genotoxicity of B[a]P. Investigating and better understanding the biological significance of low level chemical exposures will help improve human health risk assessments. The genotoxic and mutagenic effects of B[a]P were investigated using human cell lines (AHH-1, MCL-5, TK6 and HepG2) with differential expression of the CYP450 enzymes CYP1A1, 1B1 and1A2 involved in B[a]P metabolism. MCL-5 and HepG2 cells showed detectable basal expression and activity of CYP1A1, 1B1 and 1A2 than AHH-1 which only show CYP1A1 basal expression and activity. TK6 cells showed negligible expression levels of all three CYP450 enzymes. In vitro micronucleus and HPRT assays were conducted to determine the effect of B[a]P on chromosome damage and point mutation induction. After 24h exposure, linear increases in micronucleus (MN) frequency were observed in all cell lines except TK6. After 4h exposure, only the metabolically competent cell lines MCL-5 and HepG2 showed MN induction (with a threshold concentration at 25.5μM from MCL-5 cells) indicating the importance of exposure time for genotoxicity. The HPRT assay also displayed linear increases in mutant frequency in MCL-5 cells, after 4h and 24h treatments. Mutation spectra analysis of MCL-5 and AHH-1 HPRT mutants revealed frequent B[a]P induced G to T transversion mutations (72% and 44% of induced mutations in MCL-5 and AHH-1 respectively). This study therefore demonstrates a key link between metabolic capability, B[a]P exposure time and genotoxicity.

  3. Distinct Signaling Pathways After Higher or Lower Doses of Radiation in Three Closely Related Human Lymphoblast Cell Lines

    SciTech Connect

    Lu, T.-P.; Lai, L.-C.; Lin, B.-I.; Chen, L.-H.; Hsiao, T.-H.; Liber, Howard L.; Cook, John A.; Mitchell, James B.; Tsai, M.-H.; Chuang, Eric Y.

    2010-01-15

    Purpose: The tumor suppressor p53 plays an essential role in cellular responses to DNA damage caused by ionizing radiation; therefore, this study aims to further explore the role that p53 plays at different doses of radiation. Materials and Methods: The global cellular responses to higher-dose (10 Gy) and lower dose (iso-survival dose, i.e., the respective D0 levels) radiation were analyzed using microarrays in three human lymphoblast cell lines with different p53 status: TK6 (wild-type p53), NH32 (p53-null), and WTK1 (mutant p53). Total RNAs were extracted from cells harvested at 0, 1, 3, 6, 9, and 24 h after higher and lower dose radiation exposures. Template-based clustering, hierarchical clustering, and principle component analysis were applied to examine the transcriptional profiles. Results: Differential expression profiles between 10 Gy and iso-survival radiation in cells with different p53 status were observed. Moreover, distinct gene expression patterns were exhibited among these three cells after 10 Gy radiation treatment, but similar transcriptional responses were observed in TK6 and NH32 cells treated with iso-survival radiation. Conclusions: After 10 Gy radiation exposure, the p53 signaling pathway played an important role in TK6, whereas the NFkB signaling pathway appeared to replace the role of p53 in WTK1. In contrast, after iso-survival radiation treatment, E2F4 seemed to play a dominant role independent of p53 status. This study dissected the impacts of p53, NFkB and E2F4 in response to higher or lower doses of gamma-irradiation.

  4. Gene conversion is strongly induced in human cells by double-strand breaks and is modulated by the expression of BCL-x(L)

    NASA Technical Reports Server (NTRS)

    Wiese, Claudia; Pierce, Andrew J.; Gauny, Stacey S.; Jasin, Maria; Kronenberg, Amy; Chatterjee, A. (Principal Investigator)

    2002-01-01

    Homology-directed repair (HDR) of DNA double-strand breaks (DSBs) contributes to the maintenance of genomic stability in rodent cells, and it has been assumed that HDR is of similar importance in DSB repair in human cells. However, some outcomes of homologous recombination can be deleterious, suggesting that factors exist to regulate HDR. We demonstrated previously that overexpression of BCL-2 or BCL-x(L) enhanced the frequency of X-ray-induced TK1 mutations, including loss of heterozygosity events presumed to arise by mitotic recombination. The present study was designed to test whether HDR is a prominent DSB repair pathway in human cells and to determine whether ectopic expression of BCL-x(L) affects HDR. Using TK6-neo cells, we find that a single DSB in an integrated HDR reporter stimulates gene conversion 40-50-fold, demonstrating efficient DSB repair by gene conversion in human cells. Significantly, DSB-induced gene conversion events are 3-4-fold more frequent in TK6 cells that stably overexpress the antiapoptotic protein BCL-X(L). Thus, HDR plays an important role in maintaining genomic integrity in human cells, and ectopic expression of BCL-x(L) enhances HDR of DSBs. This is the first study to highlight a function for BCL-x(L) in modulating DSB repair in human cells.

  5. Quantification of the 2-deoxyribonolactone and nucleoside 5’-aldehyde products of 2-deoxyribose oxidation in DNA and cells by isotope-dilution gas chromatography mass spectrometry: Differential effects of γ-radiation and Fe2+-EDTA

    PubMed Central

    Chan, Wan; Chen, Bingzi; Wang, Lianrong; Taghizadeh, Koli; Demott, Michael S.; Dedon, Peter C.

    2010-01-01

    % and 24% of total 2-deoxyribose oxidation, respectively, with frequencies of 10 lesions per 106 nt per Gy (G-value, 13 nmol/J) and 2.4 lesions per 106 nt per µM. Studies in TK6 human lymphoblastoid cells, in which the analytical data were corrected for losses sustained during DNA isolation, revealed background levels of 2-deoxyribonolactone and nucleoside 5’-aldehyde of 9.7 and 73 lesions per 106 nt, respectively. γ-Irradiation of the cells caused increases of 0.045 and 0.22 lesions per 106 nt per Gy, respectively, which represents a ~250-fold quenching effect of the cellular environment similar to that observed in previous studies. The proportions of the various 2-deoxyribose oxidation products generated by γ-radiation are similar for purified DNA and cells. These results are consistent with solvent exposure as a major determinant of hydroxyl radical reactivity with 2-deoxyribose in DNA, but the large differences between γ-radiation and Fe2+-EDTA suggest that factors other than hydroxyl radical reactivity govern DNA oxidation chemistry. PMID:20377226

  6. Genomic instability in human lymphoid cells exposed to 1 GeV/amu Fe ions

    NASA Technical Reports Server (NTRS)

    Grosovsky, A.; Bethel, H.; Parks, K.; Ritter, L.; Giver, C.; Gauny, S.; Wiese, C.; Kronenberg, A.

    2001-01-01

    The goal of this study was to assess whether charged particle radiations of importance to spaceflight elicit genomic instability in human TK6 lymphoblasts. The incidence of genomic instability in TK6 cells was assessed 21 days after exposure to 2, 4, or 6 Fe ions (1 GeV/amu, LET= 146 keV/micrometers). Three indices of instability were used: intraclonal karyotypic heterogeneity, mutation rate analysis at the thymidine kinase (TK1) locus, and re-cloning efficiency. Fifteen of sixty clones demonstrated karyotypic heterogeneity. Five clones had multiple indicators of karyotypic change. One clone was markedly hypomutable and polyploid. Six clones were hypomutable, while 21 clones were mutators. Of these, seven were karyotypically unstable. Six clones had low re-cloning efficiencies, one of which was a mutator. All had normal karyotypes. In summary, many clones that survived exposure to a low fluence of Fe ions manifested one or more forms of genomic instability that may hasten the development of neoplasia through deletion or by recombination.

  7. Mutagenesis in human cells with accelerated H and Fe ions

    NASA Technical Reports Server (NTRS)

    Kronenberg, Amy

    1994-01-01

    The overall goals of this research were to determine the risks of mutation induction and the spectra of mutations induced by energetic protons and iron ions at two loci in human lymphoid cells. During the three year grant period the research has focused in three major areas: (1) the acquisition of sufficient statistics for human TK6 cell mutation experiments using Fe ions (400 MeV/amu), Fe ions (600 MeV/amu) and protons (250 MeV/amu); (2) collection of thymidine kinase- deficient (tk) mutants or hypoxanthine phosphoribosyltransferase deficient (hprt) mutants induced by either Fe 400 MeV/amu, Fe 600 MeV/amu, or H 250 MeV/amu for subsequent molecular analysis; and (3) molecular characterization of mutants isolated after exposure to Fe ions (600 MeV/amu). As a result of the shutdown of the BEVALAC heavy ion accelerator in December 1992, efforts were rearranged somewhat in time to complete our dose-response studies and to complete mutant collections in particular for the Fe ion beams prior to the shutdown. These goals have been achieved. A major effort was placed on collection, re-screening, and archiving of 3 different series of mutants for the various particle beam exposures: tk-ng mutants, tk-sg mutants, and hprt-deficient mutants. Where possible, groups of mutants were isolated for several particle fluences. Comparative analysis of mutation spectra has occured with characterization of the mutation spectrum for hprt-deficient mutants obtained after exposure of TK6 cells to Fe ions (600 MeV/amu) and a series of spontaneous mutants.

  8. Standardized cell sources and recommendations for good cell culture practices in genotoxicity testing.

    PubMed

    Lorge, E; Moore, M M; Clements, J; O'Donovan, M; Fellows, M D; Honma, M; Kohara, A; Galloway, S; Armstrong, M J; Thybaud, V; Gollapudi, B; Aardema, M J; Tanir, J Y

    2016-10-01

    Good cell culture practice and characterization of the cell lines used are of critical importance in in vitro genotoxicity testing. The objective of this initiative was to make continuously available stocks of the characterized isolates of the most frequently used mammalian cell lines in genotoxicity testing anywhere in the world ('IVGT' cell lines). This project was organized under the auspices of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing. First, cell isolates were identified that are as close as possible to the isolate described in the initial publications reporting their use in genotoxicity testing. The depositors of these cell lines managed their characterization and their expansion for preparing continuously available stocks of these cells that are stored at the European Collection of Cell Cultures (ECACC, UK) and the Japanese Collection of Research Bioresources (JCRB, Japan). This publication describes how the four 'IVGT' cell lines, i.e. L5178Y TK(+/-) 3.7.2C, TK6, CHO-WBL and CHL/IU, were prepared for deposit at the ECACC and JCRB cell banks. Recommendations for handling these cell lines and monitoring their characteristics are also described. The growth characteristics of these cell lines (growth rates and cell cycles), their identity (karyotypes and genetic status) and ranges of background frequencies of select endpoints are also reported to help in the routine practice of genotoxicity testing using these cell lines.

  9. Towards a more reliable comet assay: optimising agarose concentration, unwinding time and electrophoresis conditions.

    PubMed

    Azqueta, Amaya; Gutzkow, Kristine B; Brunborg, Gunnar; Collins, Andrew R

    2011-09-18

    The comet assay is now the method of choice for measuring most kinds of DNA damage in cells. However, due to the lack of a standardised protocol inter-laboratory comparisons are of limited value. The aim of this paper is to demonstrate how small changes in comet-assay variables may significantly affect the results. We examined the effect of varying agarose concentrations, alkaline unwinding time, electrophoresis time, voltage and current, by use of two cell types, viz. human peripheral blood lymphocytes and the lymphoblastoid cell line TK-6. All these variables have marked effects on assay performance and, therefore, on the determination of DNA damage. Here we identify factors of particular importance.

  10. Correlation of In Vivo Versus In Vitro Benchmark Doses (BMDs) Derived From Micronucleus Test Data: A Proof of Concept Study.

    PubMed

    Soeteman-Hernández, Lya G; Fellows, Mick D; Johnson, George E; Slob, Wout

    2015-12-01

    In this study, we explored the applicability of using in vitro micronucleus (MN) data from human lymphoblastoid TK6 cells to derive in vivo genotoxicity potency information. Nineteen chemicals covering a broad spectrum of genotoxic modes of action were tested in an in vitro MN test using TK6 cells using the same study protocol. Several of these chemicals were considered to need metabolic activation, and these were administered in the presence of S9. The Benchmark dose (BMD) approach was applied using the dose-response modeling program PROAST to estimate the genotoxic potency from the in vitro data. The resulting in vitro BMDs were compared with previously derived BMDs from in vivo MN and carcinogenicity studies. A proportional correlation was observed between the BMDs from the in vitro MN and the BMDs from the in vivo MN assays. Further, a clear correlation was found between the BMDs from in vitro MN and the associated BMDs for malignant tumors. Although these results are based on only 19 compounds, they show that genotoxicity potencies estimated from in vitro tests may result in useful information regarding in vivo genotoxic potency, as well as expected cancer potency. Extension of the number of compounds and further investigation of metabolic activation (S9) and of other toxicokinetic factors would be needed to validate our initial conclusions. However, this initial work suggests that this approach could be used for in vitro to in vivo extrapolations which would support the reduction of animals used in research (3Rs: replacement, reduction, and refinement).

  11. Gender differences in the metabolism of 1,3-butadiene to butadiene diepoxide in Sprague-Dawley rats

    SciTech Connect

    Thornton-Manning, J.R.; Dahl, A.R.; Bechtold, W.E.

    1995-12-01

    1,3-Butadiene (BD), a gaseous compound used in the production of rubber, is a potent carcinogen in mice and a weak carcinogen in rats. The mechanism of BD-induced carcinogenicity is thought to involve genotoxic effects of its reactive epoxide metabolites butadiene monoepoxide (BDO) and butadiene diepoxide (BDO{sub 2}). Studies in our laboratory have shown that levels of the epoxides, particularly BDO{sub 2}, are greater in mice-the more sensitive species-than rats. While both epoxides are genotoxic in a number of assays, BDO{sub 2} is mutagenic in TK6 human lymphoblastoid cells at concentrations approximately 100-fold lower than BDO. Species differences in carcinogenicity of BD have posed a dilemma to investigators deciding which animal model is most appropriate for BD risk assessment.

  12. Lymphotoxin is an autocrine growth factor for Epstein-Barr virus- infected B cell lines

    PubMed Central

    1993-01-01

    Because human lymphotoxin (LT) was originally isolated from a lymphoblastoid cell line, we investigated the role of this molecule in three newly established Epstein-Barr virus (EBV)-infected human B cell lines. These lines were derived from acute lymphoblastic leukemia (Z- 6), myelodysplastic syndrome (Z-43), and acute myelogenous leukemia (Z- 55) patients who had a prior EBV infection. Each lymphoblastoid cell line had a karyotype that was different from that of the original parent leukemic cells, and all expressed B cell, but not T cell or myeloid surface markers. In all three lines, rearranged immunoglobulin heavy chain joining region (JH) bands were found, and the presence of EBV DNA was confirmed by Southern blotting. Z-6, Z-43, and Z-55 cell lines constitutively produced 192, 48, and 78 U/ml LT, respectively, as assessed by a cytotoxicity assay and antibody neutralization. Levels of tumor necrosis factor (TNF) were undetectable. Scatchard analysis revealed that all the cell lines expressed high-affinity TNF/LT receptors with receptor densities of 4197, 1258, and 1209 sites/cell on Z-6, Z-43, and Z-55, respectively. Furthermore, labeled TNF binding could be reversed by both unlabeled TNF, as well as by LT. Studies with p60 and p80 receptor-specific antibodies revealed that the three lines expressed primarily the p80 form of the TNF receptor. When studied in a clonogenic assay, exogenous LT stimulated proliferation of all three cell lines in a dose-dependent fashion at concentrations ranging from 25 to 500 U/ml. Similar results were obtained with [3H]TdR incorporation. Monoclonal anti-LT neutralizing antibodies at concentrations of 25-500 U/ml inhibited cellular multiplication in a dose-dependent manner. It is interesting that in spite of a common receptor, TNF (1,000 U/ml) had no direct effect on Z-55 cell growth, whereas it partially reversed the stimulatory effect of exogenous LT. In addition, TNF inhibited Z-6 and Z-43 cell proliferation, and its

  13. Diverse delayed effects in human lymphoblastoid cells surviving exposure to high-LET (56)Fe particles or low-LET (137)Cs gamma radiation

    NASA Technical Reports Server (NTRS)

    Evans, H. H.; Horng, M. F.; Ricanati, M.; Diaz-Insua, M.; Jordan, R.; Schwartz, J. L.

    2001-01-01

    To obtain information on the origin of radiation-induced genomic instability, we characterized a total of 166 clones that survived exposure to (56)Fe particles or (137)Cs gamma radiation, isolated approximately 36 generations after exposure, along with their respective control clones. Cytogenetic aberrations, growth alterations, responses to a second irradiation, and mutant frequencies at the Na(+)/K(+) ATPase and thymidine kinase loci were determined. A greater percentage of clones that survived exposure to (56)Fe particles exhibited instability (defined as clones showing one or more outlying characteristics) than in the case of those that survived gamma irradiation. The phenotypes of the unstable clones that survived exposure to (56)Fe particles were also qualitatively different from those of the clones that survived gamma irradiation. A greater percentage (20%) of the unstable clones that survived gamma irradiation than those that survived exposure to (56)Fe particles (4%) showed an altered response to the second irradiation, while an increase in the percentage of clones that had an outlying frequency of ouabain-resistant and thymidine kinase mutants was more evident in the clones exposed to (56)Fe particles than in those exposed to gamma rays. Growth alterations and increases in dicentric chromosomes were found only in clones with more than one alteration. These results underscore the complex nature of genomic instability and the likelihood that radiation-induced genomic instability arises from different original events.

  14. TP53-dependent chromosome instability is associated with transient reductions in telomere length in immortal telomerase-positive cell lines

    NASA Technical Reports Server (NTRS)

    Schwartz, J. L.; Jordan, R.; Liber, H.; Murnane, J. P.; Evans, H. H.

    2001-01-01

    Telomere shortening in telomerase-negative somatic cells leads to the activation of the TP53 protein and the elimination of potentially unstable cells. We examined the effect of TP53 gene expression on both telomere metabolism and chromosome stability in immortal, telomerase-positive cell lines. Telomere length, telomerase activity, and chromosome instability were measured in multiple clones isolated from three related human B-lymphoblast cell lines that vary in TP53 expression; TK6 cells express wild-type TP53, WTK1 cells overexpress a mutant form of TP53, and NH32 cells express no TP53 protein. Clonal variations in both telomere length and chromosome stability were observed, and shorter telomeres were associated with higher levels of chromosome instability. The shortest telomeres were found in WTK1- and NH32-derived cells, and these cells had 5- to 10-fold higher levels of chromosome instability. The primary marker of instability was the presence of dicentric chromosomes. Aneuploidy and other stable chromosome alterations were also found in clones showing high levels of dicentrics. Polyploidy was found only in WTK1-derived cells. Both telomere length and chromosome instability fluctuated in the different cell populations with time in culture, presumably as unstable cells and cells with short telomeres were eliminated from the growing population. Our results suggest that transient reductions in telomere lengths may be common in immortal cell lines and that these alterations in telomere metabolism can have a profound effect on chromosome stability. Copyright 2000 Wiley-Liss, Inc.

  15. Parkinson's disease and Alzheimer's disease: hypersensitivity to X rays in cultured cell lines.

    PubMed Central

    Robbins, J H; Otsuka, F; Tarone, R E; Polinsky, R J; Brumback, R A; Nee, L E

    1985-01-01

    Fibroblast and/or lymphoblastoid lines from patients with several inherited primary neuronal degenerations are hypersensitive to DNA-damaging agents. Therefore, lymphoblastoid lines were irradiated from patients with sporadic Parkinson's disease (PD), Alzheimer's disease, and amyotrophic lateral sclerosis. The mean survival values of the eight Parkinson's disease and of the six Alzheimer's disease lines, but not of the five amyotrophic lateral sclerosis lines, were less than that of the 28 normal lines. Our results with Parkinson's disease and Alzheimer's disease cells can be explained by a genetic defect arising as a somatic mutation during embryogenesis, causing defective repair of the X-ray type of DNA damage. Such a DNA repair defect could cause an abnormal accumulation of spontaneously occurring DNA damage in Parkinson's disease and Alzheimer's disease neurons in vivo, resulting in their premature death. PMID:3876409

  16. Ultraviolet mutagenesis in a plasmid vector replicated in lymphoid cells from patient with the melanoma-prone disorder dysplastic nevus syndrome

    SciTech Connect

    Seetharam, S.; Waters, H.L.; Seidman, M.M.; Kraemer, K.H. )

    1989-11-01

    The hereditary dysplastic nevus syndrome (DNS) is an autosomal dominant disorder in which affected individuals have increased numbers of dysplastic (premalignant) nevi and a greater than 100-fold increased risk of developing cutaneous melanoma. Epstein-Barr virus-transformed lymphoblastoid cell lines from patients with hereditary DNS have been shown to be hypermutable to UV radiation. To examine the mechanism involved in this UV hypermutability, we used a shuttle vector plasmid, pZ189, which carries a 160-base pair marker gene, supF, and can replicate in human cells. pZ189 was treated with UV radiation and transfected into DNS6BE, a lymphoblastoid cell line from a patient with hereditary DNS. Plasmid survival after UV was similar with the DNS6BE line and with a lymphoblastoid cell line from a normal donor. Plasmid mutation frequency was greater with the DNS line in accord with the DNS cellular hypermutability. Base sequence analysis was performed on 69 mutated plasmids recovered from the DNS line. There were significantly more plasmids with single base substitution mutations (P less than 0.01) in comparison to UV-treated plasmids passed through normal fibroblasts. pZ189 hypermutability and an increased frequency of single base substitutions was previously found with a cell line from a melanoma-prone xeroderma pigmentosum patient. These differences may be related to the increased melanoma susceptibility in both DNS and xeroderma pigmentosum.

  17. Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells

    NASA Technical Reports Server (NTRS)

    Plymale, D. R.; Tang, D. S.; Comardelle, A. M.; Fermin, C. D.; Lewis, D. E.; Garry, R. F.

    1999-01-01

    BACKGROUND: Data currently available on HIV-1-induced cytopathology is unclear regarding the mechanism of cell killing. OBJECTIVE: To clarify the extent to which apoptosis or necrosis is involved in HIV-1-induced cell death in view of conflicting existing data. METHODS: T lymphoblastoid cells or peripheral blood mononuclear cells were infected by various strains of HIV-1 and the numbers of apoptotic or necrotic cells were quantified at various times after infection using video-image analysis techniques; the results were compared with the amount of fragmented DNA using a quantitative method. Measurement of mitochondrial transmembrane potential (deltapsi(m)) and intracellular calcium concentrations [Ca2+]i was performed with fluorescent probes and fluorescence concentration analysis (FCA). RESULTS: Although lymphoblastoid and monocytoid cells acutely infected by HIV-1 had increased levels of fragmented DNA, a marker of apoptotic cell death, few (<12%) had condensed chromatin and fragmented nuclei, the morphological features of apoptosis. The predominant alterations in acutely infected cells were distended endoplasmic reticulum and abnormal mitochondria; these ultrastructural changes are consistent with necrosis, although some infected cells simultaneously displayed features of both necrosis and apoptosis. Viability of cells persistently infected by HIV-1 was only minimally reduced from that of uninfected cells. This reduction was accounted for by an increased propensity of the persistently infected cells to die by apoptosis. Alterations in [Ca2+]i and deltapsi(m) occurred in both acutely and persistently infected cells. CONCLUSION: Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells.

  18. Videomicrofluorometry on living cells and discriminant factorial analysis to study cell cycle distributions.

    PubMed

    Savatier, J; Gbankoto, A; Vigo, J; Salmon, J M

    2004-01-01

    After a rapid overview of the approaches used to study cell cycle, a fluorescent digital imaging microscopy method is proposed. This method is improved by a factorial analysis relying on the evaluation of several parameters recorded on each living cell. Single lympho-blastoid living cells are labeled with three fluorescent markers: Hoechst 33342 for nuclear DNA, Rhodamine 123 for mitochondria and Nile Red for plasma membrane. For each cell, morphological and functional information parameters are obtained. A typological analysis is used to separate control cells into four groups: G0-G1, S, G2+M and polyploid cells Gn. These control cells define a learning population used to analyze untreated and adriamycine treated cells as supplementary individuals in a discriminant factorial analysis. Such an approach allows to accurately evidence the change of the values of some cellular parameters.

  19. Up-regulation of the embryonic self-renewal network through reversible polyploidy in irradiated p53-mutant tumour cells

    SciTech Connect

    Salmina, Kristine; Jankevics, Eriks; Huna, Anda; Perminov, Dmitry; Radovica, Ilze; Klymenko, Tetyana; Ivanov, Andrey; Jascenko, Elina; Scherthan, Harry; Cragg, Mark; Erenpreisa, Jekaterina

    2010-08-01

    We have previously documented that transient polyploidy is a potential cell survival strategy underlying the clonogenic re-growth of tumour cells after genotoxic treatment. In an attempt to better define this mechanism, we recently documented the key role of meiotic genes in regulating the DNA repair and return of the endopolyploid tumour cells (ETC) to diploidy through reduction divisions after irradiation. Here, we studied the role of the pluripotency and self-renewal stem cell genes NANOG, OCT4 and SOX2 in this polyploidy-dependent survival mechanism. In irradiation-resistant p53-mutated lymphoma cell-lines (Namalwa and WI-L2-NS) but not sensitive p53 wild-type counterparts (TK6), low background expression of OCT4 and NANOG was up-regulated by ionising radiation with protein accumulation evident in ETC as detected by OCT4/DNA flow cytometry and immunofluorescence (IF). IF analysis also showed that the ETC generate PML bodies that appear to concentrate OCT4, NANOG and SOX2 proteins, which extend into complex nuclear networks. These polyploid tumour cells resist apoptosis, overcome cellular senescence and undergo bi- and multi-polar divisions transmitting the up-regulated OCT4, NANOG and SOX2 self-renewal cassette to their descendents. Altogether, our observations indicate that irradiation-induced ETC up-regulate key components of germ-line cells, which potentially facilitate survival and propagation of the tumour cell population.

  20. Complement-dependent antibody cytotoxicity test of chicken antibody with duck complement used against cells of a Marek's disease lymphoma-derived cell line (MSB-1).

    PubMed

    Sugimoto, C; Kodama, H; Mikami, T

    1979-01-01

    Complement-dependent antibody cytotoxicity (CDAC) against cells of a lymphoblastoid cell line (MSB-1) derived from Marek's disease lymphoma was investigated in a chicken antibody system using complements from several animal species. Cytotoxicity was seldom observed when rabbit, guinea pig, or chicken complements were used in the presence of hyperimmune chicken serum against MSB-1. With duck complement, however, cytotoxicity was always observed. Therefore, duck complement appears to be suitable for assay of hyperimmune chicken serum against MSB-1 cells by the CDAC test.

  1. High-Throughput Screening Platform for Engineered Nanoparticle-Mediated Genotoxicity Using CometChip Technology

    PubMed Central

    2015-01-01

    The likelihood of intentional and unintentional engineered nanoparticle (ENP) exposure has dramatically increased due to the use of nanoenabled products. Indeed, ENPs have been incorporated in many useful products and have enhanced our way of life. However, there are many unanswered questions about the consequences of nanoparticle exposures, in particular, with regard to their potential to damage the genome and thus potentially promote cancer. In this study, we present a high-throughput screening assay based upon the recently developed CometChip technology, which enables evaluation of single-stranded DNA breaks, abasic sites, and alkali-sensitive sites in cells exposed to ENPs. The strategic microfabricated, 96-well design and automated processing improves efficiency, reduces processing time, and suppresses user bias in comparison to the standard comet assay. We evaluated the versatility of this assay by screening five industrially relevant ENP exposures (SiO2, ZnO, Fe2O3, Ag, and CeO2) on both suspension human lymphoblastoid (TK6) and adherent Chinese hamster ovary (H9T3) cell lines. MTT and CyQuant NF assays were employed to assess cellular viability and proliferation after ENP exposure. Exposure to ENPs at a dose range of 5, 10, and 20 μg/mL induced dose-dependent increases in DNA damage and cytotoxicity. Genotoxicity profiles of ZnO > Ag > Fe2O3 > CeO2 > SiO2 in TK6 cells at 4 h and Ag > Fe2O3 > ZnO > CeO2 > SiO2 in H9T3 cells at 24 h were observed. The presented CometChip platform enabled efficient and reliable measurement of ENP-mediated DNA damage, therefore demonstrating the efficacy of this powerful tool in nanogenotoxicity studies. PMID:24617523

  2. Structural units important for activity of a novel-type phosphoserine phosphatase from Hydrogenobacter thermophilus TK-6 revealed by crystal structure analysis.

    PubMed

    Chiba, Yoko; Horita, Shoichiro; Ohtsuka, Jun; Arai, Hiroyuki; Nagata, Koji; Igarashi, Yasuo; Tanokura, Masaru; Ishii, Masaharu

    2013-04-19

    Novel-type serine-synthesizing enzymes, termed metal-independent phosphoserine phosphatases (iPSPs), were recently identified and characterized from Hydrogenobacter thermophilus, a chemolithoautotrophic bacterium belonging to the order Aquificales. iPSPs are cofactor-dependent phosphoglycerate mutase (dPGM)-like phosphatases that have significant amino acid sequence similarity to dPGMs but lack phosphoglycerate mutase activity. Genes coding dPGM-like phosphatases have been identified in a broad range of organisms; however, predicting the function of the corresponding proteins based on sequence information alone is difficult due to their diverse substrate preferences. Here, we determined the crystal structure of iPSP1 from H. thermophilus in the apo-form and in complex with its substrate L-phosphoserine to find structural units important for its phosphatase activity toward L-phosphoserine. Structural and biochemical characterization of iPSP1 revealed that the side chains of His(85) and C-terminal region characteristic of iPSP1 are responsible for the PSP activity. The importance of these structural units for PSP activity was confirmed by high PSP activity observed in two novel dPGM-like proteins from Cyanobacteria and Chloroflexus in which the two structural units were conserved. We anticipate that our present findings will facilitate understanding of the serine biosynthesis pathways of organisms that lack gene(s) encoding conventional PSPs, as the structural information revealed here will help to identify iPSP from sequence databases.

  3. An application of LOH analysis for detecting the genetic influences of space environmental radiation

    NASA Astrophysics Data System (ADS)

    Yatagai, F.; Umebayashi, Y.; Honma, M.; Abe, T.; Suzuki, H.; Shimazu, T.; Ishioka, N.; Iwaki, M.

    To detect the genetic influence of space environmental radiation at the chromosome level we proposed an application of loss of heterozygosity LOH analysis system for the mutations induced in human lymphoblastoid TK6 cells Surprisingly we succeeded the mutation detection in the frozen dells which were exposed to a low-dose 10 cGy of carbon-ion beam irradiation Mutation assays were performed within a few days or after about one month preservation at --80 r C following irradiation The results showed an increase in mutation frequency at the thymidine kinase TK gene locus 1 6-fold 2 5 X 10 -6 to 3 9 X 10 -6 and 2 1-fold 2 5 X 10 -6 to 5 3 X 10 -6 respectively Although the relative distributions of mutation classes were not changed by the radiation exposure in either assay an interesting characteristic was detected using this LOH analysis system two TK locus markers and eleven microsatellite loci spanning chromosome 17 The radiation-specific patterns of interstitial deletions were observed in the hemizygous LOH mutants which were considered as a result of end-joining repair of carbon ion-induced DNA double-strand breaks These results clearly demonstrate that this analysis can be used for the detection of low-dose ionizing radiation effects in the frozen cells In addition we performed so called adaptive response experiments in which TK6 cells were pre-irradiated with low-dose 2 5 sim 10 cGy of X-ray and then exposed to challenging dose 2Gy of X-rays Interestingly the

  4. Genotoxicity of silver nanoparticles evaluated using the Ames test and in vitro micronucleus assay.

    PubMed

    Li, Yan; Chen, David H; Yan, Jian; Chen, Ying; Mittelstaedt, Roberta A; Zhang, Yongbin; Biris, Alexandru S; Heflich, Robert H; Chen, Tao

    2012-06-14

    Silver nanoparticles (AgNPs) have antimicrobial properties, which have contributed to their widespread use in consumer products. A current issue regarding nanomaterials is the extent to which existing genotoxicity assays are useful for evaluating the risks associated with their use. In this study, the genotoxicity of 5 nm AgNPs was assessed using two standard genotoxicity assays, the Salmonella reverse mutation assay (Ames test) and the in vitro micronucleus assay. Using the preincubation version of the Ames assay, Salmonella strains TA102, TA100, TA1537, TA98, and TA1535 were treated with 0.15-76.8 μg/plate of the AgNPs. Toxicity limited the doses that could be assayed to 2.4-38.4 μg/plate; no increases in mutant frequency over the vehicle control were found for the concentrations that could be assayed. Human lymphoblastoid TK6 cells were treated with 10-30 μg/ml AgNPs, and additional cells were treated with water and 0.73 gy X-rays as vehicle and positive controls. Micronucleus frequency was increased by the AgNP treatment in a dose-dependent manner. At a concentration of 30 μg/ml (with 45.4% relative population doubling), AgNPs induced a significant, 3.17-fold increase with a net increase of 1.60% in micronucleus frequency over the vehicle control, a weak positive response by our criteria. These results demonstrate that the 5 nm AgNP are genotoxic in TK6 cells. Also, the data suggest that the in vitro micronucleus assay may be more appropriate than the Ames test for evaluating the genotoxicity of the AgNPs.

  5. Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

    NASA Technical Reports Server (NTRS)

    Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

    2002-01-01

    Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

  6. Correlation of In  Vivo Versus In Vitro Benchmark Doses (BMDs) Derived From Micronucleus Test Data: A Proof of Concept Study

    PubMed Central

    Soeteman-Hernández, Lya G.; Fellows, Mick D.; Johnson, George E.; Slob, Wout

    2015-01-01

    In this study, we explored the applicability of using in vitro micronucleus (MN) data from human lymphoblastoid TK6 cells to derive in vivo genotoxicity potency information. Nineteen chemicals covering a broad spectrum of genotoxic modes of action were tested in an in vitro MN test using TK6 cells using the same study protocol. Several of these chemicals were considered to need metabolic activation, and these were administered in the presence of S9. The Benchmark dose (BMD) approach was applied using the dose-response modeling program PROAST to estimate the genotoxic potency from the in vitro data. The resulting in vitro BMDs were compared with previously derived BMDs from in vivo MN and carcinogenicity studies. A proportional correlation was observed between the BMDs from the in vitro MN and the BMDs from the in vivo MN assays. Further, a clear correlation was found between the BMDs from in vitro MN and the associated BMDs for malignant tumors. Although these results are based on only 19 compounds, they show that genotoxicity potencies estimated from in vitro tests may result in useful information regarding in vivo genotoxic potency, as well as expected cancer potency. Extension of the number of compounds and further investigation of metabolic activation (S9) and of other toxicokinetic factors would be needed to validate our initial conclusions. However, this initial work suggests that this approach could be used for in vitro to in vivo extrapolations which would support the reduction of animals used in research (3Rs: replacement, reduction, and refinement). PMID:26443842

  7. Assessing the concentration of phthalate esters (PAEs) and bisphenol A (BPA) and the genotoxic potential of treated wastewater (final effluent) in Saudi Arabia.

    PubMed

    Al-Saleh, Iman; Elkhatib, Rola; Al-Rajoudi, Tahreer; Al-Qudaihi, Ghofran

    2017-02-01

    Plasticizers such as phthalate esters (PAEs) and bisphenol A (BPA) are highly persistent organic pollutants that tend to bio-accumulate in humans through the soil-plant-animal food chain. Some studies have reported the potential carcinogenic and teratogenic effects in addition to their estrogenic activities. Water resources are scarce in Saudi Arabia, and several wastewater treatment plants (WTPs) have been constructed for agricultural and industrial use. This study was designed to: (1) measure the concentrations of BPA and six PAEs, dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), bis (2-ethylhexyl) phthalate (DEHP) and dioctyl phthalate (DOP), in secondary- and tertiary-treated wastewater collected from five WTPs in three Saudi cities for four to five weeks and (2) test their potential genotoxicity. Three genotoxicological parameters were used: % tail DNA (%T), tail moment (TM) and percentage micronuclei (%MN). Both DBP and DEHP were detected in all treated wastewater samples. DMP, DEP, BBP, DOP, and BPA were found in 83.3, 84.2, 79, 73.7 and 97.4% of the samples, respectively. The levels of DMP (p<0.001), DOP (p<0.001) and BPA (p=0.001) were higher in tertiary- treated wastewater than secondary-treated wastewater, perhaps due to the influence of the molecular weight and polarity of the chemicals. Both weekly sampling frequency and WTP locations significantly affected the variability in our data. Treated wastewater from Wadi Al-Araj was able to induce DNA damage (%T and TM) in human lymphoblastoid TK6 cells that was statistically higher than wastewater from all other WTPs and in untreated TK6 cells (negative control). %MN in samples from both Wadi Al-Araj and Manfouah did not differ statistically but was significantly higher than in the untreated TK6 cells. This study also showed that the samples of tertiary-treated wastewater had a higher genotoxicological potential to induce DNA damage than the samples of

  8. Broad target cell selectivity of Kaposi's sarcoma-associated herpesvirus glycoprotein-mediated cell fusion and virion entry

    SciTech Connect

    Kaleeba, Johnan A.R.; Berger, Edward A. . E-mail: edward_berger@nih.gov

    2006-10-10

    The molecular mechanism of Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) entry is poorly understood. We tested a broad variety of cell types of diverse species and tissue origin for their ability to function as targets in a quantitative reporter gene assay for KSHV-glycoprotein-mediated cell fusion. Several human, non-human primate, and rabbit cell lines were efficient targets, whereas rodent and all human lymphoblastoid cell lines were weak targets. Parallel findings were obtained with a virion entry assay using a recombinant KSHV encoding a reporter gene. No correlation was observed between target cell activity and surface expression of {alpha}3{beta}1 integrin, a proposed KSHV receptor. We hypothesize that target cell permissiveness in both the cell fusion and virion entry assays reflects the presence of a putative KSHV fusion-entry receptor.

  9. Influence of fusion cell ratio and cell plating density on production of human-human hybridomas secreting anti-DNA autoantibodies from patients with systemic lupus erythematosus.

    PubMed

    Massicotte, H; Rauch, J; Shoenfeld, Y; Tannenbaum, H

    1984-01-01

    The utilization of one human lymphoblastoid cell line in fusion experiments with peripheral blood lymphocytes from patients with systemic lupus erythematosus (SLE) has made it possible to define efficient and reproducible conditions for the production of anti-DNA-secreting human-human hybridomas. This investigation, using the human lymphoblastoid cell line GM 4672 fused in the presence of 44% polyethylene glycol with lymphocytes from SLE patients, demonstrated a maximal yield of 22.8% hybridomas, 17% of which produced anti-DNA antibodies. We were able to define, in two independent laboratories, that the maximal yield of hybridomas occurred when the lymphocyte to GM 4672 cell ratio was 1:1 and cells were seeded in 2.0 ml wells at a concentration of 4 X 10(5) cells/well. This report demonstrates the reproducibility of human-human hybridoma production with the GM 4672 cell line and the establishment of efficient conditions for the production of anti-DNA autoantibodies from SLE patients.

  10. Reduced DNA topoisomerase II activity in ataxia-telangiectasia cells.

    PubMed Central

    Singh, S P; Mohamed, R; Salmond, C; Lavin, M F

    1988-01-01

    Considerable evidence supports a defect at the level of chromatin structure or recognition of that structure in cells from patients with the human genetic disorder ataxia-telangiectasia. Accordingly, we have investigated the activities of enzymes that alter the topology of DNA in Epstein Barr Virus-transformed lymphoblastoid cells from patients with this syndrome. Reduced activity of DNA topoisomerase II, determined by unknotting of P4 phage DNA, was observed in partially purified extracts from 5 ataxia-telangiectasia cell lines. The levels of enzyme activity was reduced substantially in 4 of these cell lines and to a lesser extent in the other cell line compared to controls. DNA topoisomerase I, assayed by relaxation of supercoiled DNA, was found to be present at comparable levels in both cell types. Reduced activity of topoisomerase II in ataxia-telangiectasia is compatible with the molecular, cellular and clinical changes described in this syndrome. Images PMID:2836804

  11. [Detection of hybrid DQ molecules by the use of T cell clone and 2D-gel analyses].

    PubMed

    Hawkin, S

    1986-11-01

    The HLA-D region incorporates three subregions, DR, DQ and DP, encoding for three sets of Ia molecules. Whereas DR antigens consist of a constant alpha chain and an extremely polymorphic beta chain, both of alpha and beta chain of DQ antigens show moderate polymorphism. This indicated us the existence of hybrid HLA-DQ molecules in HLA-D heterozygous cells, resulting from the association of an alpha chain and a beta chain encoded by genes located on the two separate haplotypes. In this report, hybrid DQ antigens were demonstrated by using cytotoxic T cell-clone. A cytotoxic T cell clone, which was generated by mixed lymphocyte reaction against a lymphoblastoid B cell line, EBV-Fuk (HLA-DR1/4, DQw1/Wa), recognized only heterogenous lymphoblastoid B cell lines (HLA-DR1/4, DQw1/Wa). Cytotoxic T cell clone, however, didn't react with B cell lines which are homozygous for HLA-DR1, DQw1 or DR4/DQWa. This suggests the T cell clone recognized the hybrid DQ molecules expressed only on heterozygous cell lines. Further confirmation was obtained by inhibition test using monoclonal antibody and biochemically by 2-D gel analyses. Biological significance of hybrid DQ antigens were discussed.

  12. A hexane fraction of american ginseng suppresses mouse colitis and associated colon cancer: anti-inflammatory and pro-apoptotic mechanisms

    PubMed Central

    Poudyal, Deepak; Le, Phuong Mai; Davis, Tia; Hofseth, Anne B.; Chumanevich, Alena; Chumanevich, Alexander A.; Wargovich, Michael J.; Nagarkatti, Mitzi; Nagarkatti, Prakash S.; Windust, Anthony; Hofseth, Lorne J.

    2012-01-01

    Ulcerative colitis (UC) is a chronic inflammatory condition associated with a high colon cancer risk. We have previously reported that American Ginseng (AG) extract significantly reduced the inflammatory parameters of chemically induced colitis. The aim of this study was to further delineate the components of AG that suppress colitis and prevent colon cancer. Among five different fractions of AG (Butanol, Hexane, Ethylacetate, Dicholoromethane and Water), a Hexane Fraction has particularly potent anti-oxidant and pro-apoptotic properties. The effects of this fraction were shown in a mouse macrophage cell line (ANA-1 cells), in a human lymphoblastoid cell line (TK6), and in an ex-vivo model (CD4+/CD25− primary effector T cells). A key in vivo finding was that compared with the whole AG extract, the Hexane Fraction of AG was more potent in treating colitis in a dextran sulfate sodium (DSS) mouse model, as well as suppressing azoxymethane (AOM)/DSS-induced colon cancer. Furthermore, TUNEL labeling of inflammatory cells within the colonic mesenteric lymph nodes (MLN) was elevated in mice consuming DSS + the Hexane Fraction of AG. Results are consistent with our in vitro data, and with the hypothesis that the Hexane Fraction of AG has antiinflammatory properties, and drives inflammatory cell apoptosis in vivo, providing a mechanism by which this fraction protects from colitis in this DSS mouse model. This study moves us closer to understanding the molecular components of AG that suppress colitis, and prevent colon cancer associated with colitis. PMID:22293630

  13. Two dechlorinated chlordecone derivatives formed by in situ chemical reduction are devoid of genotoxicity and mutagenicity and have lower proangiogenic properties compared to the parent compound.

    PubMed

    Legeay, Samuel; Billat, Pierre-André; Clere, Nicolas; Nesslany, Fabrice; Bristeau, Sébastien; Faure, Sébastien; Mouvet, Christophe

    2017-02-16

    Chlordecone (CLD) is a chlorinated hydrocarbon insecticide, now classified as a persistent organic pollutant. Several studies have previously reported that chronic exposure to CLD leads to hepatotoxicity, neurotoxicity, raises early child development and pregnancy complications, and increases the risk of liver and prostate cancer. In situ chemical reduction (ISCR) has been identified as a possible way for the remediation of soils contaminated by CLD. In the present study, the objectives were (i) to evaluate the genotoxicity and the mutagenicity of two CLD metabolites formed by ISCR, CLD-5a-hydro, or CLD-5-hydro (5a- or 5- according to CAS nomenclature; CLD-1Cl) and tri-hydroCLD (CLD-3Cl), and (ii) to explore the angiogenic properties of these molecules. Mutagenicity and genotoxicity were investigated using the Ames's technique on Salmonella typhimurium and the in vitro micronucleus micromethod with TK6 human lymphoblastoid cells. The proangiogenic properties were evaluated on the in vitro capillary network formation of human primary endothelial cells. Like CLD, the dechlorinated derivatives of CLD studied were devoid of genotoxic and mutagenic activity. In the assay targeting angiogenic properties, significantly lower microvessel lengths formed by endothelial cells were observed for the CLD-3Cl-treated cells compared to the CLD-treated cells for two of the three tested concentrations. These results suggest that dechlorinated CLD derivatives are devoid of mutagenicity and genotoxicity and have lower proangiogenic properties than CLD.

  14. Human lymphocyte markers defined by antibodies derived from somatic cell hybrids. I. A hybridoma secreting antibody against a marker specific for human B lymphocytes.

    PubMed Central

    Brooks, D A; Beckman, I; Bradley, J; McNamara, P J; Thomas, M E; Zola, H

    1980-01-01

    A hybridoma has been isolated from the products of fusion of a myeloma cell line with spleen cells from mice immunized with a human B cell line. After cloning, the hybridoma secretes antibody with the following properties: (i) Human B-lymphoblastoid cell lines react with the antibody while T and null cell lines do not. (ii) The antibody reacts with the majority of leucocytes in the blood of patients with CLL, but with a minority of cells in the blood of patients with AML or ALL of the null or T type. (iii) The antibody reacts with 9-21% of mononuclear cells in normal peripheral blood. The reacting cells are not T cells and overlap extensively with cells identified as B cells by other markers. The antigen identified by this antibody appears to be distinct from known B cell markers, and is put forward as a new B cell marker with diagnostic potential. PMID:6966995

  15. Genotoxicity analysis of two halonitromethanes, a novel group of disinfection by-products (DBPs), in human cells treated in vitro

    SciTech Connect

    Liviac, Danae; Creus, Amadeu; Marcos, Ricard

    2009-04-15

    Halonitromethanes (HNMs) constitute an emerging class of disinfection by-products (DBPs) produced when chlorine and/or ozone are used for water treatment. The HNMs are structurally similar to halomethanes, but have a nitro-group in place of hydrogen bonded to the central carbon atom. Since little information exists on the genotoxic potential of HNMs, a study has been carried out with two HNM compounds, namely trichloronitromethane (TCNM) and bromonitromethane (BNM) by using human cells. Primary damage induction has been measured with the Comet assay, which is used to determine both the repair kinetics of the induced damage and the proportion of induced oxidative damage. In addition, the fixed DNA damage has been evaluated by using the micronucleus (MN) assay. The results obtained indicate that both compounds are genotoxic, inducing high levels of DNA breaks in the Comet assay, and that this DNA damage repairs well over time. In addition, oxidized bases constitute a high proportion of DNA-induced damage (50-75%). Contrarily, no positive effects were observed in the frequency of micronucleus, which measures both clastogenic and aneugenic effects, neither using TK6 cells nor peripheral blood lymphocytes. This lack of fixed genetic damage would minimize the potential mutagenic risk associated with HNMs exposure.

  16. Lymphocyte culture: induction of colonies by conditioned medium from human lymphoid cell lines.

    PubMed

    Galbraith, R M; Goust, J M; Fudenberg, H H

    1977-12-01

    The presence of phytohemagglutinin or pokeweed mitogen in cultures of human peripheral blood mononuclear cells in agar is known to stimulate the formation of lymphoid colonies. We now report that similar colonies can be induced in the absence of plant lectins upon addition of filtered and ultracentrifuged conditioned medium (CM) obtained from certain human lymphoblastoid cell lines. Colony formation required at least 6 X 10(5) mononuclear cells per milliliter, and optimum results were obtained at concentrations of 1 X 10(6) cells/ml in the presence of 20% CM (50-500 colonies per 10(6) cells cultured). Individual cells within colonies displayed uniform morphological characteristics of lymphoid cells, and the majority formed rosettes with sheep erythrocytes, suggesting that they were of T-cell type.

  17. Poor recognition of O6-isopropyl dG by MGMT triggers double strand break-mediated cell death and micronucleus induction in FANC-deficient cells

    PubMed Central

    Hashimoto, Kiyohiro; Sharma, Vyom; Sasanuma, Hiroyuki; Tian, Xu; Takata, Minoru; Takeda, Shunichi; Swenberg, James A.; Nakamura, Jun

    2016-01-01

    Isopropyl methanesulfonate (IPMS) is the most potent genotoxic compound among methanesulfonic acid esters. The genotoxic potential of alkyl sulfonate esters is believed to be due to their alkylating ability of the O6 position of guanine. Understanding the primary repair pathway activated in response to IPMS-induced DNA damage is important to profile the genotoxic potential of IPMS. In the present study, both chicken DT40 and human TK6 cell-based DNA damage response (DDR) assays revealed that dysfunction of the FANC pathway resulted in higher sensitivity to IPMS compared to EMS or MMS. O6-alkyl dG is primarily repaired by methyl guanine methyltransferase (MGMT), while isopropyl dG is less likely to be a substrate for MGMT. Comparison of the cytotoxic potential of IPMS and its isomer n-propyl methanesulfonate (nPMS) revealed that the isopropyl moiety avoids recognition by MGMT and leads to higher cytotoxicity. Next, the micronucleus (MN) assay showed that FANC deficiency increases the sensitivity of DT40 cells to MN induction by IPMS. Pretreatment with O6-benzyl guanine (OBG), an inhibitor of MGMT, increased the MN frequency in DT40 cells treated with nPMS, but not IPMS. Lastly, IPMS induced more double strand breaks in FANC-deficient cells compared to wild-type cells in a time-dependent manner. All together, these results suggest that IPMS-derived O6-isopropyl dG escapes recognition by MGMT, and the unrepaired DNA damage leads to double strand breaks, resulting in MN induction. FANC, therefore, plays a pivotal role in preventing MN induction and cell death caused by IPMS. PMID:27486975

  18. CD80 (B7.1) and CD86 (B7.2) induce EBV-transformed B cell apoptosis through the Fas/FasL pathway.

    PubMed

    Park, Ga Bin; Kim, Yeong Seok; Lee, Hyun-Kyung; Cho, Dae-Ho; Kim, Daejin; Hur, Dae Young

    2013-11-01

    CD80 and CD86 expression is strongly regulated in B cells and is induced by various stimuli (e.g., cytokines, ligation of MHC class II and CD40 ligand). Epstein-Barr virus (EBV) infection activates B lymphocytes and transforms them into lymphoblastoid cells. However, the role of CD80 and CD86 in EBV infection of B cells remains unclear. Here, we observed that cross-linking of CD80 and CD86 in EBV-transformed B cells induced apoptosis through caspase-dependent release of apoptosis-related molecules, cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, because Z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) and N-acetylcysteine (NAC) blocked apoptosis and disruption of mitochondria. Stimulation of CD80 and CD86 induced expression of Fas ligand (FasL) on EBV-transformed B cells and upregulated Fas and FasL expression in IM-9 cells. Apoptosis through Fas-FasL interactions was blocked by treatment of cells with ZB4, an antagonistic anti-Fas antibody. These results suggest that the co-stimulatory molecules CD80 and CD86 induced by EBV infection stimulate apoptosis of EBV-transformed lymphoblastoid B cells via the Fas/FasL pathway.

  19. Epstein-Barr virus latently infected cells are selectively deleted in simulated-microgravity cultures.

    PubMed

    Long, J P; Hughes, J H

    2001-04-01

    Rotating-wall vessels (RWVs) allow for the cultivation of cells in simulated microgravity. Previously, we showed that the cultivation of lymphoblastoid cells in simulated microgravity results in the suppression of Epstein-Barr virus (EBV) reactivation. To determine if the suppression generated by simulated microgravity could be reversed by changing to static culture conditions, cells were cultured in an RRWV for 5 d, and then switched to static conditions. Following the switch to static conditions, viral reactivation remained suppressed (significantly lower) relative to static control cultures over a 4-d period. Additionally, experiments were conducted to determine if chemical treatment could induce viral reactivation in cells from simulated-microgravity cultures. Cells were cultured in static flask cultures and in simulated microgravity in RWVs for 4-7 d. The cells were then transferred to 50-cm3 tubes, and treated with 3 mM n-butyrate for 48 h, or 18 ng/ml of phorbol ester, viz., 12-0-tetradecanoylphorbol-13 acetate (TPA) for either 2 or 48 h, under static conditions. Although EBV was inducible, the cells from simulated-microgravity cultures treated with n-butyrate displayed significantly lower levels of viral-antigen expression compared with the treated cells from static cultures. Also, incubation with TPA for 2-3 h, but not for 48 h, reactivated EBV in cells from RWV cultures. In contrast, EBV was inducible in cells from static cultures treated for either 2-3 or 48 h with TPA. TPA reactivation of EBV following a 2-3-h period of treatment indicates that the protein kinase C signal-transduction pathway is not impaired in lymphoblastoid cells cultured in simulated microgravity. However, the exposure of B-lymphoblastoid cells from simulated-microgravity cultures to TPA for more than 3-4 h triggered a lytic event (apoptosis or necrosis), which prevented replication of the virus. Thus, EBV-infected cells in simulated microgravity were negatively selected in the

  20. From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.

    PubMed

    Marinov, Georgi K; Williams, Brian A; McCue, Ken; Schroth, Gary P; Gertz, Jason; Myers, Richard M; Wold, Barbara J

    2014-03-01

    Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30-100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states.

  1. Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay.

    PubMed

    Speit, Günter; Schütz, Petra; Bausinger, Julia

    2016-06-01

    The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15μM). APC was either added for 2h together with the mutagen or cells were pre-incubated for 30min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay.

  2. High-level expression of a cloned HLA heavy chain gene introduced into mouse cells on a bovine papillomavirus vector.

    PubMed

    DiMaio, D; Corbin, V; Sibley, E; Maniatis, T

    1984-02-01

    A gene encoding the heavy chain of an HLA human histocompatibility antigen was isolated from a library of human DNA by recombination and selection in vivo. After insertion into a bovine papillomavirus (BPV) DNA expression vector, the gene was introduced into cultured mouse cells. Cells transformed with the HLA-BPV plasmids did not appear to contain extrachromosomal viral DNA, whereas BPV recombinants usually replicated as plasmids in transformed cell lines. Large amounts of HLA RNA were produced by the transformed cells, and the rate of synthesis of human heavy chain was several-fold higher than in the JY cell line, a well-characterized human lymphoblastoid cell line which expresses high levels of surface HLA antigen. Substantial amounts of human heavy chain accumulated in the transformed cells, and HLA antigen was present at the cell surface. These observations establish the feasibility of using BPV vectors to study the structure and function of HLA antigens and the expression of cloned HLA genes.

  3. Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells.

    PubMed

    Sánchez-Martínez, Diego; Lanuza, Pilar M; Gómez, Natalia; Muntasell, Aura; Cisneros, Elisa; Moraru, Manuela; Azaceta, Gemma; Anel, Alberto; Martínez-Lostao, Luis; Villalba, Martin; Palomera, Luis; Vilches, Carlos; García Marco, José A; Pardo, Julián

    2016-01-01

    Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.

  4. Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells

    PubMed Central

    Sánchez-Martínez, Diego; Lanuza, Pilar M.; Gómez, Natalia; Muntasell, Aura; Cisneros, Elisa; Moraru, Manuela; Azaceta, Gemma; Anel, Alberto; Martínez-Lostao, Luis; Villalba, Martin; Palomera, Luis; Vilches, Carlos; García Marco, José A.; Pardo, Julián

    2016-01-01

    Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments. PMID:27833611

  5. Comparison of DNA fragmentation and color thresholding for objective quantitation of apoptotic cells

    NASA Technical Reports Server (NTRS)

    Plymale, D. R.; Ng Tang, D. S.; Fermin, C. D.; Lewis, D. E.; Martin, D. S.; Garry, R. F.

    1995-01-01

    Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.

  6. Assay of immunoglobulins in supernatants of lymphoid cell lines by conventional laser nephelometry.

    PubMed

    Virella, G; Muñoz, J; Robinson, J E; Goust, J M

    1979-03-01

    An adaptation of the nephelometric assay for serum immunoglobulins has been developed for detection and quantitation of extracellular immunoglobulins in cultures of lymphoblastoid cell lines. This assay employs the standard equipment for laser nephelometry and commercial reagents for immunoglobulin quantitation. By adjusting dilutions of controls and sample volumes of culture supernatants, amounts of IgG and IgM below 1 microgram/ml can be detected in culture supernatants. At concentrations between 1 and 4 microgram/ml, day-to-day and within-run variations for IgM assays were 16 and 11% respectively. The possibility of measuring immunoglobulins secreted by cell lines by conventional laser nephelometry opens several areas of application in the study of the functional activity of B cells and of cell-cell interactions.

  7. The effect of bleomycin on DNA synthesis in ataxia telangiectasia lymphoid cells

    SciTech Connect

    Cohen, M.M.; Simpson, S.J.

    1982-01-01

    Bleomycin, a radiomimetic glycopeptide, inhibits de novo DNA synthesis in ataxia telangiectasia lymphoblastoid B cells to a markedly lesser extent than in normal and xeroderma pigmentosum lymphoid cells. This observation is similar to that following ionizing radiation; however, the effect is slower following the chemical treatment. Recovery of the normal cells occurs 15-18 hours after treatment, whereas the ataxia telangiectasia lines do not attain normal levels of DNA synthesis during the entire 24-hour observation period. Similar differences were not observed following treatment with mitomycin C, a bifunctional alkylating agent, indicating a specific effect of bleomycin on DNA synthesis in ataxia telangiectasia cells. Following bleomycin treatment and preincubation with hydroxyurea, residual DNA synthesis in ataxia telangiectasia cells was similar to that in both normal and xeroderma pigmentosum lymphoid lines, suggesting that the capacity to repair the induced DNA lesion is present.

  8. Engineering safer-by-design, transparent, silica-coated ZnO nanorods with reduced DNA damage potential

    PubMed Central

    Sotiriou, Georgios A.; Watson, Christa; Murdaugh, Kimberly M.; Darrah, Thomas H.; Pyrgiotakis, Georgios; Elder, Alison; Brain, Joseph D.; Demokritou, Philip

    2014-01-01

    Zinc oxide (ZnO) nanoparticles absorb UV light efficiently while remaining transparent in the visible light spectrum rendering them attractive in cosmetics and polymer films. Their broad use, however, raises concerns regarding potential environmental health risks and it has been shown that ZnO nanoparticles can induce significant DNA damage and cytotoxicity. Even though research on ZnO nanoparticle synthesis has made great progress, efforts on developing safer ZnO nanoparticles that maintain their inherent optoelectronic properties while exhibiting minimal toxicity are limited. Here, a safer-by-design concept was pursued by hermetically encapsulating ZnO nanorods in a biologically inert, nanothin amorphous SiO2 coating during their gas-phase synthesis. It is demonstrated that the SiO2 nanothin layer hermetically encapsulates the core ZnO nanorods without altering their optoelectronic properties. Furthermore, the effect of SiO2 on the toxicological profile of the core ZnO nanorods was assessed using the Nano-Cometchip assay by monitoring DNA damage at a cellular level using human lymphoblastoid cells (TK6). Results indicate significantly lower DNA damage (>3 times) for the SiO2-coated ZnO nanorods compared to uncoated ones. Such an industry-relevant, scalable, safer-by-design formulation of nanostructured materials can liberate their employment in nano-enabled products and minimize risks to the environment and human health. PMID:24955241

  9. An approach to estimate radioadaptation from DSB repair efficiency.

    PubMed

    Yatagai, Fumio; Sugasawa, Kaoru; Enomoto, Shuichi; Honma, Masamitsu

    2009-09-01

    In this review, we would like to introduce a unique approach for the estimation of radioadaptation. Recently, we proposed a new methodology for evaluating the repair efficiency of DNA double-strand breaks (DSB) using a model system. The model system can trace the fate of a single DSB, which is introduced within intron 4 of the TK gene on chromosome 17 in human lymphoblastoid TK6 cells by the expression of restriction enzyme I-SceI. This methodology was first applied to examine whether repair of the DSB (at the I-SceI site) can be influenced by low-dose, low-dose rate gamma-ray irradiation. We found that such low-dose IR exposure could enhance the activity of DSB repair through homologous recombination (HR). HR activity was also enhanced due to the pre-IR irradiation under the established conditions for radioadaptation (50 mGy X-ray-6 h-I-SceI treatment). Therefore, radioadaptation might account for the reduced frequency of homozygous loss of heterozygosity (LOH) events observed in our previous experiment (50 mGy X-ray-6 h-2 Gy X-ray). We suggest that the present evaluation of DSB repair using this I-SceI system, may contribute to our overall understanding of radioadaptation.

  10. Analysis of 75 marketed pharmaceuticals using the GADD45a-GFP 'GreenScreen HC' genotoxicity assay.

    PubMed

    Hastwell, Paul W; Webster, Thomas W; Tate, Matthew; Billinton, Nicholas; Lynch, Anthony M; Harvey, James S; Rees, Robert W; Walmsley, Richard M

    2009-09-01

    The GADD45a-GFP (GreenScreen HC) reporter assay detects genotoxic damage in the human lymphoblastoid TK6 cell line and gives positive results for all classes of genotoxin, including mutagens, aneugens and clastogens. In this study, a collection of 75 marketed pharmaceuticals were tested in the assay. Compounds in the collection represent a broad range of chemical structures, pharmacologies and therapeutic indications, including neoplasia and viral infection where positive genotoxicity results are often associated with the pharmacological activity. Based on the results of this study, two main conclusions can be drawn: (i) the GreenScreen HC is more predictive of in vivo genotoxicity (88%) and genotoxic carcinogenicity (93%) data than the any of the other regulatory in vitro genotoxicity assay and (ii) no compounds were uniquely positive in the GADD45a-GFP assay. This analysis therefore provides additional evidence to support the use of the GADD45a-GFP assay as an effective tool either in early genotoxic liability identification or non-clinical safety assessment of candidate pharmaceuticals during development.

  11. Lithium response in bipolar disorder: No difference in GADL1 gene expression between cell lines from excellent-responders and non-responders.

    PubMed

    Moreira, Jeverson; Courtin, Cindie; Geoffroy, Pierre A; Curis, Emmanuel; Bellivier, Frank; Marie-Claire, Cynthia

    2017-05-01

    Previous association studies have shown mixed results between glutamic acid decarboxylase like-1 (GADL1) gene polymorphism and prophylactic lithium response in bipolar disorder (BD) patients. In the present study, GADL1 gene expression was investigated in regard to lithium response, using Alda scale, in lymphoblastoid cells (LCLs) of 36 Caucasian BD patients. No difference in GADL1 expression was observed among LCLs from excellent-responders, non-responders or controls. Furthermore, lithium did not induce significant changes in GADL1 expression levels after 4 or 8 days. These results did not support an association of GADL1 expression in the determination of a lithium response in BD patients.

  12. Replication of the resident Marek's Disease virus genome in synchronized nonproducer MKT-1 cells.

    PubMed

    Lau, R Y; Nonoyama, M

    1980-02-01

    MKT-1, a virus nonproducer lymphoblastoid cell line established from a Marek's disease tumor, was synchronized by double thymidine block to determine the sequence of events in the synthesis of cellular and latent marek's disease virus DNA. Cellular DNA synthesis was measured by incorporation of [3H]thymidine, whereas viral DNA synthesis was determined by DNA-DNA reassociation kinetics. The results of these studies indicate that the resident Marek's disease viral DNA in MKT-1 cells replicates during the early S phase of the cell cycle, before the onset of active cellular DNA synthesis. This observation is similar to that seen in the replication of resident Epstein-Barr virus DNA in synchronized Raji cells.

  13. A supporting role of Chinese National Immortalized Cell Bank in life science research.

    PubMed

    Chongfeng, Xu; Ziyuan, Duan

    2017-01-20

    A biorepository of human samples is essential to support the research of life science. Lymphoblastoid B cell line (LCL), which is easy to be prepared and can reproduce indefinitely, is a convenient form of sample preservation. LCLs are established from human B cells transformed by Epstein-Barr virus (EBV). Chinese National Immortalized Cell Bank has preserved human LCLs from different ethnic groups in China. As there are many studies on the nature of LCLs and public available resources with genome-wide data for LCLs, they have been widely applied in genetics, immunology, pharmacogenetics/genomics, regenerative medicine, cancer pathogenesis and immunotherapy, screening and generation of fully human neutralizing monoclonal antibodies and study on EBV pathogenesis. Here, we review the characteristics of LCLs and their contributions to scientific research, and introduce preserved samples in Chinese National Immortalized Cell Bank to the scientific community. We hope this bank can support more areas in the scientific research.

  14. DNA damage in dihydroartemisinin-resistant Molt-4 cells.

    PubMed

    Park, Jungsoo; Lai, Henry C; Sasaki, Tomikazu; Singh, Narendra P

    2015-03-01

    Artemisinin generates carbon-based free radicals when it reacts with iron, and induces molecular damage and apoptosis. Its toxicity is more selective toward cancer cells because cancer cells contain a higher level of intracellular free iron. Dihydroartemisinin (DHA), an analog of artemisinin, has selective cytotoxicity toward Molt-4 human lymphoblastoid cells. A major concern is whether cancer cells could develop resistance to DHA, thus limiting its therapeutic efficacy. We have developed a DHA-resistant Molt-4 cell line (RTN) and found out that these cells exhibited resistance to DHA but no significant cross- resistance to artemisinin-tagged holotransferrin (ART-TF), a synthetic artemisinin compound. In the present study, we investigated DNA damage induced by DHA and ART-TF in both Molt-4 and RTN cells using the comet assay. RTN cells exhibited a significantly lower level of basal and X-ray-induced DNA damage compared to Molt-4 cells. Both DHA and ART-TF induced DNA damage in Molt-4 cells, whereas DNA damage was induced in RTN cells by ART-TF, and not DHA. The result of this study shows that by the cell selection method, it is possible to generate a Molt-4 cell line which is not sensitive to DHA, but sensitive to ART-TF, as measured by DNA damage.

  15. Xeroderma Pigmentosum Group A Suppresses Mutagenesis Caused by Clustered Oxidative DNA Adducts in the Human Genome.

    PubMed

    Sassa, Akira; Kamoshita, Nagisa; Kanemaru, Yuki; Honma, Masamitsu; Yasui, Manabu

    2015-01-01

    Clustered DNA damage is defined as multiple sites of DNA damage within one or two helical turns of the duplex DNA. This complex damage is often formed by exposure of the genome to ionizing radiation and is difficult to repair. The mutagenic potential and repair mechanisms of clustered DNA damage in human cells remain to be elucidated. In this study, we investigated the involvement of nucleotide excision repair (NER) in clustered oxidative DNA adducts. To identify the in vivo protective roles of NER, we established a human cell line lacking the NER gene xeroderma pigmentosum group A (XPA). XPA knockout (KO) cells were generated from TSCER122 cells derived from the human lymphoblastoid TK6 cell line. To analyze the mutagenic events in DNA adducts in vivo, we previously employed a system of tracing DNA adducts in the targeted mutagenesis (TATAM), in which DNA adducts were site-specifically introduced into intron 4 of thymidine kinase genes. Using the TATAM system, one or two tandem 7,8-dihydro-8-oxoguanine (8-oxoG) adducts were introduced into the genomes of TSCER122 or XPA KO cells. In XPA KO cells, the proportion of mutants induced by a single 8-oxoG (7.6%) was comparable with that in TSCER122 cells (8.1%). In contrast, the lack of XPA significantly enhanced the mutant proportion of tandem 8-oxoG in the transcribed strand (12%) compared with that in TSCER122 cells (7.4%) but not in the non-transcribed strand (12% and 11% in XPA KO and TSCER122 cells, respectively). By sequencing the tandem 8-oxoG-integrated loci in the transcribed strand, we found that the proportion of tandem mutations was markedly increased in XPA KO cells. These results indicate that NER is involved in repairing clustered DNA adducts in the transcribed strand in vivo.

  16. Xeroderma Pigmentosum Group A Suppresses Mutagenesis Caused by Clustered Oxidative DNA Adducts in the Human Genome

    PubMed Central

    Sassa, Akira; Kamoshita, Nagisa; Kanemaru, Yuki; Honma, Masamitsu; Yasui, Manabu

    2015-01-01

    Clustered DNA damage is defined as multiple sites of DNA damage within one or two helical turns of the duplex DNA. This complex damage is often formed by exposure of the genome to ionizing radiation and is difficult to repair. The mutagenic potential and repair mechanisms of clustered DNA damage in human cells remain to be elucidated. In this study, we investigated the involvement of nucleotide excision repair (NER) in clustered oxidative DNA adducts. To identify the in vivo protective roles of NER, we established a human cell line lacking the NER gene xeroderma pigmentosum group A (XPA). XPA knockout (KO) cells were generated from TSCER122 cells derived from the human lymphoblastoid TK6 cell line. To analyze the mutagenic events in DNA adducts in vivo, we previously employed a system of tracing DNA adducts in the targeted mutagenesis (TATAM), in which DNA adducts were site-specifically introduced into intron 4 of thymidine kinase genes. Using the TATAM system, one or two tandem 7,8-dihydro-8-oxoguanine (8-oxoG) adducts were introduced into the genomes of TSCER122 or XPA KO cells. In XPA KO cells, the proportion of mutants induced by a single 8-oxoG (7.6%) was comparable with that in TSCER122 cells (8.1%). In contrast, the lack of XPA significantly enhanced the mutant proportion of tandem 8-oxoG in the transcribed strand (12%) compared with that in TSCER122 cells (7.4%) but not in the non-transcribed strand (12% and 11% in XPA KO and TSCER122 cells, respectively). By sequencing the tandem 8-oxoG-integrated loci in the transcribed strand, we found that the proportion of tandem mutations was markedly increased in XPA KO cells. These results indicate that NER is involved in repairing clustered DNA adducts in the transcribed strand in vivo. PMID:26559182

  17. Assay for mutagenesis in heterozygous diploid human lymphoblasts

    DOEpatents

    Skopek, Thomas R.; Liber, Howard L.; Penman, Bruce W.; Thilly, William G.; Hoppe, IV, Henry

    1981-01-01

    An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay.

  18. Human lymphocyte markers defined by antibodies derived from somatic cell hybrids. II. A hybridoma secreting antibody against an antigen expressed by human B and null lymphocytes.

    PubMed

    Beckman, I G; Bradley, J; Brooks, D A; Kupa, A; McNamara, P J; Thomas, M E; Zola, H

    1980-06-01

    A hybridoma (FMC4) has been derived which secretes antibody showing selective reaction with human B lymphocytes, monocytes and some null lymphocytes. Few, if any, T lymphocytes in normal blood are stained, although stimulation of lymphocytes with PHA leads to an increase in the proportion of cells reacting with the hybridoma antibody. The antibody reacts with B and null lymphoblastoid cell lines but not with T cell lines. B chronic lymphocytic leukaemia (CLL) cells but not T-CLLs are stained and null-type acute lymphoblastic leukaemia (ALL) cells but not T-type ALL also react. Normal blood myeloid cells do not react with FMC4 supernatant whilst some myeloid leukaemias do. The expression of the antigen reacting with FMC4 supernatant suggests that FMC4 may secrete an antibody against the human equivalent of the Ia antigen.

  19. Human lymphocyte markers defined by antibodies derived from somatic cell hybrids. II. A hybridoma secreting antibody against an antigen expressed by human B and null lymphocytes.

    PubMed Central

    Beckman, I G; Bradley, J; Brooks, D A; Kupa, A; McNamara, P J; Thomas, M E; Zola, H

    1980-01-01

    A hybridoma (FMC4) has been derived which secretes antibody showing selective reaction with human B lymphocytes, monocytes and some null lymphocytes. Few, if any, T lymphocytes in normal blood are stained, although stimulation of lymphocytes with PHA leads to an increase in the proportion of cells reacting with the hybridoma antibody. The antibody reacts with B and null lymphoblastoid cell lines but not with T cell lines. B chronic lymphocytic leukaemia (CLL) cells but not T-CLLs are stained and null-type acute lymphoblastic leukaemia (ALL) cells but not T-type ALL also react. Normal blood myeloid cells do not react with FMC4 supernatant whilst some myeloid leukaemias do. The expression of the antigen reacting with FMC4 supernatant suggests that FMC4 may secrete an antibody against the human equivalent of the Ia antigen. PMID:6968260

  20. G{sub 2}/M-phase arrest and release in ataxia telangiectasia and normal cells after exposure to ionizing radiation

    SciTech Connect

    Hong, J.H.; Gatti, R.A.; Huo, Y.K.; Chiang, C.S.; McBride, W.H.

    1994-10-01

    Cells from patients with ataxia telangiectasia (AT) are abnormal in their response to irradiation as judged by clonogenic survival and accumulation in G{sub 2} phase. The relationship of the results of these two assays, however, is still a matter of controversy. Flow cytometry was used to measure the distribution of cells in the phases of the cell cycle after 2 Gy irradiation in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) and SV40-transformed fibroblasts. AT cells showed increased and prolonged accumulation in G{sub 2}/M phase regardless of the cell type (lymphoblastoid or fibroblast) or complementation group (A, C or D). To test the hypothesis that prolonged accumulation of AT cells in G{sub 2} phase after irradiation was not simply a reflection of their radiosensitivity, we gave iso-survival radiation doses to SV40-transformed fibroblasts of two AT and one control cell lines. The two AT cell lines exited from the G{sub 2}/M-phase block more slowly than control cells after each dose tested. This implies that prolonged accumulation in G{sub 2}/M phase in AT cells is not directly related to radiosensitivity as measured by clonogenic survival, but that factors involved in the exit from G{sub 2} phase after irradiation may be abnormally regulated. We found that G{sub 2}-phase arrest of AT cells did not necessarily result in a fatal consequence in the first cell cycle after irradiation. Furthermore, G{sub 2}-phase arrest did not lead to detectable DNA fragmentation characteristic of apoptosis as judged by gel electrophoresis. 37 refs., 6 figs.

  1. Effects of Laser Printer–Emitted Engineered Nanoparticles on Cytotoxicity, Chemokine Expression, Reactive Oxygen Species, DNA Methylation, and DNA Damage: A Comprehensive in Vitro Analysis in Human Small Airway Epithelial Cells, Macrophages, and Lymphoblasts

    PubMed Central

    Pirela, Sandra V.; Miousse, Isabelle R.; Lu, Xiaoyan; Castranova, Vincent; Thomas, Treye; Qian, Yong; Bello, Dhimiter; Kobzik, Lester; Koturbash, Igor; Demokritou, Philip

    2015-01-01

    Background Engineered nanomaterials (ENMs) incorporated into toner formulations of printing equipment become airborne during consumer use. Although information on the complex physicochemical and toxicological properties of both toner powders and printer-emitted particles (PEPs) continues to grow, most toxicological studies have not used the actual PEPs but rather have primarily used raw toner powders, which are not representative of current exposures experienced at the consumer level during printing. Objectives We assessed the biological responses of a panel of human cell lines to PEPs. Methods Three physiologically relevant cell lines—small airway epithelial cells (SAECs), macrophages (THP-1 cells), and lymphoblasts (TK6 cells)—were exposed to PEPs at a wide range of doses (0.5–100 μg/mL) corresponding to human inhalation exposure durations at the consumer level of 8 hr or more. Following treatment, toxicological parameters reflecting distinct mechanisms were evaluated. Results PEPs caused significant membrane integrity damage, an increase in reactive oxygen species (ROS) production, and an increase in pro-inflammatory cytokine release in different cell lines at doses equivalent to exposure durations from 7.8 to 1,500 hr. Furthermore, there were differences in methylation patterns that, although not statistically significant, demonstrate the potential effects of PEPs on the overall epigenome following exposure. Conclusions The in vitro findings obtained in this study suggest that laser printer–emitted engineered nanoparticles may be deleterious to lung cells and provide preliminary evidence of epigenetic modifications that might translate to pulmonary disorders. Citation Pirela SV, Miousse IR, Lu X, Castranova V, Thomas T, Qian Y, Bello D, Kobzik L, Koturbash I, Demokritou P. 2016. Effects of laser printer–emitted engineered nanoparticles on cytotoxicity, chemokine expression, reactive oxygen species, DNA methylation, and DNA damage: a comprehensive in

  2. Synergy of 2-deoxy-D-glucose combined with berberine in inducing the lysosome/autophagy and transglutaminase activation-facilitated apoptosis.

    PubMed

    Halicka, H Dorota; Garcia, Jorge; Li, Jiangwei; Zhao, Hong; Darzynkiewicz, Zbigniew

    2017-02-01

    Utilizing a variety of flow cytometric methods evidence was obtained indicating that a combination of the glucose analog 2-deoxy-D-glucose (2-dG) and the plant alkaloid berberine (BRB) produces synergistic effect in the induction of apoptosis in human lymphoblastoid TK6 cells. The synergistic effect is seen at concentrations of the drugs at which each of them alone shows no cytotoxicity at all. The data suggest that the combination of these drugs, which are known in terms of their overall toxicity, side effects and pharmacokinetics may be considered for further studies as chemopreventive and cancer treatment modalities. Of interest are results indicating that rapamycin, which similarly to BRB, suppresses mTOR signaling, when combined with 2-dG shows no synergistic properties. Metformin, on other hand, requires much higher concentration to show the synergy with 2-dG. Also of interest are the findings pertaining to the methodology of the present study. Specifically, dynamic assessment of cellular viability was performed by using the DRAQ7 cell exclusion fluorochrome present in cultures from 0 to 72 h. Concurrent measurement of lysosomal proton pump using acridine orange as the probe shows activation of lysosomes in the cells treated with 2-dG or BRB alone as well as with the drugs combined. Apoptosis was assessed by measuring DNA fragmentation, cell cycle, activation of caspase-3 and tissue transglutaminase (Tgase). A novel cytometric method was developed based on analysis of lysosomal (acidic vesicles) proton pump in live cells followed by cell lysis with detergent and fluorochrome labeling of proteins and DNA to analyze Tgase activation concurrently with cell cycle, in same population of cells. The data show that the cell subpopulation undergoing apoptosis has increased side (right-angle) light scatter likely due to the presence of the crosslinked (solid state) proteins, the consequence Tgase activation.

  3. Characterization of null cells in chronic lymphocytic leukaemia with B-cell allo- and hetero-antisera.

    PubMed

    Kirov, S M; Kwant, W O; Fernandez, L A; MacSween, J M; Langley, G R

    1980-02-01

    Chronic lymphocytic leukaemia peripheral blood mononuclear cells (CLL-PBMN) were separated into B, T and Null-enriched lymphocyte sub-populations using sequential mouse and sheep red blood cell rosetting depletions on Hypaque-Ficoll gradients. The procedure produced viable cell populations with mean percentage purities of 90, 87 and 75 for B, T and non-rosetting (Null-enriched) sub-populations, respectively. More than 80% of PBMN cells were generally accounted for by mouse and sheep rosetting. The purified lymphocyte sub-populations were examined with a panel of B-cell specific alloantisera obtained from kidney transplant recipients and a rabbit antiserum to B cell antigen isolated from a human B-lymphoblastoid line. The results illustrated that the antigens detected by these sera also have potential as a marker for characterizing the CLL population. Where conventional markers were weak or absent, B cell antigens were readily detected in both fluorescent and cytotoxic tests. The majority of the non-rosetting cells (less than 90%) in CLL followed similar patterns of reactivity to the purified B cells, suggesting they are a subset of B cells. A small residual population (0--5% of PBMN) did not react with the antisera, the significance of which is unknown.

  4. Induction of proliferation in vitro of resting human natural killer cells

    SciTech Connect

    London, L.

    1986-01-01

    Experiments examined the cellular and humoral factors necessary to induce proliferation of purified NK cells in vitro and analyzed the phenotypic characteristics of these proliferating cells. The authors experiments demonstrated that NK cells do not proliferate in response to typical T cell mitogens or to allogeneic stimulation. However, NK cells are readily induced to proliferate in response to either natural or recombinant IL-2. The proliferative response of NK cells to IL-2 is enhanced in the presence of irradiated B lymphoblastoid ell lines. Proliferating NK cells maintain the expression of surface markers characteristic of freshly isolated NK cells which newly expressing surface activation antigens including the IL-2 and transferric receptors and the HLA-DR antigen. The majority of NK cells initiate proliferation in response to IL-2. Greater than 50 U/ml of IL-2 is necessary to induce maximal tritiated thymidine (/sup 3/H-TdR) incorporation by NK cells, and the interaction of IL-2 with the Tac IL-2 receptor is required for the maintenance of NK cell proliferation. NK cells do not proliferate in response to irradiated Daudi cells alone, which, in the presence of IL-2, may act by maintaining continuous proliferation of the cells originally responsive to IL-2. Unlike NK cells, the authors have shown that only a minor subset of T cells proliferate in response to IL-2 alone.

  5. Ultraviolet light-induced chromosomal aberrations in cultured cells from Cockayne syndrome and complementation group C xeroderma pigmentosum patients: lack of correlation with cancer susceptibility

    SciTech Connect

    Seguin, L.R.; Tarone, R.E.; Liao, K.H.; Robbins, J.H.

    1988-03-01

    Both Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are inherited diseases with defective repair of damage induced in DNA by UV. Patients with XP, but not those with CS, have an increased susceptibility to formation of sunlight-induced skin tumors. We determined the frequency of UV-induced chromosomal aberrations in cultured lymphoblastoid cell lines from five CS patients and three complementation-group-C XP patients to determine whether such aberrations were abnormally increased only in the XP cells. We found that CS cells had the same abnormally increased number of induced aberrations as the XP cells, indicating that the number of UV-induced aberrations in XP group C cells does not account for the susceptibility of these XP patients to sunlight-induced skin cancer.

  6. Interaction of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) with owl monkey kidney cells in enhancing the yields of Herpesvirus saimiri (HVS) and its antigens

    SciTech Connect

    Faggioni, A.; Ablashi, D.V.; Dahlberg, J.; Armstrong, G.; Sundar, S.K.

    1984-05-01

    Pre- and posttreatment with N-methyl-N'-nitro-nitrosoguanidine (MNNG) of owl monkey kidney (OMK) cells infected with Herpesvirus saimiri (HVS) resulted in one to three logs higher yields of virus, depending upon the dose of MNNG. A higher percentage of cells also showed HVS early antigen (EA) and late antigen (LA) by immunofluorescence when OMK cells infected with HVS were fed with medium containing MNNG. The high yields of HVS were also observed by electron microscopy. MNNG did not induce HVS-EA in HVS nonproducer lymphoblastoid T cells, nor did it enhance TPA-induced EA to LA. The data suggest that MNNG could be useful in obtaining high yields of virus and/or antigen-producing cells for immunofluorescence or other biomedical experiments, especially from those strains of HVS which grow poorly in vitro. The interaction of MNNG and HVS could also be useful for in vitro transformation or in vivo enhancement of the malignant process.

  7. Identification of a novel gene expressed in activated natural killer cells and T cells

    SciTech Connect

    Dahl, C.A.; Schall, R.P.; He, H.; Cairns, J.S. )

    1992-01-15

    The authors have isolated a cDNA clone from a human activated NK cell-derived cDNA library that identifies a transcript [NK4] that is selectively expressed in lymphocytes. The expression of this transcript is increased after activation of T cells by mitogens or activation of NK cells by IL-2 (lymphokine-activated killer cells). The transcript levels demonstrated by Northern blot analysis increase by 12 h after activation, remain high for at least 48 h, and require protein synthesis for expression. Southern blot analysis of B lymphoblastoid lines derived from 18 unrelated individuals reveal variable banding patterns suggestive of polymorphism within the NK4 gene. No homology was found between the sequence of the coding region of this transcript and any sequences in the GenBank data base. Sequence homology to the U1 small nuclear RNA was found within the 3[prime] untranslated region immediately upstream of the site of polyadenylation, suggesting a possible role for U1 in the polyadenylation process. Sequence analysis indicates the transcript would encode a protein having a mass of 27 kDa. The presence of a signal sequence and lack of a transmembrane region suggests that the protein is secreted. In addition, the protein contains an RGD sequence that may be involved in cellular adhesion. This transcript appears to encode a novel product common to the activation pathways of both NK cells and T cells. 50 refs., 8 figs.

  8. DNA double-strand breaks measured in individual cells subjected to gel electrophoresis

    SciTech Connect

    Olive, P.L.; Wlodek, D.; Banath, J.P. )

    1991-09-01

    Microscopic examination of individual mammalian cells embedded in agarose, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye provides a novel way of measuring DNA damage and more importantly, of assessing heterogeneity in DNA damage within a mixed population of cells. With this method, DNA double-strand breaks can be detected in populations of cells exposed to X-ray doses as low as 5 Gy. The radiation dose-response relationship for initial formation of double-strand breaks was identical for cell lines irradiated in G1, regardless of their sensitivity to killing by ionizing radiation. However, for cells irradiated in S phase, DNA migration was significantly reduced. For Chinese hamster V79 cells, Chinese hamster ovary cells, WiDr human colon carcinoma cells, and L5178Y-R mouse lymphoblastoid cells, S-phase DNA appeared to be about 3 times less sensitive to X-ray damage than DNA from other phases of the cell cycle. However, for the very radiosensitive L5178Y-S cells, the migration of replicating DNA was reduced only slightly. For Chinese hamster V79 and Chinese hamster ovary cells, damage was repaired at a similar rate in all cells of the population, and 85% of the breaks were rejoined within 2 h after irradiation. The radiosensitive L5178Y-S cells repaired damage more slowly than V79 or Chinese hamster ovary cells; 2 h after exposure to 50 Gy, approximately 50% of the damage was still present.

  9. Isolation and establishment in in vitro culture of a Theileria annulata--infected cell line from Spain.

    PubMed

    Viseras, J; García-Fernández, P; Adroher, F J

    1997-01-01

    The isolation of Theileria annulata-infected lymphocytes using blood from an animal suffering from Mediterranean theileriosis as a source of parasites is described. The present work reports the first isolation and establishment in in vitro culture of a T. annulata-infected cell line from southwestern Europe, where Mediterranean theileriosis causes important economic losses, especially in southern Spain. The parasite was identified by staining of cells from culture with Giemsa, by immunofluorescent antibody techniques (IFAT), and by isoenzyme characterization. The possibility of using this T. annulata-infected lymphoblastoid cell line to obtain an antigen for diagnosis of Mediterranean theileriosis by IFAT and to develop a tissue-culture vaccine against this disease in our geographic area shows the significance of this isolation and culture.

  10. Cytotoxicity of dihydroartemisinin toward Molt-4 cells attenuated by N-tert-butyl-alpha-phenylnitrone and deferoxamine.

    PubMed

    Chan, Ho Wing; Singh, Narendra P; Lai, Henry C

    2013-10-01

    Derivatives of artemisinin, a compound extracted from the wormwood Artemisia annua L, have potent anticancer properties. The anticancer mechanisms of artemisinin derivatives have not been fully-elucidated. We hypothesize that the cytotoxicity of these compounds is due to the free radicals formed by interaction of their endoperoxide moiety with intracellular iron in cancer cells. The effects of N-tert-butyl-alpha-phenylnitrone (PBN), a spin-trap free radical scavenger, and deferoxamine (DX), an iron chelating agent, on the in vitro cytotoxicity of dihyroartemisinin (DHA) toward Molt-4 human T-lymphoblastoid leukemia cells were investigated in the present study. Dihydroartemisinin effectively killed Molt-4 cells in vitro. Its cytotoxicity was significantly attenuated by PBN and DX. Based on the data of our present and previous studies, we conclude that one anticancer mechanism of dihydroartemisinin is the formation of toxic-free radicals via an iron-mediated process.

  11. Mutagenic Effects of Iron Oxide Nanoparticles on Biological Cells.

    PubMed

    Dissanayake, Niluka M; Current, Kelley M; Obare, Sherine O

    2015-09-30

    In recent years, there has been an increased interest in the design and use of iron oxide materials with nanoscale dimensions for magnetic, catalytic, biomedical, and electronic applications. The increased manufacture and use of iron oxide nanoparticles (IONPs) in consumer products as well as industrial processes is expected to lead to the unintentional release of IONPs into the environment. The impact of IONPs on the environment and on biological species is not well understood but remains a concern due to the increased chemical reactivity of nanoparticles relative to their bulk counterparts. This review article describes the impact of IONPs on cellular genetic components. The mutagenic impact of IONPs may damage an organism's ability to develop or reproduce. To date, there has been experimental evidence of IONPs having mutagenic interactions on human cell lines including lymphoblastoids, fibroblasts, microvascular endothelial cells, bone marrow cells, lung epithelial cells, alveolar type II like epithelial cells, bronchial fibroblasts, skin epithelial cells, hepatocytes, cerebral endothelial cells, fibrosarcoma cells, breast carcinoma cells, lung carcinoma cells, and cervix carcinoma cells. Other cell lines including the Chinese hamster ovary cells, mouse fibroblast cells, murine fibroblast cells, Mytilus galloprovincialis sperm cells, mice lung cells, murine alveolar macrophages, mice hepatic and renal tissue cells, and vero cells have also shown mutagenic effects upon exposure to IONPs. We further show the influence of IONPs on microorganisms in the presence and absence of dissolved organic carbon. The results shed light on the OPEN ACCESS Int. J. Mol. Sci. 2015, 16 23483 transformations IONPs undergo in the environment and the nature of the potential mutagenic impact on biological cells.

  12. Mutagenic Effects of Iron Oxide Nanoparticles on Biological Cells

    PubMed Central

    Dissanayake, Niluka M.; Current, Kelley M.; Obare, Sherine O.

    2015-01-01

    In recent years, there has been an increased interest in the design and use of iron oxide materials with nanoscale dimensions for magnetic, catalytic, biomedical, and electronic applications. The increased manufacture and use of iron oxide nanoparticles (IONPs) in consumer products as well as industrial processes is expected to lead to the unintentional release of IONPs into the environment. The impact of IONPs on the environment and on biological species is not well understood but remains a concern due to the increased chemical reactivity of nanoparticles relative to their bulk counterparts. This review article describes the impact of IONPs on cellular genetic components. The mutagenic impact of IONPs may damage an organism’s ability to develop or reproduce. To date, there has been experimental evidence of IONPs having mutagenic interactions on human cell lines including lymphoblastoids, fibroblasts, microvascular endothelial cells, bone marrow cells, lung epithelial cells, alveolar type II like epithelial cells, bronchial fibroblasts, skin epithelial cells, hepatocytes, cerebral endothelial cells, fibrosarcoma cells, breast carcinoma cells, lung carcinoma cells, and cervix carcinoma cells. Other cell lines including the Chinese hamster ovary cells, mouse fibroblast cells, murine fibroblast cells, Mytilus galloprovincialis sperm cells, mice lung cells, murine alveolar macrophages, mice hepatic and renal tissue cells, and vero cells have also shown mutagenic effects upon exposure to IONPs. We further show the influence of IONPs on microorganisms in the presence and absence of dissolved organic carbon. The results shed light on the transformations IONPs undergo in the environment and the nature of the potential mutagenic impact on biological cells. PMID:26437397

  13. Evaluation of EBV transformation of human memory B-cells isolated by FACS and MACS techniques.

    PubMed

    Sadreddini, Sanam; Jadidi-Niaragh, Farhad; Younesi, Vahid; Pourlak, Tala; Afkham, Amir; Shokri, Fazel; Yousefi, Mehdi

    2016-07-01

    Several studies have been performed to develop effective neutralizing monoclonal antibodies. The Epstein-Barr virus (EBV) can efficiently immortalize B-cells to establish lymphoblastoid cell lines (LCL) and so it has been used extensively for transformation of B-cells to produce and secrete immunoglobulin. The present study addressed the effect of TLR7/8 agonist (R848), feeder cells layer and fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) cell separation methods on the transformation efficiency of antibody-producing memory B-cells. For these studies, the antigen used for analyses of antibody formation was the tetanus neurotoxin (TeNT) derived from Clostridium tetani. The results here showed that employing an HFFF.PI6 feeder cell layer, R848 agonist and FACS-mediated purification of memory B-cells led to increased transformation efficiency. Altogether, the effects of the R848 and the feeder cells provided an efficient method for EBV transformation of human B-cells. Moreover, there was an advantage in using FACS sorting of B-cells over the MACS method in the context of EBV transformation and immortalization of precursors of antigen-specific B-cells.

  14. A hexane fraction of American ginseng suppresses mouse colitis and associated colon cancer: anti-inflammatory and proapoptotic mechanisms.

    PubMed

    Poudyal, Deepak; Le, Phuong Mai; Davis, Tia; Hofseth, Anne B; Chumanevich, Alena; Chumanevich, Alexander A; Wargovich, Michael J; Nagarkatti, Mitzi; Nagarkatti, Prakash S; Windust, Anthony; Hofseth, Lorne J

    2012-04-01

    Ulcerative colitis is a chronic inflammatory condition associated with a high colon cancer risk. We have previously reported that American ginseng extract significantly reduced the inflammatory parameters of chemically induced colitis. The aim of this study was to further delineate the components of American ginseng that suppress colitis and prevent colon cancer. Among five different fractions of American ginseng (butanol, hexane, ethylacetate, dichloromethane, and water), a hexane fraction has particularly potent antioxidant and proapoptotic properties. The effects of this fraction were shown in a mouse macrophage cell line (ANA-1 cells), in a human lymphoblastoid cell line (TK6), and in an ex vivo model (CD4(+)/CD25(-) primary effector T cells). A key in vivo finding was that compared with the whole American ginseng extract, the hexane fraction of American ginseng was more potent in treating colitis in a dextran sodium sulfate (DSS) mouse model, as well as suppressing azoxymethane/DSS-induced colon cancer. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) labeling of inflammatory cells within the colonic mesenteric lymph nodes was elevated in mice consuming DSS + the hexane fraction of American ginseng. Results are consistent with our in vitro data and with the hypothesis that the hexane fraction of American ginseng has anti-inflammatory properties and drives inflammatory cell apoptosis in vivo, providing a mechanism by which this fraction protects from colitis in this DSS mouse model. This study moves us closer to understanding the molecular components of American ginseng that suppress colitis and prevent colon cancer associated with colitis.

  15. Human cell mutagenicity of chlorinated and unchlorinated water and the disinfection byproduct 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX).

    PubMed

    Woodruff, N W; Durant, J L; Donhoffner, L L; Penman, B W; Crespi, C L

    2001-08-22

    Extracts of three water samples--humic acid-enriched water-both peatland water and drinking water, both with and without chlorination were tested for mutagenicity at the tk locus in MCL-5 cells, a line of human B-lymphoblastoid cells that express cytochrome P450 enzymes and microsomal epoxide hydrolase. Our results show that chlorination caused a 5.5-fold increase (P<0.0001) in the mutagenicity of the humic acid-enriched water. The unchlorinated peatland water was mutagenic at the two highest doses (240 and 480 microg equivalent total organic carbon (TOC)/ml), possibly due to polycyclic aromatic hydrocarbons (PAH) that were measured in the peat. In contrast, the chlorinated peatland water was non-mutagenic at low doses, while at the highest dose (240 microg equivalent TOC/ml) the sample was so toxic that an insufficient number of cells survived treatment to allow plating. The chlorinated and unchlorinated drinking water were both non-mutagenic. 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent bacterial mutagen and chlorine-disinfection byproduct, was also tested in MCL-5 cells as well as in two other human B-lymphoblastoid cell-lines, AHH-1 TK+/- and h1A1v2 cells, which differ from each other and from MCL-5 cells in the amounts of cytochrome P450 enzymes they can express. MX was mutagenic to all three cell-lines, but there was no apparent correlation between cytochrome P450 enzyme expression and the mutagenicity of MX. Overall, our results show that samples of chlorinated humic acid-enriched water and MX, a model chlorine-disinfection byproduct, are moderately mutagenic to human cells.

  16. [Mechanisms of gamma-inducible death of Jurkat cells line].

    PubMed

    Gamkrelidze, M M; Bezhitashvili, N D; Pavliashvili, A T; Mchedlishvili, T V; Sanikidze, T V

    2008-06-01

    Mechanisms of radio-inducible death of Jurkat cells were investigated. Human lymphoblastoid T-cell line Jurkat is widely established model for studying apoptosis mechanisms. The cell was radiated by "Teragam" (Czech Republic) by dose 2 g during 1 minute. After radiation cells were incubated at standard conditions during 24 hours. After gamma radiation in cell population amount of cells in gaplois (apoptotic G 0) stage was increased 8,2 folds, in diplois (G 0/G1) stage - by 17%, in synthetic (S) stage decreased by 35% and tetraploid (G2/M) stage by 73% in comparison to control group. It was revealed intensive production of free radicals of oxygen and nitric oxide and decreasing activity of antioxidant enzymes (superoxidismutasa, catalasa and glutathione peroxidase). Revealed dependence between intensification of apoptosis and radiation-induced arrest of cell cycle G2/M phase may be determined by excess amount of free oxygen and nitrogen radicals generated in Jurkat cells as a result of nondirect effects of low doses of gamma radiation.

  17. Relationships among micronuclei, nucleoplasmic bridges and nuclear buds within individual cells in the cytokinesis-block micronucleus assay.

    PubMed

    Cheong, Han S J; Seth, Isheeta; Joiner, Michael C; Tucker, James D

    2013-07-01

    Micronuclei have been used extensively in studies as an easily evaluated indicator of DNA damage but little is known about their association with other types of damage such as nucleoplasmic bridges and nuclear buds. Here, radiation-induced clastogenic events were evaluated via the cytokinesis-block micronucleus assay in two normal human lymphoblastoid cell lines exposed to neutrons or γ-radiation. DNA damage induced by the chemical agents mitomycin C and phleomycin was also evaluated in two normal and two mitochondrial mutant human lymphoblastoid cell lines. In addition to micronuclei, nucleoplasmic bridges and nuclear buds were enumerated by recording the coincident presence of these end points within individual cells, and the associations among these three end points were evaluated for all treatment conditions. The common odds ratios for micronuclei and nucleoplasmic bridges were found to be significantly larger than unity, indicating that the presence of one or more micronuclei in a cell imposes a significant risk of having one or more nucleoplasmic bridges in that same cell, and vice versa. The strength of this association did not change significantly with radiation dose or concentration of the chemical clastogens. Common odds ratios for association between micronuclei and buds, and between bridges and buds were also found to be significantly higher than unity. However, associations between micronuclei and buds could not be calculated for some treatments due to heterogeneity in the odds ratios and hence may depend on chemical clastogen concentration or radiation dose. This study provides evidence of how paired analyses among genetic end points in the cytokinesis-block micronucleus assay can provide information concerning abnormalities of cell division and possibly about structural chromosomal rearrangements induced by clastogens.

  18. Mitotic slippage underlies the relationship between p53 dysfunction and the induction of large micronuclei by colcemid.

    PubMed

    Hashimoto, Kiyohiro; Todo, Takeshi

    2013-07-01

    Micronuclei induced by aneugens are larger than those induced by clastogens in both in vitro and in vivo micronucleus (MN) assays. p53 dysfunction increases the formation of large micronuclei following treatment with aneugens; this study sought to identify the mechanisms responsible for this. Treatment with colcemid, both a mitotic inhibitor and an aneugen, induced MN containing two or more chromosomes more frequently in NH32 cells, in which p53 function is compromised, than in TK6 cells, in which p53 is functional. This indicates that p53 dysfunction enhances aneugen-induced chromosome loss or perturbs apoptosis, resulting in the formation of large MN. To examine the former hypothesis, the incidence of chromosome malsegregation in colcemid-treated TK6 and NH32 cells was compared using the cytokinesis-block MN assay. The incidence of chromosome non-disjunction was higher in NH32 cells than in TK6 cells, whereas the incidence of MN containing two or more chromosomes was similar between the two cell lines. To address the involvement of apoptosis in cell cycle progression, examination of chromosome 8 distribution revealed that more mononuclear NH32 than TK6 cells were tetraploid after prolonged mitotic inhibition, which indicated that the more number of NH32 cells may have bypassed the spindle assembly checkpoint via mitotic slippage and progression into the next interphase. Cells that underwent mitotic slippage were likely to contain lagging chromosomes formed via chromosome malsegregation, resulting in MN separated from the main nucleus. The number of TK6 cells containing large MN following colcemid treatment was increased by treatment with a caspase inhibitor in a dose-dependent manner, indicating that TK6 cells with MN normally undergo apoptosis. In conclusion, these findings indicate that mitotic slippage and perturbed apoptosis contribute to the induction of large MN in p53-compromised cells following treatment with colcemid.

  19. Estimation by limiting dilution analysis of human IL 2-secreting T cells: detection of IL 2 produced by single lymphokine-secreting T cells

    SciTech Connect

    Vie, H.; Miller, R.A.

    1986-05-01

    We present here a culture method for the estimation, in human blood, of the number of lymphocytes that can respond to mitogen by producing interleukin 2 (IL 2). T cells are cultured at limiting dilutions with PHA or Con A in the presence of Epstein Barr virus-transformed human lymphoblastoid cells (EB-LCL), and supernatants are tested 3 days later for IL 2 content by a cell proliferation assay. The distribution of negative wells follows the expected Poisson single-hit relationship, suggesting that the assay is sensitive to single cells of a single limiting cell type. On average, 16.3% of peripheral blood mononuclear cells can produce IL 2 in such clonal cultures (mean of 12 determinations; SD = 5.6%). Surprisingly, irradiation (up to 2000 rad) of the titrated responder cell population diminishes the estimated frequencies by less than 50%. The ability to detect IL 2 levels in cultures containing only a single, nonproliferating T lymphocyte allows us to estimate the amount of IL 2 generated by an individual effector cell during a 3-day culture interval after mitogen stimulation. The average responding, irradiated T cell generates 0.92 pg of IL 2 (median) within 3 days. The method presented provides a straightforward way to provide independent estimates of responding cell number and of lymphokine production per cell in a variety of clinical situations.

  20. Differential effects of p53 on bystander phenotypes induced by gamma ray and high LET heavy ion radiation.

    PubMed

    He, Mingyuan; Dong, Chen; Konishi, Teruaki; Tu, Wenzhi; Liu, Weili; Shiomi, Naoko; Kobayashi, Alisa; Uchihori, Yukio; Furusawa, Yoshiya; Hei, Tom K; Dang, Bingrong; Shao, Chunlin

    2014-04-01

    High LET particle irradiation has several potential advantages over γ-rays such as p53-independent response. The purpose of this work is to disclose the effect of p53 on the bystander effect induced by different LET irradiations and underlying mechanism. Lymphocyte cells of TK6 (wild type p53) and HMy2.CIR (mutated p53) were exposed to either low or high LET irradiation, then their mitochondrial dysfunction and ROS generation were detected. The micronuclei (MN) induction in HL-7702 hepatocytes co-cultured with irradiated lymphocytes was also measured. It was found that the mitochondrial dysfunction, p66(Shc) activation, and intracellular ROS were enhanced in TK6 but not in HMy2.CIR cells after γ-ray irradiation, but all of them were increased in both cell lines after carbon and iron irradiation. Consistently, the bystander effect of MN formation in HL-7702 cells was only triggered by γ-irradiated TK6 cells but not by γ-irradiated HMy2.CIR cells. But this bystander effect was induced by both lymphocyte cell lines after heavy ion irradiation. PFT-μ, an inhibitor of p53, only partly inhibited ROS generation and bystander effect induced by 30 keV/μm carbon-irradiated TK6 cells but failed to suppress the bystander effect induced by the TK6 cells irradiated with either 70 keV/μm carbon or 180 keV/μm iron. The mitochondrial inhibitors of rotenone and oligomycin eliminated heavy ion induced ROS generation in TK6 and HMy2.CIR cells and hence diminished the bystander effect on HL-7702 cells. These results clearly demonstrate that the bystander effect is p53-dependent for low LET irradiation, but it is p53-independent for high LET irradiation which may be because of p53-independent ROS generation due to mitochondrial dysfunction.

  1. Differential effects of p53 on bystander phenotypes induced by gamma ray and high LET heavy ion radiation

    NASA Astrophysics Data System (ADS)

    He, Mingyuan; Dong, Chen; Konishi, Teruaki; Tu, Wenzhi; Liu, Weili; Shiomi, Naoko; Kobayashi, Alisa; Uchihori, Yukio; Furusawa, Yoshiya; Hei, Tom K.; Dang, Bingrong; Shao, Chunlin

    2014-04-01

    High LET particle irradiation has several potential advantages over γ-rays such as p53-independent response. The purpose of this work is to disclose the effect of p53 on the bystander effect induced by different LET irradiations and underlying mechanism. Lymphocyte cells of TK6 (wild type p53) and HMy2.CIR (mutated p53) were exposed to either low or high LET irradiation, then their mitochondrial dysfunction and ROS generation were detected. The micronuclei (MN) induction in HL-7702 hepatocytes co-cultured with irradiated lymphocytes was also measured. It was found that the mitochondrial dysfunction, p66Shc activation, and intracellular ROS were enhanced in TK6 but not in HMy2.CIR cells after γ-ray irradiation, but all of them were increased in both cell lines after carbon and iron irradiation. Consistently, the bystander effect of MN formation in HL-7702 cells was only triggered by γ-irradiated TK6 cells but not by γ-irradiated HMy2.CIR cells. But this bystander effect was induced by both lymphocyte cell lines after heavy ion irradiation. PFT-μ, an inhibitor of p53, only partly inhibited ROS generation and bystander effect induced by 30 keV/μm carbon-irradiated TK6 cells but failed to suppress the bystander effect induced by the TK6 cells irradiated with either 70 keV/μm carbon or 180 keV/μm iron. The mitochondrial inhibitors of rotenone and oligomycin eliminated heavy ion induced ROS generation in TK6 and HMy2.CIR cells and hence diminished the bystander effect on HL-7702 cells. These results clearly demonstrate that the bystander effect is p53-dependent for low LET irradiation, but it is p53-independent for high LET irradiation which may be because of p53-independent ROS generation due to mitochondrial dysfunction.

  2. Development of a dihydroartemisinin-resistant Molt-4 leukemia cell line.

    PubMed

    Park, Jungsoo; Lai, Henry C; Singh, Mallika; Sasaki, Tomikazu; Singh, Narendra P

    2014-06-01

    Artemisinin generates cytotoxic free radicals when it reacts with iron. Its toxicity is more selective toward cancer cells because cancer cells contain a higher level of intracellular-free iron. We previously reported that dihydroartemisinin (DHA), an active metabolite of artemisinin, has selective cytotoxicity toward Molt-4 human lymphoblastoid cells. A concern is whether cancer cells could develop resistance to DHA after repeated administration, thus limiting its therapeutic efficacy. In the present study, we developed a DHA-resistant Molt-4 cell line (RTN) by exposing Molt-4 cells to gradually increasing concentrations of DHA in vitro. The half-maximal inhibitory concentration (IC50) of DHA for RTN cells is 7.1-times higher than that of Molt-4 cells. RTN cells have a higher growth rate than Molt-4 cells. In addition, we investigated the toxicities of two more potent synthetic artemisinin compounds, artemisinin dimer-alcohol and artemisinin-tagged holotransferrin toward RTN cells; RTN cells showed no significant cross-resistance to these compounds.

  3. The role of urinary pH in o-phenylphenol-induced cytotoxicity and chromosomal damage in the bladders of F344 rats.

    PubMed

    Balakrishnan, S; Hasegawa, L; Eastmond, D A

    2016-04-01

    o-Phenylphenol (OPP) is a widely used fungicide and antibacterial agent that at high doses has been shown to cause bladder cancer in male F344 rats. The mechanisms underlying OPP-induced bladder carcinogenicity remain unclear but it has been proposed that a non-enzymatic pH-dependent autoxidation of phenylhydroquinone (PHQ), a primary metabolite of OPP, may be a key step in OPP-induced rat bladder carcinogenesis. To investigate this mechanism and to provide insights into the potential human health relevance of OPP-induced cancer, a series of in vitro and in vivo experiments were conducted. In human lymphoblastoid TK-6 cells and rat bladder epithelial NBT-II cells, strong increases in cytotoxicity were seen at a constant concentration of PHQ by increasing the buffer pH as well as by increasing concentrations of PHQ at a constant pH. In in vivo studies, male rats were administered OPP (4,000 and 8,000 ppm) in a diet supplemented with either 1% ammonium chloride or 3% sodium bicarbonate to produce acidic and alkaline urinary pH, respectively. Significant increases in cell proliferation as detected by 5-bromo-2'-deoxyuridine incorporation and micronucleus formation were seen in the bladder cells of OPP-treated rats with neutral or alkaline urinary pH but not in animals with the acidified urine. The results from these in vitro and in vivo studies provide support for the autoxidation hypothesis of bioactivation, and provide additional evidence that urinary pH can significantly influence the genotoxicity and carcinogenicity of this important agent.

  4. Transplantability of human lymphoid cell line, lymphoma, and leukemia in splenectomized and/or irradiated nude mice

    SciTech Connect

    Watanabe, S.; Shimosato, Y.; Kuroki, M.; Sato, Y.; Nakajima, T.

    1980-07-01

    The effects of splenectomy and/or whole-body irradiation of nude mice before xenotransplantation of lymphoid cell lines, lymphoma, and leukemia were studied. Transplantation after whole-body irradiation resulted in the increased ''take'' rate of three cultured cell lines (two of T-cell-derived acute lymphocytic leukemia and one of B-cell derived acute lymphocytic leukemia) and in the tumorous growth of Burkitt-derived Raji and spontaneously transformed lymphoblastoid cell lines. With splenectomy plus irradiation as a pretreatment, tumorous growth occurred in four other cell lines which were not transplantable after irradiation only (two cell lines of Epstein-Barr virus-transformed cord blood cells and one each of null acute lymphocytic leukemia and nodular lymphoma-derived cell lines). Direct transplantation of leukemia and lymphoma cells into the pretreated mice was successful in 7 of 24 cases (29%). B-cell-derived diffuse large lymphoid lymphoma was transplantable in three of seven cases (43%). However, lymphoma and leukemia of peripheral T-cell origin was difficult to transplant even with pretreatment, and only one pleomorphic T-cell lymphoma grew to a significant size (2 cm). One tumor each of B-cell-derived diffuse large lymphoid and T-cell diffuse lymphoblastic lymphoma became transplantable.

  5. Generation of EBV-specific T cells for adoptive immunotherapy: a novel protocol using formalin-fixed stimulator cells to increase biosafety.

    PubMed

    Hammer, Markus H; Brestrich, Gordon; Mittenzweig, Alexa; Roemhild, Andy; Zwinger, Sandra; Subklewe, Marion; Beier, Carola; Kurtz, Andreas; Babel, Nina; Volk, Hans-Dieter; Reinke, Petra

    2007-01-01

    Adoptive immunotherapy with in vitro generated Epstein-Barr virus (EBV)-specific T cells is a safe and effective treatment in patients with EBV-related complications after transplantation. More frequent use of EBV-specific T cells is held back by their cost and time-intensive generation under good manufacturing practice (GMP) conditions. Currently, EBV-specific T cells are produced by repetitive stimulation of peripheral blood mononuclear cells with EBV-infected lymphoblastoid cell lines (LCLs), a protocol that requires several open GMP-handling steps. The aim of the present study was to improve T-cell generation under GMP conditions. We introduce a novel generation protocol that replaces repetitive with short-term LCL stimulation of PMBCs. Vital and formalin-fixed LCLs were used to further increase biosafety. Stimulated T cells were selected by the clinically approved cytokine secretion assay followed by nonspecific expansion. Sufficient numbers of EBV-specific T-cell lines were generated with all protocols. Specific recognition and killing of EBV-infected targets was found and was independent of the generation protocol applied. The novel protocol based on formalin-fixed cells, selection, and expansion reduced open GMP-handling steps and increased biosafety. Furthermore, fixation will allow the use of transgenic LCLs (eg, with cytomegalovirus or tumor antigens) and thereby facilitate the generation of antigen-specific T cells directed against pathogens other than EBV.

  6. Caffeic Acid Phenylethyl Ester and MG-132 Have Apoptotic and Antiproliferative Effects on Leukemic Cells But Not on Normal Mononuclear Cells12

    PubMed Central

    Cavaliere, Victoria; Papademetrio, Daniela L; Lorenzetti, Mario; Valva, Pamela; Preciado, María Victoria; Gargallo, Patricia; Larripa, Irene; Monreal, Mariela B; Pardo, María Laura; Hajos, Silvia E; Blanco, Guillermo AC; Álvarez, Élida MC

    2009-01-01

    Chemotherapy aims to limit proliferation and induce apoptotic cell death in tumor cells. Owing to blockade of signaling pathways involved in cell survival and proliferation, nuclear factor κB (NF-κB) inhibitors can induce apoptosis in a number of hematological malignancies. The efficacy of conventional chemotherapeutic drugs, such as vincristine (VCR) and doxorubicine (DOX), may be enhanced with combined therapy based on NF-κB modulation. In this study, we evaluated the effect of caffeic acid phenylethyl ester (CAPE) and MG-132, two nonspecific NF-κB inhibitors, and conventional chemotherapeutics drugs DOX and VCR on cell proliferation and apoptosis induction on a lymphoblastoid B-cell line, PL104, established and characterized in our laboratory. CAPE and MG-132 treatment showed a strong antiproliferative effect accompanied by clear cell cycle deregulation and apoptosis induction. Doxorubicine and VCR showed antiproliferative effects similar to those of CAPE and MG-132, although the latter drugs showed an apoptotic rate two-fold higher than DOX and VCR. None of the four compounds showed cytotoxic effect on peripheral mononuclear cells from healthy volunteers. CAPE- and MG-132-treated bone marrow cells from patients with myeloid and lymphoid leukemias showed 69% (P < .001) and 25% decrease (P < .01) in cell proliferation and 42% and 34% (P < .01) apoptosis induction, respectively. Overall, our results indicate that CAPE and MG-132 had a strong and selective apoptotic effect on tumor cells that may be useful in future treatment of hematological neoplasias. PMID:19252751

  7. Radiation sensitivity of Merkel cell carcinoma cell lines

    SciTech Connect

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

    1995-07-30

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

  8. A Metabolic Biofuel Cell: Conversion of Human Leukocyte Metabolic Activity to Electrical Currents

    PubMed Central

    2011-01-01

    An investigation of the electrochemical activity of human white blood cells (WBC) for biofuel cell (BFC) applications is described. WBCs isolated from whole human blood were suspended in PBS and introduced into the anode compartment of a proton exchange membrane (PEM) fuel cell. The cathode compartment contained a 50 mM potassium ferricyanide solution. Average current densities between 0.9 and 1.6 μA cm-2 and open circuit potentials (Voc) between 83 and 102 mV were obtained, which were both higher than control values. Cyclic voltammetry was used to investigate the electrochemical activity of the activated WBCs in an attempt to elucidate the mechanism of electron transfer between the cells and electrode. Voltammograms were obtained for the WBCs, including peripheral blood mononuclear cells (PBMCs - a lymphocyte-monocyte mixture isolated on a Ficoll gradient), a B lymphoblastoid cell line (BLCL), and two leukemia cell lines, namely K562 and Jurkat. An oxidation peak at about 363 mV vs. SCE for the PMA (phorbol ester) activated primary cells, with a notable absence of a reduction peak was observed. Oxidation peaks were not observed for the BLCL, K562 or Jurkat cell lines. HPLC confirmed the release of serotonin (5-HT) from the PMA activated primary cells. It is believed that serotonin, among other biochemical species released by the activated cells, contributes to the observed BFC currents. PMID:21569243

  9. Identification of a Cell Surface Protein, p97, in Human Melanomas and Certain Other Neoplasms

    NASA Astrophysics Data System (ADS)

    Woodbury, Richard G.; Brown, Joseph P.; Yeh, Ming-Yang; Hellstrom, Ingegerd; Hellstrom, Karl Erik

    1980-04-01

    BALB/c mice were immunized with a human melanoma cell line, SK-MEL 28, and their spleen cells were fused with mouse NS-1 myeloma cells. Hybrid cells were tested in an indirect 125I-labeled protein A assay for production of antibodies that bound to surface antigens of SK-MEL 28 melanoma cells but not to autologous skin fibroblasts. One hybridoma, designated 4.1, had the required specificity. It was cloned and grown in mice as an ascites tumor. The monoclonal IgG1 antibody produced by the hybridoma was purified from the ascites fluid and labeled with 125I. The labeled antibody bound, at significant levels, to approximately 90% of the melanomas tested and to approximately 55% of other tumor cells, but not to three B-lymphoblastoid cell lines or to cultivated fibroblasts from 15 donors. Immunoprecipitation and sodium dodecyl sulfate gel electrophoresis were used to detect the target antigen in 125I-labeled cell membranes of both cultivated cells and tumor biopsy samples. A protein with a molecular weight of 97,000 was identified. This protein, designated p97, was present in both cultured cells and biopsy material from melanomas and certain other tumors, but it was not detected in eight different samples of normal adult epithelial or mesenchymal tissues obtained from five donors.

  10. {sub p}53-Dependent Adaptive Responses in Human Cells Exposed to Space Radiations

    SciTech Connect

    Takahashi, Akihisa; Su Xiaoming; Suzuki, Hiromi; Omori, Katsunori; Seki, Masaya; Hashizume, Toko; Shimazu, Toru; Ishioka, Noriaki; Iwasaki, Toshiyasu; Ohnishi, Takeo

    2010-11-15

    Purpose: It has been reported that priming irradiation or conditioning irradiation with a low dose of X-rays in the range of 0.02-0.1 Gy induces a p53-dependent adaptive response in mammalian cells. The aim of the present study was to clarify the effect of space radiations on the adaptive response. Methods and Materials: Two human lymphoblastoid cell lines were used; one cell line bears a wild-type p53 (wtp53) gene, and another cell line bears a mutated p53 (mp53) gene. The cells were frozen during transportation on the space shuttle and while in orbit in the International Space Station freezer for 133 days between November 15, 2008 and March 29, 2009. After the frozen samples were returned to Earth, the cells were cultured for 6 h and then exposed to a challenging X-ray-irradiation (2 Gy). Cellular sensitivity, apoptosis, and chromosome aberrations were scored using dye-exclusion assays, Hoechst33342 staining assays, and chromosomal banding techniques, respectively. Results: In cells exposed to space radiations, adaptive responses such as the induction of radioresistance and the depression of radiation-induced apoptosis and chromosome aberrations were observed in wtp53 cells but not in mp53 cells. Conclusion: These results have confirmed the hypothesis that p53-dependent adaptive responses are apparently induced by space radiations within a specific range of low doses. The cells exhibited this effect owing to space radiations exposure, even though the doses in space were very low.

  11. Comparison of toxicogenomics and traditional approaches to inform mode of action and points of departure in human health risk assessment of benzo[a]pyrene in drinking water

    PubMed Central

    Labib, Sarah; Bourdon-Lacombe, Julie; Kuo, Byron; Buick, Julie K.; Lemieux, France; Williams, Andrew; Halappanavar, Sabina; Malik, Amal; Luijten, Mirjam; Aubrecht, Jiri; Hyduke, Daniel R.; Fornace, Albert J.; Swartz, Carol D.; Recio, Leslie; Yauk, Carole L.

    2015-01-01

    Toxicogenomics is proposed to be a useful tool in human health risk assessment. However, a systematic comparison of traditional risk assessment approaches with those applying toxicogenomics has never been done. We conducted a case study to evaluate the utility of toxicogenomics in the risk assessment of benzo[a]pyrene (BaP), a well-studied carcinogen, for drinking water exposures. Our study was intended to compare methodologies, not to evaluate drinking water safety. We compared traditional (RA1), genomics-informed (RA2) and genomics-only (RA3) approaches. RA2 and RA3 applied toxicogenomics data from human cell cultures and mice exposed to BaP to determine if these data could provide insight into BaP's mode of action (MOA) and derive tissue-specific points of departure (POD). Our global gene expression analysis supported that BaP is genotoxic in mice and allowed the development of a detailed MOA. Toxicogenomics analysis in human lymphoblastoid TK6 cells demonstrated a high degree of consistency in perturbed pathways with animal tissues. Quantitatively, the PODs for traditional and transcriptional approaches were similar (liver 1.2 vs. 1.0 mg/kg-bw/day; lung 0.8 vs. 3.7 mg/kg-bw/day; forestomach 0.5 vs. 7.4 mg/kg-bw/day). RA3, which applied toxicogenomics in the absence of apical toxicology data, demonstrates that this approach provides useful information in data-poor situations. Overall, our study supports the use of toxicogenomics as a relatively fast and cost-effective tool for hazard identification, preliminary evaluation of potential carcinogens, and carcinogenic potency, in addition to identifying current limitations and practical questions for future work. PMID:25605026

  12. Comparison of toxicogenomics and traditional approaches to inform mode of action and points of departure in human health risk assessment of benzo[a]pyrene in drinking water.

    PubMed

    Moffat, Ivy; Chepelev, Nikolai L; Labib, Sarah; Bourdon-Lacombe, Julie; Kuo, Byron; Buick, Julie K; Lemieux, France; Williams, Andrew; Halappanavar, Sabina; Malik, Amal I; Luijten, Mirjam; Aubrecht, Jiri; Hyduke, Daniel R; Fornace, Albert J; Swartz, Carol D; Recio, Leslie; Yauk, Carole L

    2015-01-01

    Toxicogenomics is proposed to be a useful tool in human health risk assessment. However, a systematic comparison of traditional risk assessment approaches with those applying toxicogenomics has never been done. We conducted a case study to evaluate the utility of toxicogenomics in the risk assessment of benzo[a]pyrene (BaP), a well-studied carcinogen, for drinking water exposures. Our study was intended to compare methodologies, not to evaluate drinking water safety. We compared traditional (RA1), genomics-informed (RA2) and genomics-only (RA3) approaches. RA2 and RA3 applied toxicogenomics data from human cell cultures and mice exposed to BaP to determine if these data could provide insight into BaP's mode of action (MOA) and derive tissue-specific points of departure (POD). Our global gene expression analysis supported that BaP is genotoxic in mice and allowed the development of a detailed MOA. Toxicogenomics analysis in human lymphoblastoid TK6 cells demonstrated a high degree of consistency in perturbed pathways with animal tissues. Quantitatively, the PODs for traditional and transcriptional approaches were similar (liver 1.2 vs. 1.0 mg/kg-bw/day; lungs 0.8 vs. 3.7 mg/kg-bw/day; forestomach 0.5 vs. 7.4 mg/kg-bw/day). RA3, which applied toxicogenomics in the absence of apical toxicology data, demonstrates that this approach provides useful information in data-poor situations. Overall, our study supports the use of toxicogenomics as a relatively fast and cost-effective tool for hazard identification, preliminary evaluation of potential carcinogens, and carcinogenic potency, in addition to identifying current limitations and practical questions for future work.

  13. Heparanase regulates secretion, composition, and function of tumor cell-derived exosomes.

    PubMed

    Thompson, Camilla A; Purushothaman, Anurag; Ramani, Vishnu C; Vlodavsky, Israel; Sanderson, Ralph D

    2013-04-05

    Emerging evidence indicates that exosomes play a key role in tumor-host cross-talk and that exosome secretion, composition, and functional capacity are altered as tumors progress to an aggressive phenotype. However, little is known regarding the mechanisms that regulate these changes. Heparanase is an enzyme whose expression is up-regulated as tumors become more aggressive and is associated with enhanced tumor growth, angiogenesis, and metastasis. We have discovered that in human cancer cells (myeloma, lymphoblastoid, and breast cancer), when expression of heparanase is enhanced or when tumor cells are exposed to exogenous heparanase, exosome secretion is dramatically increased. Heparanase enzyme activity is required for robust enhancement of exosome secretion because enzymatically inactive forms of heparanase, even when present in high amounts, do not dramatically increase exosome secretion. Heparanase also impacts exosome protein cargo as reflected by higher levels of syndecan-1, VEGF, and hepatocyte growth factor in exosomes secreted by heparanase-high expressing cells as compared with heparanase-low expressing cells. In functional assays, exosomes from heparanase-high cells stimulated spreading of tumor cells on fibronectin and invasion of endothelial cells through extracellular matrix better than did exosomes secreted by heparanase-low cells. These studies reveal that heparanase helps drive exosome secretion, alters exosome composition, and facilitates production of exosomes that impact both tumor and host cell behavior, thereby promoting tumor progression.

  14. Gene Transfer from Targeted Liposomes to Specific Lymphoid Cells by Electroporation

    NASA Astrophysics Data System (ADS)

    Machy, Patrick; Lewis, Florence; McMillan, Lynette; Jonak, Zdenka L.

    1988-11-01

    Large unilamellar liposomes, coated with protein A and encapsulating the gene that confers resistance to mycophenolic acid, were used as a model system to demonstrate gene transfer into specific lymphoid cells. Protein A, which selectively recognizes mouse IgG2a antibodies, was coupled to liposomes to target them specifically to defined cell types coated with IgG2a antibody. Protein A-coated liposomes bound human B lymphoblastoid cells preincubated with a mouse IgG2a anti-HLA monoclonal antibody but failed to adhere to cells challenged with an irrelevant (anti-H-2) antibody of the same isotype or to cells incubated in the absence of antibody. Transfection of target cells bound to protein A-coated liposomes was achieved by electroporation. This step was essential since only electroporated cells survived in a selective medium containing mycophenolic acid. Transfection efficiency with electroporation and targeted liposomes was as efficient as conventional procedures that used unencapsulated plasmids free in solution but, in the latter case, cell selectivity is not possible. This technique provides a methodology for introducing defined biological macromolecules into specific cell types.

  15. Discovery of novel hematopoietic cell adhesion molecules from human bone marrow stromal cell membrane protein extracts by a new cell-blotting technique.

    PubMed

    Seshi, B

    1994-05-01

    In an attempt to define the role of cell adhesion molecules (CAMs) within the bone marrow (BM) microenvironment in normal hematopoiesis and in leukemia development, a novel cell-blotting technique that involved cell adhesion to protein bands after separation by lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) and blotting onto polyvinylidene difluoride (PVDF) membrane has been developed. Human BM stromal cell membrane fractions have been prepared from Dexter-type cultures after cell lysis by sonification and differential centrifugations of the sonification contents. The 20,000 g pellets representing membrane fractions have been solubilized by 2% Triton X-100, 0.575% LDS, and 8 mol/L urea in sequential order. The protein extracts are fractionated by LDS-PAGE and screened for CAMs by the new cell-blotting technique. This led to identification of nine protein bands in lanes containing LDS extracts showing adhesion of KG1a (CD34+ progenitor myeloid) cells. Evidence that the BM proteins exhibiting KG1a cell adhesion are novel CAMs is based on the observations that these proteins, in comparison with known CAMs, specifically VCAM-1, CD54, and CD44, show (1) contrasting detergent-solubility properties, (2) different temperature requirement for mediating cell adhesion function, and (3) markedly distinct electrophoretic mobilities. The various cell types tested, notably KG1a, NALM-6, WIL-2, Ramos, HS-Sultan, K562, JY B lymphoblastoid cells, and T lymphoblasts, showed distinctive patterns of binding to different subsets of BM CAMs. These results demonstrate a new approach to studies of molecular mechanisms that may determine specificity of hematopoietic cellular localization within BM microenvironment and may play an important role in controlling hematopoiesis.

  16. Two classes of single-stranded regions evident in deproteinized preparations of replicating DNA isolated from mammalian cells

    SciTech Connect

    Stewart, B.W.; Kavallaris, M.; Catchpoole, D.; Norris, M.D. )

    1991-02-01

    In DNA isolated from proliferating human lymphoblastoid CCRF-CEM cells which had been pulse-labeled by exposure to (3H)thymidine for periods from 30 s to 10 min, single-stranded regions were analyzed by caffeine-gradient elution from benzoylated DEAE-cellulose. Two classes of structural defect were evident. Some replicating DNA exhibited single-stranded regions of approximately 200 nucleotides, while most newly incorporated radioactivity was associated with DNA containing single-stranded regions from 900 to approximately 4000 nucleotides. The distribution of thymidine-derived radioactivity did not suggest sequential or preferential labeling of these DNA fractions as the incorporation time was varied. The findings may be correlated with recent proposals regarding the structural basis of eukaryotic DNA replication.

  17. Major histocompatibility complex-unrestricted cytolytic activity of human T cells: analysis of precursor frequency and effector phenotype

    SciTech Connect

    Patel, S.S.; Thiele, D.L.; Lipsky, P.E.

    1987-12-01

    The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. All of the 198 clones generated by this method were T cells (CD2/sup +/, CD3/sup +/, CD4/sup +/ or CD2/sup +/, CD3/sup +/, CD8/sup +/) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis, measured by /sup 51/Cr release, was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur of K562 was not mediated by a soluble factor secreted by the clones. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD 16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype.

  18. Thymoquinone efficiently inhibits the survival of EBV-infected B cells and alters EBV gene expression.

    PubMed

    Zihlif, Malek A; Mahmoud, Ismail S; Ghanim, Majd T; Zreikat, Manar S; Alrabadi, Nasr; Imraish, Amer; Odeh, Fadwa; Abbas, Manal A; Ismail, Said I

    2013-05-01

    Epstein--Barr virus (EBV) is a human virus with oncogenic potentials that is implicated in various human diseases and malignancies. In this study, the modulator activity of the potent herbal extract drug thymoquinone on EBV was assessed in vitro. Thymoquinone was tested for cytotoxicity on human cells of lymphoblastoid cells, Raji Burkitt's lymphoma, DG-75 Burkitt's lymphoma, peripheral blood mononuclear cells, and periodontal ligament fibroblast. Apoptosis induction was analyzed via TUNEL assay and activity studies of caspase-3. The effect of thymoquinone on EBV gene expression was determined using real-time polymerase chain reaction. We report here, for the first time, a promising selective inhibitory affect of thymoquinone on EBV-infected B cell lines in vitro, compared with lower activity on EBV negative B cell line and very low toxicity on human peripheral blood mononuclear cells and periodontal ligament fibroblasts. Moreover, the drug was found to efficiently suppress the RNA expression of EBNA2, LMP1, and EBNA1 genes. Specifically, EBNA2 expression levels were the most affected indicating that this gene might have a major contribution to thymoquinone potency against EBV infected cells. Overall, our results suggest that thymoquinone has the potential to suppress the growth of EBV-infected B cells efficiently.

  19. Epstein-Barr virus-carrying B cells are large, surface IgM, IgD-bearing cells in normal individuals and acute malaria patients.

    PubMed Central

    Lam, K M; Whittle, H; Grzywacz, M; Crawford, D H

    1994-01-01

    In this study the presence of Epstein-Barr virus (EBV) carrying B lymphocytes in different B-cell subpopulations from peripheral blood was determined by spontaneous outgrowth which gives rise to lymphoblastoid cell lines. In healthy seropositive adults, the EBV-carrying B cell was predominantly within the IgM- and IgD-positive but not the IgG-positive subpopulations. Furthermore, these B lymphocytes were in the low-density (large cell) Percoll fraction. The IgM- and IgD-positive B cell phenotype suggests the EBV-carrying B cells to be circulating virgin B cells recently released from the bone marrow. These B cells have an estimated life span of only 6-8 weeks suggesting that long-term EBV persistence in the body may be the result of infection of a more primitive B-cell type. Similar experiments were carried out in children with acute malaria from the Gambia, West Africa, where Burkitt lymphoma (BL) is endemic in order to determine whether a population of EBV-carrying B cells could be identified which had a similar phenotype to the BL cell. The EBV-carrying B cells in this patient group were also found in the IgM-positive, IgG-negative B-cell subpopulation. The majority of these cells were found in the low-density (large cell) Percoll fraction although in some patients a proportion was derived from the high-density (small cell) fraction. This cellular phenotype is not representative of a BL cell. PMID:7959872

  20. Detection and quantification of 4-ABP adducts in DNA from bladder cancer patients.

    PubMed

    Zayas, Beatriz; Stillwell, Sara W; Wishnok, John S; Trudel, Laura J; Skipper, Paul; Yu, Mimi C; Tannenbaum, Steven R; Wogan, Gerald N

    2007-02-01

    We analyzed bladder DNA from 27 cancer patients for dG-C8-4-aminobiphenyl (dG-C8-ABP) adducts using the liquid chromatography tandem mass spectrometry method with a 700 attomol (1 adduct in 10(9) bases) detection limit. Hemoglobin (Hb) 4-aminobiphenyl (4-ABP) adduct levels were measured by gas chromatography-mass spectrometry. After isolation of dG-C8-ABP by immunoaffinity chromatography and further purification, deuterated (d9) dG-C8-ABP (MW=443 Da) was added to each sample. Structural evidence and adduct quantification were determined by selected reaction monitoring, based on the expected adduct ion [M+H+]+1, at m/z 435 with fragmentation to the product ion at m/z 319, and monitoring of the transition for the internal standard, m/z 444-->328. The method was validated by analysis of DNA (100 microg each) from calf thymus; livers from ABP-treated and untreated rats; human placentas; and TK6 lymphoblastoid cells. Adduct was detected at femtomol levels in DNA from livers of ABP-treated rats and calf thymus, but not in other controls. The method was applied to 41 DNA samples (200 microg each) from 27 human bladders; 28 from tumor and 14 from surrounding non-tumor tissue. Of 27 tissues analyzed, 44% (12) contained 5-80 dG-C8-ABP adducts per 10(9) bases; only 1 out of 27 (4%) contained adduct in both tumor and surrounding tissues. The Hb adduct was detected in samples from all patients, at levels of 12-1960 pg per gram Hb. There was no correlation between levels of DNA and Hb adducts. The presence of DNA adducts in 44% of the subjects and high levels of Hb adducts in these non-smokers indicate environmental sources of exposure to 4-ABP.

  1. High expression of the CC chemokine TARC in Reed-Sternberg cells. A possible explanation for the characteristic T-cell infiltratein Hodgkin's lymphoma.

    PubMed

    van den Berg, A; Visser, L; Poppema, S

    1999-06-01

    Hodgkin's lymphoma is characterized by the combination of Reed-Sternberg (R-S) cells and a prominent inflammatory cell infiltrate. One of the intriguing questions regarding this disease is what is causing the influx of T lymphocytes into the involved tissues. We applied the serial analysis of gene expression (SAGE) technique on the Hodgkin's lymphoma-derived cell line L428 and on an Epstein-Barr virus (EBV)-transformed lymphoblastoid B-cell line. A frequently expressed tag in L428 corresponded to the T-cell-directed CC chemokine TARC. Reverse transcription polymerase chain reaction analyses demonstrated expression of TARC in nodular sclerosis (NS) and mixed cellularity (MC) classical Hodgkin's lymphomas but not in NLP Hodgkin's lymphoma, anaplastic large-cell lymphomas, and large-B-cell lymphomas with CD30 positivity. Two of five cases of T-cell-rich B-cell lymphoma (TCRBCL) were TARC positive. RNA in situ hybridization (ISH) showed a strong signal for TARC in the cytoplasm of R-S cells, and immunohistochemical staining confirmed the presence of the TARC protein in the R-S cells of NS and MC Hodgkin's lymphomas. The lymphocytic and histiocytic (L&H)-type cells of nodular lymphocyte predominance Hodgkin's lymphoma and the neoplastic cells of non-Hodgkin's lymphomas with the exception of two cases of TCRBCL did not stain for TARC. TARC is known to bind to the CCR4 receptor, which is expressed on activated Th2 lymphocytes. The immunophenotype of lymphocytes surrounding R-S cells is indeed Th2-like, and by RNA ISH these lymphocytes showed a positive signal for the chemokine receptor CCR4. The findings suggest that production of TARC by the R-S cells may explain the characteristic T-cell infiltrate in classical Hodgkin's lymphoma.

  2. Defective T-cell control of Epstein–Barr virus infection in multiple sclerosis

    PubMed Central

    Pender, Michael P; Csurhes, Peter A; Burrows, Jacqueline M; Burrows, Scott R

    2017-01-01

    Mounting evidence indicates that infection with Epstein–Barr virus (EBV) has a major role in the pathogenesis of multiple sclerosis (MS). Defective elimination of EBV-infected B cells by CD8+ T cells might cause MS by allowing EBV-infected autoreactive B cells to accumulate in the brain. Here we undertake a comprehensive analysis of the T-cell response to EBV in MS, using flow cytometry and intracellular IFN-γ staining to measure T-cell responses to EBV-infected autologous lymphoblastoid cell lines and pools of human leukocyte antigen (HLA)-class-I-restricted peptides from EBV lytic or latent proteins and cytomegalovirus (CMV), in 95 patients and 56 EBV-seropositive healthy subjects. In 20 HLA-A2+ healthy subjects and 20 HLA-A2+ patients we also analysed CD8+ T cells specific for individual peptides, measured by binding to HLA-peptide complexes and production of IFN-γ, TNF-α and IL-2. We found a decreased CD8+ T-cell response to EBV lytic, but not CMV lytic, antigens at the onset of MS and at all subsequent disease stages. CD8+ T cells directed against EBV latent antigens were increased but had reduced cytokine polyfunctionality indicating T-cell exhaustion. During attacks the EBV-specific CD4+ and CD8+ T-cell populations expanded, with increased functionality of latent-specific CD8+ T cells. With increasing disease duration, EBV-specific CD4+ and CD8+ T cells progressively declined, consistent with T-cell exhaustion. The anti-EBNA1 IgG titre correlated inversely with the EBV-specific CD8+ T-cell frequency. We postulate that defective CD8+ T-cell control of EBV reactivation leads to an expanded population of latently infected cells, including autoreactive B cells. PMID:28197337

  3. Theileria-infected cell line from an African buffalo (Syncerus caffer).

    PubMed

    Zweygarth, Erich; Koekemoer, Otto; Josemans, Antoinette I; Rambritch, Natasha; Pienaar, Ronel; Putterill, John; Latif, Abdalla; Potgieter, Fred T

    2009-08-01

    Mononuclear cells were isolated from the peripheral blood of a buffalo infected with a Theileria sp. using density gradient centrifugation, and the cells were put into culture flasks covered by a monolayer of bovine endothelial cells. Twenty days after culture initiation, cells containing macroschizonts were detected in Giemsa-stained smears. The first subculture was carried out on day 45 of culture propagation. Subsequently, infected cells were subcultured twice a week, and each time 1 to 2 x 10(6) per milliliter cells were harvested. DNA was extracted from culture material and a partial polymerase chain reaction amplification of the 18S ribosomal RNA (rRNA) gene was carried out using Theileria genus-specific primers. Sequence data and phylogenetic analysis using the 18S rRNA gene indicated a close relationship to Theileria sp. buffalo, previously described in literature. Here, the first successful attempt to establish a macroschizont-infected lymphoblastoid cell line of Theileria sp. (buffalo) from an African buffalo is described.

  4. Epstein-Barr virus-positive T/NK-cell lymphoproliferative disorders.

    PubMed

    Cai, Qingqing; Chen, Kailin; Young, Ken H

    2015-01-23

    Epstein-Barr virus, a ubiquitous human herpesvirus, can induce both lytic and latent infections that result in a variety of human diseases, including lymphoproliferative disorders. The oncogenic potential of Epstein-Barr virus is related to its ability to infect and transform B lymphocytes into continuously proliferating lymphoblastoid cells. However, Epstein-Barr virus has also been implicated in the development of T/natural killer cell lymphoproliferative diseases. Epstein-Barr virus encodes a series of products that mimic several growth, transcription and anti-apoptotic factors, thus usurping control of pathways that regulate diverse homeostatic cellular functions and the microenvironment. However, the exact mechanism by which Epstein-Barr virus promotes oncogenesis and inflammatory lesion development remains unclear. Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases often have overlapping clinical symptoms as well as histologic and immunophenotypic features because both lymphoid cell types derive from a common precursor. Accurate classification of Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases is a prerequisite for appropriate clinical management. Currently, the treatment of most T/natural killer cell lymphoproliferative diseases is less than satisfactory. Novel and targeted therapies are strongly required to satisfy clinical demands. This review describes our current knowledge of the genetics, oncogenesis, biology, diagnosis and treatment of Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases.

  5. Enkephalins stimulate leukemia cell migration and surface expression of CD9.

    PubMed Central

    Heagy, W; Duca, K; Finberg, R W

    1995-01-01

    Opioid peptides have been implicated in the regulation of tumor growth and biology; however, little attention has been given to the mechanisms that are involved. In this study we show that physiological concentrations of the endogenous opioid neuropeptide methionine-enkephalin (MET-ENK) and the synthetic enkephalins D-Ala2, Me-Phe4, Gly(ol)5 and D-Ala2, D-Leu5 are stimulants for the in vitro migration of pre-B acute lymphoblastoid leukemia (ALL) cells. Activation of the human pre-B ALL cell lines NALM 6 and LAZ 221 with MET-ENK resulted in both an increase in their migration and an augmentation in the surface expression of the leukemia cell marker CD9. The opiate receptor antagonist naloxone reversed these enkephalin-induced effects on the leukemia cells. When the pre-B ALL cells were preincubated with an anti-CD9 mAb before challenge with MET-ENK their migration to the enkephalin was markedly reduced. These studies show that endogenous and synthetic opioid peptides are stimulants for pre-B ALL cell migration and suggest that CD9 is important in the regulation of leukemia cell motility. Images PMID:7657811

  6. Epstein–Barr virus-positive T/NK-cell lymphoproliferative disorders

    PubMed Central

    Cai, Qingqing; Chen, Kailin; Young, Ken H

    2015-01-01

    Epstein–Barr virus, a ubiquitous human herpesvirus, can induce both lytic and latent infections that result in a variety of human diseases, including lymphoproliferative disorders. The oncogenic potential of Epstein–Barr virus is related to its ability to infect and transform B lymphocytes into continuously proliferating lymphoblastoid cells. However, Epstein–Barr virus has also been implicated in the development of T/natural killer cell lymphoproliferative diseases. Epstein–Barr virus encodes a series of products that mimic several growth, transcription and anti-apoptotic factors, thus usurping control of pathways that regulate diverse homeostatic cellular functions and the microenvironment. However, the exact mechanism by which Epstein–Barr virus promotes oncogenesis and inflammatory lesion development remains unclear. Epstein–Barr virus-associated T/natural killer cell lymphoproliferative diseases often have overlapping clinical symptoms as well as histologic and immunophenotypic features because both lymphoid cell types derive from a common precursor. Accurate classification of Epstein–Barr virus-associated T/natural killer cell lymphoproliferative diseases is a prerequisite for appropriate clinical management. Currently, the treatment of most T/natural killer cell lymphoproliferative diseases is less than satisfactory. Novel and targeted therapies are strongly required to satisfy clinical demands. This review describes our current knowledge of the genetics, oncogenesis, biology, diagnosis and treatment of Epstein–Barr virus-associated T/natural killer cell lymphoproliferative diseases. PMID:25613730

  7. DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

    SciTech Connect

    Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D. )

    1991-07-01

    Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links.

  8. The Sonoda–Tajima Cell Collection: A Human Genetics Research Resource with Emphasis on South American Indigenous Populations

    PubMed Central

    Danjoh, Inaho; Saijo, Kaoru; Hiroyama, Takashi

    2011-01-01

    The Sonoda–Tajima Cell Collection includes cell samples obtained from a range of ethnic minority groups across the world but in particular from South America. The collection is made all the more valuable by the fact that some of these ethnic populations have since died out, and thus it will be impossible to prepare a similar cell collection again. The collection was donated to our institute, a public cell bank in Japan, by Drs Sonoda and Tajima to make it available to researchers throughout the world. The original cell collection was composed of cryopreserved peripheral blood samples that would obviously have been rapidly exhausted if used directly. We, therefore, immortalized some samples with the Epstein–Barr virus and established B-lymphoblastoid cell lines (B-LCLs). As there is continuing controversy over whether the B-LCL genome is stably maintained, we performed an array comparative genomic hybridization (CGH) analysis to confirm the genomic stability of the cell lines. The array CGH analysis of the B-LCL lines and their parental B cells demonstrated that genomic stability was maintained in the long-term cell cultures. The B-LCLs of the Sonoda–Tajima Collection will therefore be made available to interested scientists around the world. At present, 512 B-LCLs have been developed, and we are willing to increase the number if there is sufficient demand. PMID:21383383

  9. 1,3-butadiene: cancer, mutations, and adducts. Part II: Roles of two metabolites of 1,3-butadiene in mediating its in vivo genotoxicity.

    PubMed

    Recio, L; Saranko, C J; Steen, A M

    2000-03-01

    1,3-Butadiene (BD) is carcinogenic in mice and rats, with mice being more susceptible than rats to its carcinogenic effects. 1,3-Butadiene is mutagenic in the bone marrow and spleen cells of B6C3F1 lacI transgenic mice. The goal of this research was to assess the roles of two BD metabolites, 1,2-epoxy-3-butene (BDO) and 1,2,3,4-diepoxybutane (BDO2), in the mutagenicity and mutational spectrum of the parent compound BD by determining the mutagenicity and mutational spectra of BDO and BDO2 in human and rodent cells in vitro and in vivo. In human TK6 lymphoblastoid cells (TK6 cells), BDO exposure increased the frequency of G.C-->A.T transitions and A.T-->T.A transversions (Fisher exact test; p < 0.05). The most striking difference in the type of base-substitution mutations between BDO-exposed and BDO-unexposed TK6 cells was the 19-fold increase in A.T-->T.A transversions. 1,2,3,4-Diepoxybutane increased the frequency of A.T-->T.A transversions (Fisher exact test; p < 0.05) and the frequency of deletions in exposed TK6 cells compared with unexposed controls. Exposure of Rat2 lacI transgenic fibroblasts (Rat2 cells) to BDO increased the frequency of three types of base-substitution mutations: G.C-->A.T transitions, G.C-->T.A transversions, and A.T-->T.A transversions. Exposure of Rat2 cells to BDO2-induced dose-dependent increases in micronuclei at exposure levels that apparently did not induce mutagenicity at the lacI transgene. The lack of detectable mutagenicity at the lacI transgene in Rat2 cells exposed to BDO2 probably reflects the poor recovery of large deletions by this lambda phage-based mutagenicity assay. Inhalation exposure of B6C3F1 lacI transgenic mice (lacI mice) and F344 lacI transgenic rats (lacI rats) to BDO (29.9 parts per million [ppm]; 6 hours/day; 5 days/week for 2 weeks) did not increase the lacI mutant frequency (MF) in bone marrow or spleen cells of mice and rats, but in the cells of mouse lung (a tumor target organ for BD), significant

  10. Genotoxic effects of high-energy iron particles in human lymphoblasts differing in radiation sensitivity

    NASA Technical Reports Server (NTRS)

    Evans, H. H.; Horng, M. F.; Evans, T. E.; Jordan, R.; Schwartz, J. L.

    2001-01-01

    The effects of (56)Fe particles and (137)Cs gamma radiation were compared in TK6 and WTK1 human lymphoblasts, two related cell lines which differ in TP53 status and in the ability to rejoin DNA double-strand breaks. Both cell lines were more sensitive to the cytotoxic and clastogenic effects of (56)Fe particles than to those of gamma rays. However, the mutagenicity of (56)Fe particles and gamma rays at the TK locus was the same per unit dose and was higher for gamma rays than for (56)Fe particles at isotoxic doses. The respective RBEs for TK6 and WTK1 cells were 1.5 and 1.9 for cytotoxicity and 2.5 and 1.9 for clastogenicity, but only 1 for mutagenicity. The results indicate that complex lesions induced by (56)Fe particles are repaired less efficiently than gamma-ray-induced lesions, leading to fewer colony-forming cells, a slightly higher proportion of aberrant cells at the first division, and a lower frequency of viable mutants at isotoxic doses. WTK1 cells (mutant TP53) were more resistant to the cytotoxic effects of both gamma rays and (56)Fe particles, but showed greater cytogenetic and mutagenic damage than TK6 cells (TP53(+)). A deficiency in the number of damaged TK6 cells (a) reaching the first mitosis after exposure and (b) forming viable mutants can explain these results.

  11. Generation of tumor-specific, HLA class I-restricted human Th1 and Tc1 cells by cell engineering with tumor peptide-specific T-cell receptor genes.

    PubMed

    Tsuji, Takemasa; Yasukawa, Masaki; Matsuzaki, Junko; Ohkuri, Takayuki; Chamoto, Kenji; Wakita, Daiko; Azuma, Taichi; Niiya, Hironari; Miyoshi, Hiroyuki; Kuzushima, Kiyotaka; Oka, Yoshihiro; Sugiyama, Haruo; Ikeda, Hiroaki; Nishimura, Takashi

    2005-07-15

    Tumor antigen-specific CD4+ and CD8+ T lymphocytes, especially interferon-gamma (IFN-gamma)-producing type-1 helper T (Th1) and type-1 cytotoxic T (Tc1) cells, play a crucial role in tumor eradication. Adoptive transfer using tumor-specific Th1 and Tc1 cells is a promising therapeutic strategy for tumor immunotherapy. However, its clinical application has been hampered because of difficulties in generating tumor-specific Th1 cells from patients with tumors. To overcome this problem, we have developed an efficient method to prepare tumor-specific Th1 and Tc1 cells. T-cell receptor (TCR) alpha and beta genes obtained from an HLA-A24-restricted, Wilms tumor 1 (WT1) peptide-specific Tc clone were lentivirally transduced to polyclonally activated Th1 and Tc1 cells. As expected, TCR gene-modified Tc1 cells showed cytotoxicity and IFN-gamma production in response to peptide-loaded lymphoblastoid cell lines, WT1 gene-transduced cells, and freshly isolated leukemia cells expressing both WT1 and HLA-A24. Surprisingly, we further demonstrated that Th1 cells transduced with HLA-class I-restricted TCR genes also showed both cytotoxicity and cytokine production in an HLA-A24-restricted manner. In contrast to gene-modified Tc1 cells, Th1 cells produced high amounts of interleukin-2 (IL-2) in addition to IFN-gamma, which is beneficial for induction of antitumor cellular immunity. Thus, TCR gene-modified HLA-class I-restricted Th1 and Tc1 cells are a powerful strategy for the application to adoptive immunotherapy of human cancer.

  12. Measles virus transmembrane fusion protein synthesized de novo or presented in immunostimulating complexes is endogenously processed for HLA class I- and class II-restricted cytotoxic T cell recognition

    PubMed Central

    1992-01-01

    The routes used by antigen-presenting cells (APC) to convert the transmembrane fusion glycoprotein (F) of measles virus (MV) to HLA class I and class II presentable peptides have been examined, using cloned cytotoxic T lymphocytes in functional assays. Presentation by Epstein-Barr virus-transformed B lymphoblastoid cell lines was achieved using live virus, ultraviolet light-inactivated virus, and purified MV- F delivered either as such or incorporated in immunostimulating complexes (MV-F-ISCOM). Only live virus and MV-F-ISCOM allow presentation by class I molecules, while all antigen preparations permit class II-restricted presentation. We observe presentation of MV- F from live virus and as MV-F-ISCOM by class II molecules in a fashion that is not perturbed by chloroquine. Our studies visualize novel presentation pathways of type I transmembrane proteins. PMID:1613454

  13. A T-cell specific transcriptional enhancer element 3 prime of C sub. alpha. in the human T-cell receptor. alpha. locus

    SciTech Connect

    Ho, Icheng; Yang, Lihsuan; Morle, G.; Leiden, J.M. )

    1989-09-01

    A transcriptional enhancer element has been identified 4.5 kilobases 3{prime} of C{sub {alpha}} (constant region {alpha} chain) in the human T-cell receptor (TCR) {alpha}-chain locus. This enhancer is active on both a TCR V{sub {alpha}} (variable region {alpha} chain) promoter and the minimal simian virus 40 promoter in TCR {alpha}/{beta} Jurkat and EL4 cells but is inactive on a V{sub {alpha}} promoter TCR {gamma}/{delta} PEER and Molt-13 cells, clone 13 B cells, and HeLa fibroblasts. The enhancer has been localized to a 116-base-pair BstXI/Dra I restriction enzyme fragment, which lacks immunoglobulin octamer and {kappa}B enhancer motifs but does contain a consensus cAMP-response element (CRE). DNase I footprint analyses demonstrated that the minimal enhancer contains two binding sites for Jurkat nuclear proteins. One of these sites corresponds to the CRE, while the other does not correspond to a known transcriptional enhancer motif. These data support a model in which TCR {alpha} gene transcription is regulated by a unique set of cis-acting sequences and trans-acting factors, which are differentially active in cells of the TCR {alpha}/{beta} lineage. In addition, the TCR {alpha} enhancer may play a role in activating oncogene expression in T-lymphoblastoid tumors that have previously been shown to display chromosomal translocations into the human TCR {alpha} locus.

  14. Role of EBNA-3 Family Proteins in EBV Associated B-cell Lymphomagenesis

    PubMed Central

    Bhattacharjee, Shaoni; Ghosh Roy, Shatadru; Bose, Priyanka; Saha, Abhik

    2016-01-01

    Epstein-Barr virus (EBV) is highly ubiquitous in human population and establishes a lifelong asymptomatic infection within the infected host unless the immune system is compromised. Following initial infection in the oropharyngeal epithelial cells, EBV primarily infects naive B-lymphocytes and develops a number of B-cell lymphomas particularly in immune-deficient individuals. In vitro, EBV can also infect and subsequently transform quiescent B-lymphocytes into continuously proliferating lymphoblastoid cell lines (LCLs) resembling EBV-induced lymphoproliferative disorders in which a subset of latent transcripts are detected. Genetic studies revealed that EBNA-3 family comprising of three adjacent genes in the viral genome—EBNA-3A and -3C, but not -3B, are critical for B-cell transformation. Nevertheless, all three proteins appear to significantly contribute to maintain the overall proliferation and viability of transformed cells, suggesting a critical role in lymphoma development. Apart from functioning as important viral transcriptional regulators, EBNA-3 proteins associate with many cellular proteins in different signaling networks, providing a suitable platform for lifelong survival of the virus and concurrent lymphoma development in the infected host. The chapter describes the function of each these EBV nuclear antigen 3 proteins employed by the virus as a means to understand viral pathogenesis of several EBV-associated B-cell malignancies. PMID:27092119

  15. Isolation of a T-cell clone showing HLA-DRB1*0405-restricted cytotoxicity for hematopoietic cells in a patient with aplastic anemia.

    PubMed

    Nakao, S; Takami, A; Takamatsu, H; Zeng, W; Sugimori, N; Yamazaki, H; Miura, Y; Ueda, M; Shiobara, S; Yoshioka, T; Kaneshige, T; Yasukawa, M; Matsuda, T

    1997-05-15

    The existence of T cells capable of inhibiting in vitro hematopoiesis has been shown in aplastic anemia (AA), although whether such inhibition is mediated by a specific immune reaction involving an HLA allele remained unknown. We isolated a CD4+ Vbeta21+ T-cell clone that was most dominant among Vbeta21+ T cells in the bone marrow (BM) of an AA patient whose HLA-DRB1 alleles included 1501 and 0405. The T-cell clone named NT4.2 lysed an autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) and phytohemagglutinin-stimulated lymphocytes (PHA-blasts) as well as allogeneic LCLs sharing HLA-DRB1*0405. Cytotoxicity against LCL cells and PHA-blasts by NT4.2 was blocked by anti-HLA-DR monoclonal antibody (MoAb) or anti-CD3 MoAb. NT4.2 also lysed autologous BM mononuclear cells enriched with CD34+ cells that had been cultured for one week in the presence of colony-stimulating factors as well as allogeneic CD34+ cells of a normal individual carrying HLA-DRB1*0405, cultured in the same way. Moreover, NT4.2 strongly inhibited colony formation by hematopoietic progenitor cells derived from cultured CD34+ cells sharing HLA-DRB1*0405. These results indicate that the AA patient has T cells capable of killing hematopoietic cells in an HLA-DRB1*0405-restricted manner and that such cytotoxic T cells may contribute to the pathogenesis of AA.

  16. Human Leukocyte Antigen (HLA) A*1101-Restricted Epstein-Barr Virus-Specific T-cell Receptor Gene Transfer to Target Nasopharyngeal Carcinoma.

    PubMed

    Zheng, Yong; Parsonage, Greg; Zhuang, Xiaodong; Machado, Lee R; James, Christine H; Salman, Asmaa; Searle, Peter F; Hui, Edwin P; Chan, Anthony T C; Lee, Steven P

    2015-10-01

    Infusing virus-specific T cells is effective treatment for rare Epstein-Barr virus (EBV)-associated posttransplant lymphomas, and more limited success has been reported using this approach to treat a far more common EBV-associated malignancy, nasopharyngeal carcinoma (NPC). However, current approaches using EBV-transformed lymphoblastoid cell lines to reactivate EBV-specific T cells for infusion take 2 to 3 months of in vitro culture and favor outgrowth of T cells targeting viral antigens expressed within EBV(+) lymphomas, but not in NPC. Here, we explore T-cell receptor (TCR) gene transfer to rapidly and reliably generate T cells specific for the NPC-associated viral protein LMP2. We cloned a human leukocyte antigen (HLA) A*1101-restricted TCR, which would be widely applicable because 40% of NPC patients carry this HLA allele. Studying both the wild-type and modified forms, we have optimized expression of the TCR and demonstrated high-avidity antigen-specific function (proliferation, cytotoxicity, and cytokine release) in both CD8(+) and CD4(+) T cells. The engineered T cells also inhibited LMP2(+) epithelial tumor growth in a mouse model. Furthermore, transduced T cells from patients with advanced NPC lysed LMP2-expressing NPC cell lines. Using this approach, within a few days large numbers of high-avidity LMP2-specific T cells can be generated reliably to treat NPC, thus providing an ideal clinical setting to test TCR gene transfer without the risk of autoimmunity through targeting self-antigens.

  17. Human Leukocyte Antigen (HLA) A*1101-restricted Epstein-Barr Virus-specific T-cell Receptor Gene Transfer to Target Nasopharyngeal Carcinoma

    PubMed Central

    Zheng, Yong; Parsonage, Greg; Zhuang, Xiaodong; Machado, Lee R; James, Christine H.; Salman, Asmaa; Searle, Peter F.; Hui, Edwin P.; Chan, Anthony T.C.; Lee, Steven P.

    2015-01-01

    Infusing virus-specific T cells is effective treatment for rare Epstein-Barr virus (EBV)-associated post-transplant lymphomas and more limited success has been reported using this approach to treat a far more common EBV-associated malignancy, nasopharyngeal carcinoma (NPC). However, current approaches using EBV-transformed lymphoblastoid cell lines to reactivate EBV-specific T cells for infusion take 2 to 3 months of in vitro culture and favour outgrowth of T cells targeting viral antigens expressed within EBV+ lymphomas but not in NPC. Here we explore T-cell receptor (TCR) gene transfer to rapidly and reliably generate T cells specific for the NPC-associated viral protein LMP2. We cloned a HLA A*1101-restricted TCR, which would be widely applicable since 40% of NPC patients carry this HLA allele. Studying both the wild-type and modified forms we have optimised expression of the TCR and demonstrated high avidity antigen-specific function (proliferation, cytotoxicity, cytokine release) in both CD8+ and CD4+ T cells. The engineered T cells also inhibited LMP2+ epithelial tumour growth in a mouse model. Furthermore, transduced T cells from patients with advanced NPC lysed LMP2-expressing NPC cell lines. Using this approach, within a few days large numbers of high avidity LMP2-specific T cells can be generated reliably to treat NPC, thus providing an ideal clinical setting to test TCR gene transfer without the risk of autoimmunity through targeting self-antigens. PMID:25711537

  18. Abnormal T cell subpopulations and circulating immune complexes in the Guillain-Barré syndrome and multiple sclerosis.

    PubMed

    Goust, J M; Chenais, F; Carnes, J E; Hames, C G; Fudenberg, H H; Hogan, E L

    1978-05-01

    Immunologic studies were performed in 21 patients with multiple sclerosis (MS) and 16 with the Guillain-Barré syndrome (GBS). Levels of thymus-derived (T) cells measured by "total" and "active" rosette formation between sheep erythrocytes and peripheral blood mononuclear cells (TEt, TEa) were within normal limits in all the patients, with the exception of four GBS patients, including one who also had received chemotherapy for lymphoma and three who were receiving steroids. When lymphocytes from the 21 patients were incubated with the bone-marrow-derived (B) lymphoblastoid cell line PGLC-33H, there were, for 12 of 18 MS patients and 11 of 16 GBS patients, significant decreases in a subpopulation of peripheral blood T lymphocytes that form "PGLC rosettes" (PGR) with the PGLC-33H cells. (Peripheral blood T cells from normal individuals formed PGR with 23.9 +/- 3.8 percent of PGLC-33H cells.) Using the 125l-C1q binding assay, immune complexes were detected in the serum of 14 of 19 MS patients and 15 of 16 GBS patients. An association between increased C1q binding and decreased PGR values was found in 10 of 18 MS patients and 12 of 17 GBS patients. The results suggest that in both diseases the etiology may involve a decrease in the subset of T cells that bind to the IgM-producing cell line PGLC-33H, in association with the appearance of circulating immune complexes containing the infectious viral agent.

  19. Treatment with a BH3 mimetic overcomes the resistance of latency III EBV (+) cells to p53-mediated apoptosis

    PubMed Central

    Pujals, A; Renouf, B; Robert, A; Chelouah, S; Hollville, É; Wiels, J

    2011-01-01

    P53 inactivation is often observed in Burkitt's lymphoma (BL) cells due to mutations in the p53 gene or overexpression of its negative regulator, murine double minute-2 (MDM2). This event is now considered an essential part of the oncogenic process. Epstein–Barr virus (EBV) is strongly associated with BL and is a cofactor in its development. We previously showed that nutlin-3, an antagonist of MDM2, activates the p53 pathway in BL cell lines harboring wild-type p53. However, nutlin-3 strongly induced apoptosis in EBV (−) or latency I EBV (+) cells, whereas latency III EBV (+) cells were much more resistant. We show here that this resistance to apoptosis is also observed in latency III EBV (+) lymphoblastoid cell lines. We also show that, in latency III EBV (+) cells, B-cell lymphona 2 (Bcl-2) is selectively overproduced and interacts with Bcl-2-associated X protein (Bax), preventing its activation. The treatment of these cells with the Bcl-2-homology domain 3 mimetic ABT-737 disrupts Bax/Bcl-2 interaction and allows Bax activation by nutlin-3. Furthermore, treatment with these two compounds strongly induces apoptosis. Thus, a combination of Mdm2 and Bcl-2 inhibitors might be a useful anti-cancer strategy for diseases linked to EBV infection. PMID:21796156

  20. Treatment with a BH3 mimetic overcomes the resistance of latency III EBV (+) cells to p53-mediated apoptosis.

    PubMed

    Pujals, A; Renouf, B; Robert, A; Chelouah, S; Hollville, E; Wiels, J

    2011-07-28

    P53 inactivation is often observed in Burkitt's lymphoma (BL) cells due to mutations in the p53 gene or overexpression of its negative regulator, murine double minute-2 (MDM2). This event is now considered an essential part of the oncogenic process. Epstein-Barr virus (EBV) is strongly associated with BL and is a cofactor in its development. We previously showed that nutlin-3, an antagonist of MDM2, activates the p53 pathway in BL cell lines harboring wild-type p53. However, nutlin-3 strongly induced apoptosis in EBV (-) or latency I EBV (+) cells, whereas latency III EBV (+) cells were much more resistant. We show here that this resistance to apoptosis is also observed in latency III EBV (+) lymphoblastoid cell lines. We also show that, in latency III EBV (+) cells, B-cell lymphona 2 (Bcl-2) is selectively overproduced and interacts with Bcl-2-associated X protein (Bax), preventing its activation. The treatment of these cells with the Bcl-2-homology domain 3 mimetic ABT-737 disrupts Bax/Bcl-2 interaction and allows Bax activation by nutlin-3. Furthermore, treatment with these two compounds strongly induces apoptosis. Thus, a combination of Mdm2 and Bcl-2 inhibitors might be a useful anti-cancer strategy for diseases linked to EBV infection.

  1. Neutron exposures in human cells: bystander effect and relative biological effectiveness.

    PubMed

    Seth, Isheeta; Schwartz, Jeffrey L; Stewart, Robert D; Emery, Robert; Joiner, Michael C; Tucker, James D

    2014-01-01

    Bystander effects have been observed repeatedly in mammalian cells following photon and alpha particle irradiation. However, few studies have been performed to investigate bystander effects arising from neutron irradiation. Here we asked whether neutrons also induce a bystander effect in two normal human lymphoblastoid cell lines. These cells were exposed to fast neutrons produced by targeting a near-monoenergetic 50.5 MeV proton beam at a Be target (17 MeV average neutron energy), and irradiated-cell conditioned media (ICCM) was transferred to unirradiated cells. The cytokinesis-block micronucleus assay was used to quantify genetic damage in radiation-naïve cells exposed to ICCM from cultures that received 0 (control), 0.5, 1, 1.5, 2, 3 or 4 Gy neutrons. Cells grown in ICCM from irradiated cells showed no significant increase in the frequencies of micronuclei or nucleoplasmic bridges compared to cells grown in ICCM from sham irradiated cells for either cell line. However, the neutron beam has a photon dose-contamination of 5%, which may modulate a neutron-induced bystander effect. To determine whether these low doses of contaminating photons can induce a bystander effect, cells were irradiated with cobalt-60 at doses equivalent to the percent contamination for each neutron dose. No significant increase in the frequencies of micronuclei or bridges was observed at these doses of photons for either cell line when cultured in ICCM. As expected, high doses of photons induced a clear bystander effect in both cell lines for micronuclei and bridges (p<0.0001). These data indicate that neutrons do not induce a bystander effect in these cells. Finally, neutrons had a relative biological effectiveness of 2.0 ± 0.13 for micronuclei and 5.8 ± 2.9 for bridges compared to cobalt-60. These results may be relevant to radiation therapy with fast neutrons and for regulatory agencies setting standards for neutron radiation protection and safety.

  2. Quantitative proteomic analysis of HIV-1 infected CD4+ T cells reveals an early host response in important biological pathways: Protein synthesis, cell proliferation, and T-cell activation

    SciTech Connect

    Navare, Arti T.; Sova, Pavel; Purdy, David E.; Weiss, Jeffrey M.; Wolf-Yadlin, Alejandro; Korth, Marcus J.; Chang, Stewart T.; Proll, Sean C.; Jahan, Tahmina A.; Krasnoselsky, Alexei L.; Palermo, Robert E.; Katze, Michael G.

    2012-07-20

    Human immunodeficiency virus (HIV-1) depends upon host-encoded proteins to facilitate its replication while at the same time inhibiting critical components of innate and/or intrinsic immune response pathways. To characterize the host cell response on protein levels in CD4+ lymphoblastoid SUP-T1 cells after infection with HIV-1 strain LAI, we used mass spectrometry (MS)-based global quantitation with iTRAQ (isobaric tag for relative and absolute quantification). We found 266, 60 and 22 proteins differentially expressed (DE) (P-value{<=}0.05) at 4, 8, and 20 hours post-infection (hpi), respectively, compared to time-matched mock-infected samples. The majority of changes in protein abundance occurred at an early stage of infection well before the de novo production of viral proteins. Functional analyses of these DE proteins showed enrichment in several biological pathways including protein synthesis, cell proliferation, and T-cell activation. Importantly, these early changes before the time of robust viral production have not been described before.

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

    PubMed

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

    2015-12-01

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

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

    PubMed Central

    Muller, Ryan Y.; Hammond, Ming C.

    2015-01-01

    The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion. PMID:26543439

  5. Novel radiation response genes identified in gene-trapped MCF10A mammary epithelial cells.

    PubMed

    Malone, Jennifer; Ullrich, Robert

    2007-02-01

    We have used a gene-trapping strategy to screen human mammary epithelial cells for radiation response genes. Relative mRNA expression levels of five candidate genes in MCF10A cells were analyzed, both with and without exposure to radiation. In all five cases, the trapped genes were significantly down-regulated after radiation treatment. Sequence analysis of the fusion transcripts identified the trapped genes: (1) the human androgen receptor, (2) the uncharacterized DREV1 gene, which has known homology to DNA methyltransferases, (3) the human creatine kinase gene, (4) the human eukaryotic translation elongation factor 1 beta 2, and (5) the human ribosomal protein L27. All five genes were down-regulated significantly after treatment with varying doses of ionizing radiation (0.10 to 4.0 Gy) and at varying times (2-30 h after treatment). The genes were also analyzed in human fibroblast and lymphoblastoid cell lines to determine whether the radiation response being observed was cell-type specific. The results verified that the observed radiation response was not a cell-type-specific phenomenon, suggesting that the genes play essential roles in the radiation damage control pathways. This study demonstrates the potential of the gene-trap approach for the identification and functional analysis of novel radiation response genes.

  6. Class I major histocompatibility proteins as cell surface receptors for simian virus 40.

    PubMed

    Atwood, W J; Norkin, L C

    1989-10-01

    Class I major histocompatibility complex proteins appear to be the major cell surface receptors for simian virus 40 (SV40), as implied by the following observations. Adsorption of SV40 to LLC-MK2 rhesus monkey kidney cells specifically inhibited binding of a monoclonal antibody (MAb) against class I human lymphocyte antigen (HLA) proteins. Conversely, pretreatment of LLC-MK2 cells with anti-HLA MAbs inhibited infection by SV40. The ability of anti-HLA to inhibit infection was greatly reduced when the order of addition of the anti-HLA and the virus was reversed. Infection was also inhibited by preincubating SV40 with purified soluble class I protein. Finally, human lymphoblastoid cells of the Daudi line, which do not express class I major histocompatibility complex proteins, were infected at relatively low levels with SV40 virions. In a control experiment, we found that pretreatment of cells with a MAb specific for the leukocytic-function-associated antigen LFA-3 actually enhanced infection. This finding may also support the premise that class I major histocompatibility complex proteins are receptors for SV40.

  7. Constituents of French Marigold (Tagetes patula L.) Flowers Protect Jurkat T-Cells against Oxidative Stress

    PubMed Central

    Chkhikvishvili, Irakli; Sanikidze, Tamar; Gogia, Nunu; Enukidze, Maia; Machavariani, Marine; Kipiani, Nana; Vinokur, Yakov; Rodov, Victor

    2016-01-01

    The flowers of French marigold (Tagetes patula L.) are widely used in folk medicine, in particular for treating inflammation-related disorders. However, cellular mechanisms of this activity demand further investigation. In the present work, we studied the potential of T. patula compounds to alleviate the oxidative stress in hydrogen peroxide-challenged human lymphoblastoid Jurkat T-cells. Crude extracts of marigold flowers and purified fractions containing flavonoids patuletin, quercetagetin, and quercetin and their derivatives, as well as the carotenoid lutein, were brought in contact with Jurkat cells challenged with 25 or 50 μM H2O2. Hydrogen peroxide caused oxidative stress in the cells, manifested as generation of superoxide and peroxyl radicals, reduced viability, arrested cell cycle, and enhanced apoptosis. The stress was alleviated by marigold ingredients that demonstrated high radical-scavenging capacity and enhanced the activity of antioxidant enzymes involved in neutralization of reactive oxygen species. Flavonoid fraction rich in quercetin and quercetagetin showed the highest cytoprotective activity, while patuletin in high dose exerted a cytotoxic effect associated with its anticancer potential. T. patula compounds enhanced the production of anti-inflammatory and antioxidant interleukin-10 (IL-10) in Jurkat cells. Both direct radical-scavenging capacity and stimulation of protective cellular mechanisms can underlay the anti-inflammatory properties of marigold flowers. PMID:27433287

  8. Constituents of French Marigold (Tagetes patula L.) Flowers Protect Jurkat T-Cells against Oxidative Stress.

    PubMed

    Chkhikvishvili, Irakli; Sanikidze, Tamar; Gogia, Nunu; Enukidze, Maia; Machavariani, Marine; Kipiani, Nana; Vinokur, Yakov; Rodov, Victor

    2016-01-01

    The flowers of French marigold (Tagetes patula L.) are widely used in folk medicine, in particular for treating inflammation-related disorders. However, cellular mechanisms of this activity demand further investigation. In the present work, we studied the potential of T. patula compounds to alleviate the oxidative stress in hydrogen peroxide-challenged human lymphoblastoid Jurkat T-cells. Crude extracts of marigold flowers and purified fractions containing flavonoids patuletin, quercetagetin, and quercetin and their derivatives, as well as the carotenoid lutein, were brought in contact with Jurkat cells challenged with 25 or 50 μM H2O2. Hydrogen peroxide caused oxidative stress in the cells, manifested as generation of superoxide and peroxyl radicals, reduced viability, arrested cell cycle, and enhanced apoptosis. The stress was alleviated by marigold ingredients that demonstrated high radical-scavenging capacity and enhanced the activity of antioxidant enzymes involved in neutralization of reactive oxygen species. Flavonoid fraction rich in quercetin and quercetagetin showed the highest cytoprotective activity, while patuletin in high dose exerted a cytotoxic effect associated with its anticancer potential. T. patula compounds enhanced the production of anti-inflammatory and antioxidant interleukin-10 (IL-10) in Jurkat cells. Both direct radical-scavenging capacity and stimulation of protective cellular mechanisms can underlay the anti-inflammatory properties of marigold flowers.

  9. Analysis of the cross-talk of Epstein–Barr virus-infected B cells with T cells in the marmoset

    PubMed Central

    Dunham, Jordon; van Driel, Nikki; Eggen, Bart JL; Paul, Chaitali; ‘t Hart, Bert A; Laman, Jon D; Kap, Yolanda S

    2017-01-01

    Despite the well-known association of Epstein–Barr virus (EBV), a lymphocryptovirus (LCV), with multiple sclerosis, a clear pathogenic role for disease progression has not been established. The translationally relevant experimental autoimmune encephalomyelitis (EAE) model in marmoset monkeys revealed that LCV-infected B cells have a central pathogenic role in the activation of T cells that drive EAE progression. We hypothesized that LCV-infected B cells induce T-cell functions relevant for EAE progression. In the current study, we examined the ex vivo cross-talk between lymph node mononuclear cells (MNCs) from EAE marmosets and (semi-) autologous EBV-infected B-lymphoblastoid cell lines (B-LCLs). Results presented here demonstrate that infection with EBV B95-8 has a strong impact on gene expression profile of marmoset B cells, particularly those involved with antigen processing and presentation or co-stimulation to T cells. At the cellular level, we observed that MNC co-culture with B-LCLs induced decrease of CCR7 expression on T cells from EAE responder marmosets, but not in EAE monkeys without clinically evident disease. B-LCL interaction with T cells also resulted in significant loss of CD27 expression and reduced expression of IL-23R and CCR6, which coincided with enhanced IL-17A production. These results highlight the profound impact that EBV-infected B-LCL cells can have on second and third co-stimulatory signals involved in (autoreactive) T-cell activation. PMID:28243437

  10. The effects of ethidium bromide induced loss of mitochondrial DNA on mitochondrial phenotype and ultrastructure in a human leukemia T-cell line (MOLT-4 cells).

    PubMed

    Armand, Ray; Channon, Jacqueline Y; Kintner, Jennifer; White, Kristina A; Miselis, Kristin A; Perez, Raymond P; Lewis, Lionel D

    2004-04-01

    Mitochondrial DNA-deficient (rho(0)) cells were generated following a 26-day incubation of MOLT-4 lymphoblastoid T cells in ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide). The absence of mitochondrial DNA (mtDNA) in the resultant MOLT-4 rho(0) cells was confirmed by Southern analysis and quantitative polymerase chain reaction (PCR). MOLT-4 rho(0) cells proliferated more slowly than parental cells (wild type) and produced significantly more lactate (approximately fourfold increase; P < 0.001) with concomitantly reduced oxygen consumption (12.3% vs. 100%; P < 0.001) compared with the wild type. MOLT-4 rho(0) cells also showed reduced cytochrome c oxidase activity and a reduced cytochrome c oxidase/citrate synthase activity ratio compared to parental wild-type MOLT-4 cells (P < 10(-11)). Electron microscopy showed elongated mitochondria with parallel cristae in MOLT-4 cells although the mitochondria in MOLT-4 rho(0) cells appeared enlarged, some were vacuolated with either an absent or a grossly distorted cristae pattern. Vital staining with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) was used to image mitochondria in intact cells and study mitochondrial transmembrane potential (Deltapsi(m)). Flow cytometry using JC-1 indicated that MOLT-4 rho(0) had a lower Deltapsi(m) than MOLT-4. Sodium fluoride (an inhibitor of the glycolytic pathway) at a concentration of 20 mM further reduced the Deltapsi(m) in MOLT-4-rho(0) cells. This data suggested that a glycolytic pathway product, possibly ATP, was required for the maintenance of Deltapsi(m) in MOLT-4 rho(0) cells.

  11. Reduced cell surface expression of processed human immunodeficiency virus type 1 envelope glycoprotein in the presence of Nef.

    PubMed Central

    Schwartz, O; Rivière, Y; Heard, J M; Danos, O

    1993-01-01

    nef genes from two laboratory grown human immunodeficiency virus type 1 (HIV-1) strains and from two proviruses that had not been propagated in vitro were introduced into CD4+ lymphoblastoid CEM cells. The stable expression of all four Nef proteins was associated with an almost complete abrogation of CD4 cell surface localization. The consequences of the presence of Nef on gp160 cleavage, gp120 surface localization, and envelope-induced cytopathic effect were examined in CEM cells in which the HIV-1 env gene was expressed from a vaccinia virus vector. The presence of Nef did not modify the processing of gp160 into its subunits but resulted in a significant decrease of cell surface levels of gp120, associated with a dramatic reduction of the fusion-mediated cell death. Surface levels of mutant envelope glycoproteins unable to bind CD4 were not altered in Nef-expressing cells, suggesting that the phenomenon was CD4 dependent. The intracellular accumulation of fully processed envelope glycoproteins could significantly delay the cytopathic effect associated with envelope surface expression in HIV-infected cells and may be relevant to the selective advantage associated with Nef during the in vivo infectious process. Images PMID:8497051

  12. Reovirus type 3 synthesizes proteins in interferon-treated HeLa cells without reversing the antiviral state.

    PubMed

    Feduchi, E; Esteban, M; Carrasco, L

    1988-06-01

    Treatment of HeLa cells with human lymphoblastoid interferon (IFN-alpha) does not inhibit reovirus type 3 protein synthesis during virus infection. In contrast, reovirus translation is blocked by treatment of L cells with mouse IFN-alpha. The (2'-5')A synthetase activity is induced in HeLa cells by IFN-alpha treatment and is activated after reovirus infection, since cell lysates from these cells synthesize in vitro (2'-5')A oligonucleotides. The IFN-induced protein kinase activity is also triggered in those lysates upon dsRNA addition. Thus, contrary to DNA-containing viruses, such as vaccinia virus or adenovirus, reovirus infection does not destroy or reverse the IFN-induced antiviral state. In support of this conclusion, superinfection with poliovirus or vesicular stomatitis virus of reovirus-infected HeLa cells treated with IFN leads only to a blockade of translation of the former viruses. These results provide a remarkable example where in the same cells doubly infected with two different viruses, the antiviral state induced by IFN-alpha is manifested by selectively inhibiting translation of one kind of virus (poliovirus or vesicular stomatitis virus) without affecting the translation of reovirus type 3. In addition, these results indicate that the resistance of reovirus translation to inhibition by IFN is different from the mechanism of resistance induced by DNA-containing viruses.

  13. Epstein-Barr virus (EBV) can immortalize B-cll cells activated by cytokines.

    PubMed

    Wendel-Hansen, V; Sällström, J; De Campos-Lima, P O; Kjellström, G; Sandlund, A; Siegbahn, A; Carlsson, M; Nilsson, K; Rosén, A

    1994-03-01

    B-type of chronic lymphocytic leukemia (B-CLL) cells are inert to the potent transforming action of Epstein-Barr virus (EBV). The mitogenic action of Staphylococcus aureus Cowan I (SAC), MP6-thioredoxin, and interleukin 2 (IL-2), agents previously shown to induce proliferation in normal as well as in B-CLL cells, lifted this block, and EBV-positive cell lines could be established. It was not possible to establish cell lines of leukemic origin from cultures that were incubated with EBV alone or cytokine mix alone. CLL-cells infected with EBV only, expressed the viral nuclear antigen complex (EBNA), but not the viral latent membrane protein (LMP). They were not activated as measured by cell size and 3H-thymidine incorporation. In contrast, cells incubated with EBV and cytokine mix expressed both EBNA and LMP in parallel with enlargement and increased 3H-thymidine incorporation. These results emphasize that LMP expression is a prerequisite for growth transformation and immortalization and that cytokine activation signals are required for its expression in B-CLLs. Cells incubated with SAC/MP6-thioredoxin/IL-2 did not express any of the viral antigens, but were activated with regard to the mentioned parameters. Nine cell lines were established from six patients. From each of the three patients, we obtained 'twin'-pair lines: one corresponding to the malignant cell and the other to a normal B-lymphoblastoid cell. Thus, malignant and normal B-cell counterparts, from the very same donor, are at hand for comparative studies. The cell lines have been carried out for more than 12 months in culture. We conclude that B-CLL that are refractory to EBV-transformation can be rendered susceptible through in vitro cytokine activation.

  14. Leukosialin (CD43)-major histocompatibility class I molecule interactions involved in spontaneous T cell conjugate formation

    PubMed Central

    1996-01-01

    Resting T cells spontaneously adhere in a selective manner to potent accessory cells, such as dendritic cells (DC) and lymphoblastoid B blasts (LCL). Here we demonstrate that leukosialin (CD43) and major histocompatibility complex class I molecules (MHC-I) might play a critical role in this process. T cell conjugate formation with monocyte- derived DC (md-DC) and LCL could be strongly inhibited by either preincubating T cells with Fab fragments of CD43 monoclonal antibody (mAb) 6F5 or by preincubating md-DC or LCL with MHC-I mAb W6/32. Intact CD43 mAb 6F5, in contrast to monovalent Fab fragments, enhanced T cell adhesiveness by transactivating CD2 binding to CD58 molecules. Interestingly, induction of this proadhesive signal via CD43 with intact 6F5 mAb was found to revert mAb W6/32-mediated inhibition of T cell conjugate formation. These observations indicated that CD43 cross- linkage mimics and monovalent mAb 6F5 inhibits interaction of T cell CD43 with a stimulatory ligand on opposing cells, presumably MHC-I. For the demonstration of direct physical interaction between CD43 on T cells and MHC-I-coated beads it was necessary, however, to ligate CD2 on T cells with a stimulatory pair of CD2 mAbs (VIT13 plus TS2/18). This suggests that CD2 ligation crosswise upregulates CD43 binding avidity for MHC-I and that both adhesion molecule pairs (CD43/MHC-I and CD2/CD58) act in concert to induce and mediate T cell conjugate formation with certain cell types. PMID:8920865

  15. Detection by Epitope-defined Monoclonal Antibodies of Werner DNA Helicases in the Nucleoplasm and Their Upregulation by Cell Transformation and Immortalization

    PubMed Central

    Shiratori, Miwa; Sakamoto, Sakae; Suzuki, Noriyuki; Tokutake, Yoshiki; Kawabe, Yoichi; Enomoto, Takemi; Sugimoto, Masanobu; Goto, Makoto; Matsumoto, Takehisa; Furuichi, Yasuhiro

    1999-01-01

    We prepared several monoclonal antibodies (mAbs) specific for the NH2- and COOH-terminal regions of the DNA helicase (WRN helicase) responsible for Werner's syndrome known as a premature aging disease. With these antibodies, we detected by immunoblot analysis the endogenous WRN helicase of a relative mass of 180 kD in several lines of cultured cells, but not in patient cells with a defined mutation. Immunocytochemical staining of proliferating fibroblasts and tumor cells showed that the major part of WRN helicase is in the nucleoplasm and not in the nucleolus. Similar experiments with a rat mAb specific to the mouse homologue of human WRN helicase yielded an identical conclusion. Although this nucleoplasmic staining was evident in cells in interphase, the condensed chromatin structure in metaphase was not stained by the same mAbs, suggesting that WRN helicases exist perhaps in a soluble form or bound to the unfolded chromatin structure. From quantitative immunoblot analysis, higher levels of WRN helicase were observed in all transformed cells and tumor cells examined than those of normal cells. The expression of WRN helicase was enhanced consistently in fibroblasts and B-lymphoblastoid cells by transformation with SV-40 and Epstein-Barr virus, respectively, suggesting that rapidly proliferating cells require a high copy numbers of WRN helicase. PMID:9885239

  16. Measurement of DNA damage and apoptosis in Molt-4 cells after in vitro exposure to radiofrequency radiation.

    PubMed

    Hook, Graham J; Zhang, Peng; Lagroye, I; Li, Li; Higashikubo, Ryuji; Moros, Eduardo G; Straube, William L; Pickard, William F; Baty, Jack D; Roti Roti, Joseph L

    2004-02-01

    To determine whether exposure to radiofrequency (RF) radiation can induce DNA damage or apoptosis, Molt-4 T lymphoblastoid cells were exposed with RF fields at frequencies and modulations of the type used by wireless communication devices. Four types of frequency/modulation forms were studied: 847.74 MHz code-division multiple-access (CDMA), 835.62 MHz frequency-division multiple-access (FDMA), 813.56 MHz iDEN(R) (iDEN), and 836.55 MHz time-division multiple-access (TDMA). Exponentially growing cells were exposed to RF radiation for periods up to 24 h using a radial transmission line (RTL) exposure system. The specific absorption rates used were 3.2 W/kg for CDMA and FDMA, 2.4 or 24 mW/kg for iDEN, and 2.6 or 26 mW/kg for TDMA. The temperature in the RTLs was maintained at 37 degrees C +/- 0.3 degrees C. DNA damage was measured using the single-cell gel electrophoresis assay. The annexin V affinity assay was used to detect apoptosis. No statistically significant difference in the level of DNA damage or apoptosis was observed between sham-treated cells and cells exposed to RF radiation for any frequency, modulation or exposure time. Our results show that exposure of Molt-4 cells to CDMA, FDMA, iDEN or TDMA modulated RF radiation does not induce alterations in level of DNA damage or induce apoptosis.

  17. Signal-peptide-peptidase-like 2a is required for CD74 intramembrane proteolysis in human B cells

    PubMed Central

    Schneppenheim, Janna; Hüttl, Susann; Kruchen, Anne; Fluhrer, Regina; Müller, Ingo; Saftig, Paul; Schneppenheim, Reinhard; Martin, Christa L; Schröder, Bernd

    2015-01-01

    The invariant chain (CD74) mediates targeting of the MHCII complex to endosomal compartments, where CD74 undergoes degradation allowing MHCII to acquire peptides. We demonstrated recently that intramembrane proteolysis of the final membrane-bound N-terminal fragment (NTF) of CD74 is catalysed by Signal-peptide-peptidase-like 2a (SPPL2a) and that this process is indispensable for development and function of B lymphocytes in mice. In SPPL2a−/− mice, homeostasis of these cells is disturbed by the accumulation of the unprocessed CD74 NTF. So far, evidence for this essential role of SPPL2a is restricted to mice. Nevertheless, inhibition of SPPL2a has been suggested as novel approach to target B cells for treating autoimmunity. Here, we characterize human B cell lines with a homozygous microdeletion on chromosome 15. We demonstrate that this deletion disrupts the SPPL2a genomic locus and leads to loss of SPPL2a transcript. Lymphoblastoid cell lines from patients with this deletion exhibit absence of SPPL2a at the protein level and show an accumulation of the CD74 NTF comparable to B cells from SPPL2a−/− mice. By this means, we present evidence that the role of SPPL2a in CD74 proteolysis is conserved in human B cells and provide support for modulation of SPPL2a activity as a therapeutic concept. PMID:25035924

  18. The CD8+ T Cell-Mediated Immunity Induced by HPV-E6 Uploaded in Engineered Exosomes Is Improved by ISCOMATRIXTM Adjuvant

    PubMed Central

    Manfredi, Francesco; di Bonito, Paola; Ridolfi, Barbara; Anticoli, Simona; Arenaccio, Claudia; Chiozzini, Chiara; Baz Morelli, Adriana; Federico, Maurizio

    2016-01-01

    We recently described the induction of an efficient CD8+ T cell-mediated immune response against a tumor-associated antigen (TAA) uploaded in engineered exosomes used as an immunogen delivery tool. This immune response cleared tumor cells inoculated after immunization, and controlled the growth of tumors implanted before immunization. We looked for new protocols aimed at increasing the CD8+ T cell specific response to the antigen uploaded in engineered exosomes, assuming that an optimized CD8+ T cell immune response would correlate with a more effective depletion of tumor cells in the therapeutic setting. By considering HPV-E6 as a model of TAA, we found that the in vitro co-administration of engineered exosomes and ISCOMATRIXTM adjuvant, i.e., an adjuvant composed of purified ISCOPREPTM saponin, cholesterol, and phospholipids, led to a stronger antigen cross-presentation in both B- lymphoblastoid cell lines ( and monocyte-derived immature dendritic cells compared with that induced by the exosomes alone. Consistently, the co-inoculation in mice of ISCOMATRIXTM adjuvant and engineered exosomes induced a significant increase of TAA-specific CD8+ T cells compared to mice immunized with the exosomes alone. This result holds promise for effective usage of exosomes as well as alternative nanovesicles in anti-tumor therapeutic approaches. PMID:27834857

  19. Overexpression of the human BCL-2 gene product results in growth enhancement of Epstein-Barr virus-immortalized B cells

    SciTech Connect

    Tsujimoto, Yoshihide )

    1989-03-01

    The biological activity of the human BCL-2 gene product was analyzed in an Epstein-Barr virus (EBV)-infected human lymphoblastoid B-cell line transfected with BCL-2 sequences driven by the simian virus 40 promoter and enhancer. Overproduction of the BCL-2 protein conferred a selective growth advantage to the EBV-infected B cells as compared with control transfectants in low-serum medium and also after seeding at limiting dilution but did not render the cells tumorigenic in athymic nude mice. This growth enhancement was also seen in cells transfected with the BCL-2 gene with its own promoter juxtaposed to the immunoglobulin heavy chain gene enhancer, which represents the translocated form of the BCL-2 gene observed in follicular lymphomas with the t(14;18) translocation. The growth advantage of EBV-infected B cells overproducing the BCL-2 protein is neither due to the enhanced growth factor production nor due to an enhanced sensitivity of the BCL-2 transfectants to interleukins 1 or 6, although both lymphokines are known to stimulate proliferation of EBV-infected B-cell lines. The growth advantage of EBV-infected B-cell lines. The growth advantage of EBV-infected B cells by overproduction of the BCL-2 protein suggests the direct involvement of the BCL-2 gene product in the pathogenesis of follicular lymphoma.

  20. Phorbol ester and B cell-stimulatory factor synergize to induce B-chronic lymphocytic leukemia cells to simultaneous immunoglobulin secretion and DNA synthesis.

    PubMed

    Carlsson, M; Matsson, P; Rosén, A; Sundström, C; Tötterman, T H; Nilsson, K

    1988-11-01

    This paper discusses the response of two B cell-type chronic lymphocytic leukemia (B-CLL) clones, 173 and 183, to the phorbol ester TPA combined with a B cell-stimulatory factor (BSF) derived from a T helper cell hybridoma (MP6). Previous studies with 173 and 183 cells have consistently shown that TPA alone induces differentiation but no proliferation. However, when the two clones were exposed to TPA plus BSF-MP6, not only differentiation but also DNA synthesis was observed. Compared with TPA exposure alone, the fraction of cells with induced lymphoblastoid-plasmacytoid morphology increased and Ig secretion was enhanced. By a 1-hr TPA pulse followed by BSF-MP6, the DNA synthesis was further augmented, but less maturation was observed. T cell and monocyte removal, using cell sorting, showed that the DNA synthesis induced was independent of these cell types, also under serum-free conditions. Quantitation of several cell cycle-associated surface Ags showed that the 4F2, Ba, Bac-1, and cD23 Ags increased while the CD37 decreased in expression upon addition of BSF-MP6. We conclude that B-CLLs are inducible by TPA and BSF-MP6 not only to differentiation, but also to DNA synthesis even under serum-free conditions in vitro. The results furthermore suggest that the very low proliferation activity in B-CLL tumors in vivo may reflect a relative deficiency of proper growth and differentiation factors or a subnormal response of B-CLL cells to such factors.

  1. Development of a serum-free and heat-sterilizable medium and continuous high-density cell culture.

    PubMed

    Minamoto, Y; Ogawa, K; Abe, H; Iochi, Y; Mitsugi, K

    1991-01-01

    We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium.As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth. As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth. The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37°C for 4 months. In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640+10% FBS.It has been proved that BSA and transferrin, which are also heat-labile but essential for the growth of various cell lines, can be substituted by heat-stable alpha-cyclodextrin and cholesterol, and Fe-gluconate, respectively. Insulin has also proved to be heat stable in a solution of Fe-gluconate. We thus established a new serum-free medium, all the components of which could be heat-sterilizable.Moreover, by adding EGF and BSA but without the adhesion factor included in FBS, the serum-free medium was found to support a long-term serial culture of a human diploid fibroblast.Finally, with this auotoclavable serum-free medium in a perfusion culture apparatus, we were able to continuously cultivate a human lymphoblastoid cell line. The production rate of IgM was found to be markedly increased by feeding the serum-free medium enriched by glucose, bicarbonate, L-Cys, and approtinin. The cell density reached as high as 2×10(8)/ml in the serum-free medium. Although the working volume in the reactor was only 1 1, the rate of IgM production reached 480 mg/day.The new heat-sterilizable serum-free medium has several advantages, because L-Gln peptide is a heat-stable and available precursor of L-Gln.

  2. Development of a serum-free and heat-sterilizable medium and continuous high-density cell culture.

    PubMed

    Minamoto, Y; Ogawa, K; Abe, H; Iochi, Y; Mitsugi, K

    1991-01-01

    We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium. As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth. As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth. The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37 degrees C for 4 months. In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640 + 10% FBS. It has been proved that BSA and transferrin, which are also heat-labile but essential for the growth of various cell lines, can be substituted by heat-stable alpha-cyclodextrin and cholesterol, and Fe-gluconate, respectively. Insulin has also proved to be heat stable in a solution of Fe-gluconate. We thus established a new serum-free medium, all the components of which could be heat-sterilizable. Moreover, by adding EGF and BSA but without the adhesion factor included in FBS, the serum-free medium was found to support a long-term serial culture of a human diploid fibroblast. Finally, with this auotoclavable serum-free medium in a perfusion culture apparatus, we were able to continuously cultivate a human lymphoblastoid cell line. The production rate of IgM was found to be markedly increased by feeding the serum-free medium enriched by glucose, bicarbonate, L-Cys, and approtinin. The cell density reached as high as 2 x 10(8)/ml in the serum-free medium. Although the working volume in the reactor was only 1 1, the rate of IgM production reached 480 mg/day. The new heat-sterilizable serum-free medium has several advantages, because L-Gln peptide is a heat-stable and available precursor of L-Gln.

  3. Prediction of PAH mutagenicity in human cells by QSAR classification.

    PubMed

    Papa, E; Pilutti, P; Gramatica, P

    2008-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants of high environmental concern. The experimental data of a mutagenicity test on human B-lymphoblastoid cells (alternative to the Ames bacterial test) for a set of 70 oxo-, nitro- and unsubstituted PAHs, detected in particulate matter (PM), were modelled by Quantitative Structure-Activity Relationships (QSAR) classification methods (k-NN, k-Nearest Neighbour, and CART, Classification and Regression Tree) based on different theoretical molecular descriptors selected by Genetic Algorithms. The best models were validated for predictivity both externally and internally. For external validation, Self Organizing Maps (SOM) were applied to split the original data set. The best models, developed on the training set alone, show good predictive performance also on the prediction set chemicals (sensitivity 69.2-87.1%, specificity 62.5-87.5%). The classification of PAHs according to their mutagenicity, based only on a few theoretical molecular descriptors, allows a preliminary assessment of the human health risk, and the prioritisation of these compounds.

  4. The IL-15R alpha chain signals through association with Syk in human B cells.

    PubMed

    Bulanova, E; Budagian, V; Pohl, T; Krause, H; Dürkop, H; Paus, R; Bulfone-Paus, S

    2001-12-01

    The alpha-chain of the IL-15R (IL-15Ralpha) serves as the specific, high-affinity receptor for IL-15. It is expressed by lymphoid and nonlymphoid cells, including B cell lymphoma lines. In this study, we have further explored IL-15Ralpha-mediated signaling in activated primary B cells and in Raji cells, a human B-lymphoblastoid cell line which expresses the IL-15Ralpha and IL-2Rgamma chains, but lacks the IL-2Rbeta chain. Stimulation of Raji cells with IL-15 induces their proliferation and rescues them from C2-ceramide-induced apoptosis. By immunoprecipitation and Western blotting, we show that treatment of Raji cells and activated primary B cells with IL-15 induces coprecipitation of Syk kinase with the IL-15Ralpha chain. Upon association, the activated Syk kinase phosphorylates the IL-15Ralpha chain as well as phospholipase Cgamma, which coprecipitates with Syk. Furthermore, transfection of Raji cells with stem-loop Syk antisense oligonucleotides prevents IL-15Ralpha and phospholipase Cgamma phosphorylation as well as the inhibition of apoptosis by IL-15. Mutation of a defined region of the intracellular signaling portion of IL-15Ralpha (Tyr227) abrogates both the IL-15Ralpha/Syk association and IL-15Ralpha phosphorylation. Taken together, this suggests that Syk kinase physically and functionally associates with the IL-15Ralpha chain in B cells and that Syk plays a key role in mediating IL-15-induced signal transduction, thus accounting for the distinct functional consequences of IL-15 vs IL-2 binding to B cells.

  5. Global methylation profiles in DNA from different blood cell types.

    PubMed

    Wu, Hui-Chen; Delgado-Cruzata, Lissette; Flom, Julie D; Kappil, Maya; Ferris, Jennifer S; Liao, Yuyan; Santella, Regina M; Terry, Mary Beth

    2011-01-01

    DNA methylation measured in white blood cell DNA is increasingly being used as in studies of cancer susceptibility. However, little is known about the correlation between different assays to measure global methylation and whether the source of DNA matters when examining methylation profiles in different blood cell types. Using information from 620 women, 217 and 403 women with DNA available from granulocytes (Gran), and total white blood cells (WBC), respectively, and 48 women with DNA available from four different sources (WBC, Gran, mononuclear (MN), and lymphoblastoid cell lines (LCL)), we compared DNA methylation for three repetitive elements (LINE1, Sat2, Alu) by MethyLight, luminometric methylation assay (LUMA), and [(3)H]-methyl acceptance assay. For four of the five assays, DNA methylation levels measured in Gran were not correlated with methylation in LBC, MN, or WBC; the exception was Sat2. DNA methylation in LCL was correlated with methylation in MN and WBC for the [(3)H]-methyl acceptance, LINE1, and Alu assays. Methylation in MN was correlated with methylation in WBC for the [(3)H]-methyl acceptance and LUMA assays. When we compared the five assays to each other by source of DNA, we observed statistically significant positive correlations ranging from 0.3-0.7 for each cell type with one exception (Sat2 and Alu in MN). Among the 620 women stratified by DNA source, correlations among assays were highest for the three repetitive elements (range 0.39-0.64). Results from the LUMA assay were modestly correlated with LINE1 (0.18-0.20). These results suggest that both assay and source of DNA are critical components in the interpretation of global DNA methylation patterns from WBC.

  6. Protective activity of C-geranylflavonoid analogs from Paulownia tomentosa against DNA damage in 137Cs irradiated AHH-1 cells.

    PubMed

    Moon, Hyung-In; Jeong, Min Ho; Jo, Wol Soon

    2014-09-01

    Radiotherapy is an important form of treatment for a wide range of cancers, but it can damage DNA and cause adverse effects. We investigated if the diplacone analogs of P. tomentosa were radio-protective in a human lymphoblastoid cell line (AHH-1). Four geranylated flavonoids, diplacone, 3'-O-methyl-5'-hydroxydiplacone, 3'-O-methyl-5'-O-methyldiplacone and 3'-O-methyldiplacol, were tested for their antioxidant and radio-protective effects. Diplacone analogs effectively scavenged free radicals and inhibited radiation-induced DNA strand breaks in vitro. They significantly decreased levels of reactive oxygen species and cellular DNA damage in 2 Gy-irradiated AHH-1 cells. Glutathione levels and superoxide dismutase activity in irradiated AHH-1 cells increased significantly after treatment with these analogs. The enhanced biological anti-oxidant activity and radioprotective activity of diplacone analogs maintained the survival of irradiated AHH-1 cells in a clonogenic assay. These data suggest that diplacone analogs may protect healthy tissue surrounding tumor cells during radiotherapy to ensure better control of radiotherapy and allow higher doses of radiotherapy to be employed.

  7. Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer.

    PubMed

    Rudin, Charles M; Durinck, Steffen; Stawiski, Eric W; Poirier, John T; Modrusan, Zora; Shames, David S; Bergbower, Emily A; Guan, Yinghui; Shin, James; Guillory, Joseph; Rivers, Celina Sanchez; Foo, Catherine K; Bhatt, Deepali; Stinson, Jeremy; Gnad, Florian; Haverty, Peter M; Gentleman, Robert; Chaudhuri, Subhra; Janakiraman, Vasantharajan; Jaiswal, Bijay S; Parikh, Chaitali; Yuan, Wenlin; Zhang, Zemin; Koeppen, Hartmut; Wu, Thomas D; Stern, Howard M; Yauch, Robert L; Huffman, Kenneth E; Paskulin, Diego D; Illei, Peter B; Varella-Garcia, Marileila; Gazdar, Adi F; de Sauvage, Frederic J; Bourgon, Richard; Minna, John D; Brock, Malcolm V; Seshagiri, Somasekar

    2012-10-01

    Small-cell lung cancer (SCLC) is an exceptionally aggressive disease with poor prognosis. Here, we obtained exome, transcriptome and copy-number alteration data from approximately 53 samples consisting of 36 primary human SCLC and normal tissue pairs and 17 matched SCLC and lymphoblastoid cell lines. We also obtained data for 4 primary tumors and 23 SCLC cell lines. We identified 22 significantly mutated genes in SCLC, including genes encoding kinases, G protein-coupled receptors and chromatin-modifying proteins. We found that several members of the SOX family of genes were mutated in SCLC. We also found SOX2 amplification in ∼27% of the samples. Suppression of SOX2 using shRNAs blocked proliferation of SOX2-amplified SCLC lines. RNA sequencing identified multiple fusion transcripts and a recurrent RLF-MYCL1 fusion. Silencing of MYCL1 in SCLC cell lines that had the RLF-MYCL1 fusion decreased cell proliferation. These data provide an in-depth view of the spectrum of genomic alterations in SCLC and identify several potential targets for therapeutic intervention.

  8. Ultraviolet-induced mutations in Cockayne syndrome cells are primarily caused by cyclobutane dimer photoproducts while repair of other photoproducts is normal

    SciTech Connect

    Parris, C.N.; Kraemer, K.H. )

    1993-08-01

    The authors compared the contribution to mutagenesis on Cockayne syndrome (CS) cells of the major class of UV photoproducts, the cyclobutane pyrimidine dimer, to that of other DNA photoproducts by using the mutagenesis shuttle vector pZ189. Lymphoblastoid cell lines from the DNA repair-deficient disorders CS and xeroderma pigmentosum (XP) and a normal line were transfected with UV-treated pZ189. Cyclobutane dimers were selectively removed before transfection by photoreactivation (PR), leaving nondimer photoproducts intact. After UV exposure and replication in CS and XP cells, plasmid survival was abnormally elevated. After PR, plasmid survival increased and mutation frequency in CS cells decreased to normal levels but remained abnormal in XP cells. Sequence analysis of >200 mutant plasmids showed that with CS cells a major mutational hot spot was caused by unrepaired cyclobutane dimers. These data indicate that with both CS and XP cyclobutane dimers are major photoproducts generating reduced plasmid survival and increased mutation frequency. However, unlike XP, CS cells are proficient in repair of nondimer photoproducts. Since XP but not CS patients have a high frequency of UV-induced skin cancers, the data suggest that prevention of UV-induced skin cancers is associated with proficient repair of nondimer photoproducts. 38 refs., 3 figs., 2 tabs.

  9. The c-Jun N-terminal kinase pathway is critical for cell transformation by the latent membrane protein 1 of Epstein-Barr virus

    SciTech Connect

    Kutz, Helmut; Reisbach, Gilbert; Schultheiss, Ute; Kieser, Arnd

    2008-02-20

    The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) transforms cells activating signal transduction pathways such as NF-{kappa}B, PI3-kinase, or c-Jun N-terminal kinase (JNK). Here, we investigated the functional role of the LMP1-induced JNK pathway in cell transformation. Expression of a novel dominant-negative JNK1 allele caused a block of proliferation in LMP1-transformed Rat1 fibroblasts. The JNK-specific inhibitor SP600125 reproduced this effect in Rat1-LMP1 cells and efficiently interfered with proliferation of EBV-transformed lymphoblastoid cells (LCLs). Inhibition of the LMP1-induced JNK pathway in LCLs caused the downregulation of c-Jun and Cdc2, the essential G2/M cell cycle kinase, which was accompanied by a cell cycle arrest of LCLs at G2/M phase transition. Moreover, SP600125 retarded tumor growth of LCLs in a xenograft model in SCID mice. Our data support a critical role of the LMP1-induced JNK pathway for proliferation of LMP1-transformed cells and characterize JNK as a potential target for intervention against EBV-induced malignancies.

  10. Withania somnifera Induces Cytotoxic and Cytostatic Effects on Human T Leukemia Cells

    PubMed Central

    Turrini, Eleonora; Calcabrini, Cinzia; Sestili, Piero; Catanzaro, Elena; de Gianni, Elena; Diaz, Anna Rita; Hrelia, Patrizia; Tacchini, Massimo; Guerrini, Alessandra; Canonico, Barbara; Papa, Stefano; Valdrè, Giovanni; Fimognari, Carmela

    2016-01-01

    Cancer chemotherapy is characterized by an elevated intrinsic toxicity and the development of drug resistance. Thus, there is a compelling need for new intervention strategies with an improved therapeutic profile. Immunogenic cell death (ICD) represents an innovative anticancer strategy where dying cancer cells release damage-associated molecular patterns promoting tumor-specific immune responses. The roots of Withania somnifera (W. somnifera) are used in the Indian traditional medicine for their anti-inflammatory, immunomodulating, neuroprotective, and anticancer activities. The present study is designed to explore the antileukemic activity of the dimethyl sulfoxide extract obtained from the roots of W. somnifera (WE). We studied its cytostatic and cytotoxic activity, its ability to induce ICD, and its genotoxic potential on a human T-lymphoblastoid cell line by using different flow cytometric assays. Our results show that WE has a significant cytotoxic and cytostatic potential, and induces ICD. Its proapoptotic mechanism involves intracellular Ca2+ accumulation and the generation of reactive oxygen species. In our experimental conditions, the extract possesses a genotoxic potential. Since the use of Withania is suggested in different contexts including anti-infertility and osteoarthritis care, its genotoxicity should be carefully considered for an accurate assessment of its risk–benefit profile. PMID:27187469

  11. Epstein–Barr virus–host cell interactions: an epigenetic dialog?

    PubMed Central

    Niller, Hans H.; Szenthe, Kalman; Minarovits, Janos

    2014-01-01

    Here, we wish to highlight the genetic exchange and epigenetic interactions between Epstein–Barr virus (EBV) and its host. EBV is associated with diverse lymphoid and epithelial malignancies. Their molecular pathogenesis is accompanied by epigenetic alterations which are distinct for each of them. While lymphoblastoid cell lines derived from B cells transformed by EBV in vitro are characterized by a massive demethylation and euchromatinization of the viral and cellular genomes, the primarily malignant lymphoid tumor Burkitt’s lymphoma and the epithelial tumors nasopharyngeal carcinoma and EBV-associated gastric carcinoma are characterized by hypermethylation of a multitude of cellular tumor suppressor gene loci and of the viral genomes. In some cases, the viral latency and oncoproteins including the latent membrane proteins LMP1 and LMP2A and several nuclear antigens affect the level of cellular DNA methyltransferases or interact with the histone modifying machinery. Specific molecular mechanisms of the epigenetic dialog between virus and host cell remain to be elucidated. PMID:25400657

  12. Epstein-Barr Virus oncoprotein super-enhancers control B cell growth

    PubMed Central

    Zhou, Hufeng; Schmidt, Stefanie CS; Jiang, Sizun; Willox, Bradford; Bernhardt, Katharina; Liang, Jun; Johannsen, Eric C; Kharchenko, Peter; Gewurz, Benjamin E; Kieff, Elliott; Zhao, Bo

    2015-01-01

    Summary Super-enhancers are clusters of gene-regulatory sites bound by multiple transcription factors that govern cell transcription, development, phenotype, and oncogenesis. By examining Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs), we identified four EBV oncoproteins and five EBV-activated NF-κB subunits co-occupying ~1800 enhancer sites. Of these, 187 had markedly higher and broader histone H3K27ac signals characteristic of super-enhancers, and were designated “EBV super-enhancers”. EBV super-enhancer-associated genes included the MYC and BCL2 oncogenes, enabling LCL proliferation and survival. EBV super-enhancers were enriched for B cell transcription factor motifs and had a high co-occupancy of the transcription factors STAT5 and NFAT. EBV super-enhancer-associated genes were more highly expressed than other LCL genes. Disrupting EBV super-enhancers by the bromodomain inhibitor, JQ1 or conditionally inactivating an EBV oncoprotein or NF-κB decreased MYC or BCL2 expression and arrested LCL growth. These findings provide insight into mechanisms of EBV-induced lymphoproliferation and identify potential therapeutic interventions. PMID:25639793

  13. Pre-stimulation of CD81 expression by resting B cells increases proliferation following EBV infection, but the overexpression of CD81 induces the apoptosis of EBV-transformed B cells.

    PubMed

    Park, Ga Bin; Kim, Daejin; Park, Sung Jae; Lee, Hyun-Kyung; Kim, Ji Hyun; Kim, Yeong Seok; Park, Sae-Gwang; Choi, In-Hak; Yoon, Sung Ho; Lee, Youn Jae; Paeng, Sunghwa; Hur, Dae Young

    2015-12-01

    Hepatitis C virus (HCV) E2 protein binds to CD81, which is a component of the B cell co-stimulatory complex. The E2-CD81 interaction leads to B cell proliferation, protein tyrosine phosphorylation and to the hypermutation of immunoglobulin genes. Epidemiological studies have reported a high prevalence of B cell non-Hodgkin lymphoma (NHL) in HCV-positive patients, suggesting a potential association between HCV and Epstein-Barr virus (EBV) in the genesis of B lymphocyte proliferative disorders. In the present study, in order to investigate the association between EBV and HCV in B cells, we created an in vitro EBV-induced B cell transformation model. CD81 was gradually overexpressed during transformation by EBV. B cells isolated from HCV-positive patients grew more rapidly and clumped together earlier than B cells isolated from healthy donors following EBV infection. Pre-stimulation of CD81 expressed by resting B cells with anti-CD81 monoclonal antibody (mAb) or HCV E2 accelerated the generation of lymphoblastoid cell lines (LCLs) by EBV infection. These cells proliferated prominently through the early expression of interleukin-10 and intracellular latent membrane protein (LMP)-l. By contrast, the overexpression of CD81 on EBV-transformed B cells by anti-CD81 mAb or HCV E2 protein induced apoptosis through reactive oxygen species (ROS)-mediated mitochondrial dysfunction. These results suggest that the engagement of CD81 expressed by B cells has differential effects on B cell fate (proliferation or apoptosis) according to EBV infection and the expression level of CD81.

  14. Virus and Autoantigen-Specific CD4+ T Cells Are Key Effectors in a SCID Mouse Model of EBV-Associated Post-Transplant Lymphoproliferative Disorders

    PubMed Central

    Linnerbauer, Stefanie; Behrends, Uta; Adhikary, Dinesh; Witter, Klaus; Bornkamm, Georg W.; Mautner, Josef

    2014-01-01

    Polyclonal Epstein-Barr virus (EBV)-infected B cell line (lymphoblastoid cell lines; LCL)-stimulated T-cell preparations have been successfully used to treat EBV-positive post-transplant lymphoproliferative disorders (PTLD) in transplant recipients, but function and specificity of the CD4+ component are still poorly defined. Here, we assessed the tumor-protective potential of different CD4+ T-cell specificities in a PTLD-SCID mouse model. Injection of different virus-specific CD4+ T-cell clones showed that single specificities were capable of prolonging mouse survival and that the degree of tumor protection directly correlated with recognition of target cells in vitro. Surprisingly, some CD4+ T-cell clones promoted tumor development, suggesting that besides antigen recognition, still elusive functional differences exist among virus-specific T cells. Of several EBV-specific CD4+ T-cell clones tested, those directed against virion antigens proved most tumor-protective. However, enriching these specificities in LCL-stimulated preparations conferred no additional survival benefit. Instead, CD4+ T cells specific for unknown, probably self-antigens were identified as principal antitumoral effectors in LCL-stimulated T-cell lines. These results indicate that virion and still unidentified cellular antigens are crucial targets of the CD4+ T-cell response in this preclinical PTLD-model and that enriching the corresponding T-cell specificities in therapeutic preparations may enhance their clinical efficacy. Moreover, the expression in several EBV-negative B-cell lymphoma cell lines implies that these putative autoantigen(s) might also qualify as targets for T-cell-based immunotherapy of virus-negative B cell malignancies. PMID:24853673

  15. Maintenance of imprinting and nuclear architecture in cycling cells.

    PubMed

    Teller, Kathrin; Solovei, Irina; Buiting, Karin; Horsthemke, Bernhard; Cremer, Thomas

    2007-09-18

    Dynamic gene repositioning has emerged as an additional level of epigenetic gene regulation. An early example was the report of a transient, spatial convergence (< or =2 microm) of oppositely imprinted regions ("kissing"), including the Angelman syndrome/Prader-Willi syndrome (AS/PWS) locus and the Beckwith-Wiedemann syndrome locus in human lymphocytes during late S phase. It was argued that kissing is required for maintaining opposite imprints in cycling cells. Employing 3D-FISH with a BAC contig covering the AS/PWS region, light optical, serial sectioning, and quantitative 3D-image analysis, we observed that both loci always retained a compact structure and did not form giant loops. Three-dimensional distances measured among various, homologous AS/PWS segments in 393 human lymphocytes, 132 human fibroblasts, and 129 lymphoblastoid cells from Gorilla gorilla revealed a wide range of distances at any stage of interphase and in G(0). At late S phase, 4% of nuclei showed distances < or =2 microm, 49% showed distances >6 microm, and 18% even showed distances >8 microm. A similar distance variability was found for Homo sapiens (HSA) 15 centromeres in a PWS patient with a deletion of the maternal AS/PWS locus and for the Beckwith-Wiedemann syndrome loci in human lymphocytes. A transient kiss during late S phase between loci widely separated at other stages of the cell cycle seems incompatible with known global constraints of chromatin movements in cycling cells. Further experiments suggest that the previously observed convergence of AS/PWS loci during late S phase was most likely a side effect of the convergence of nucleolus organizer region-bearing acrocentric human chromosomes, including HSA 15.

  16. Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells.

    PubMed

    Iordanskiy, Sergey; Van Duyne, Rachel; Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao; Romerio, Fabio; Kashanchi, Fatah

    2015-11-01

    The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4(+) T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4(+) T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4(+) T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the "Shock and Kill" strategy for latently HIV-1 infected cells.

  17. Dynamic Epstein-Barr virus gene expression on the path to B-cell transformation.

    PubMed

    Price, Alexander M; Luftig, Micah A

    2014-01-01

    Epstein-Barr virus (EBV) is an oncogenic human herpesvirus in the γ-herpesvirinae subfamily that contains a 170-180kb double-stranded DNA genome. In vivo, EBV commonly infects B and epithelial cells and persists for the life of the host in a latent state in the memory B-cell compartment of the peripheral blood. EBV can be reactivated from its latent state, leading to increased expression of lytic genes that primarily encode for enzymes necessary to replicate the viral genome and structural components of the virion. Lytic cycle proteins also aid in immune evasion, inhibition of apoptosis, and the modulation of other host responses to infection. In vitro, EBV has the potential to infect primary human B cells and induce cellular proliferation to yield effectively immortalized lymphoblastoid cell lines, or LCLs. EBV immortalization of B cells in vitro serves as a model system for studying EBV-mediated lymphomagenesis. While much is known about the steady-state viral gene expression within EBV-immortalized LCLs and other EBV-positive cell lines, relatively little is known about the early events after primary B-cell infection. It was previously thought that upon latent infection, EBV only expressed the well-characterized latency-associated transcripts found in LCLs. However, recent work has characterized the early, but transient, expression of lytic genes necessary for efficient transformation and delayed responses in the known latency genes. This chapter summarizes these recent findings that show how dynamic and controlled expression of multiple EBV genes can control the activation of B cells, entry into the cell cycle, the inhibition of apoptosis, and innate and adaptive immune responses.

  18. Folate metabolite profiling of different cell types and embryos suggests variation in folate one-carbon metabolism, including developmental changes in human embryonic brain.

    PubMed

    Leung, Kit-Yi; De Castro, Sandra C P; Cabreiro, Filipe; Gustavsson, Peter; Copp, Andrew J; Greene, Nicholas D E

    2013-06-01

    Folates act as co-factors for transfer of one-carbon units for nucleotide production, methylation and other biosynthetic reactions. Comprehensive profiling of multiple folates can be achieved using liquid chromatography tandem mass spectrometry, enabling determination of their relative abundance that may provide an indication of metabolic differences between cell types. For example, cell lines exposed to methotrexate showed a dose-dependent elevation of dihydrofolate, consistent with inhibition of dihydrofolate reductase. We analysed the folate profile of E. coli sub-types as well as cell lines and embryonic tissue from both human and mouse. The folate profile of bacteria differed markedly from those of all the mammalian samples, most notably in the greater abundance of formyl tetrahydrofolate. The overall profiles of mouse and human fibroblasts and mid-gestation mouse embryos were broadly similar, with specific differences. The major folate species in these cell types was 5-methyl tetrahydrofolate, in contrast to lymphoblastoid cell lines in which the predominant form was tetrahydrofolate. Analysis of embryonic human brain revealed a shift in folate profile with increasing developmental stage, with a decline in relative abundance of dihydrofolate and increase in 5-methyl tetrahydrofolate. These cell type-specific and developmental changes in folate profile may indicate differential requirements for the various outputs of folate metabolism.

  19. Multiple Sclerosis Risk Allele in CLEC16A Acts as an Expression Quantitative Trait Locus for CLEC16A and SOCS1 in CD4+ T Cells.

    PubMed

    Leikfoss, Ingvild S; Keshari, Pankaj K; Gustavsen, Marte W; Bjølgerud, Anja; Brorson, Ina S; Celius, Elisabeth G; Spurkland, Anne; Bos, Steffan D; Harbo, Hanne F; Berge, Tone

    2015-01-01

    For multiple sclerosis, genome wide association studies and follow up studies have identified susceptibility single nucleotide polymorphisms located in or near CLEC16A at chromosome 16p13.13, encompassing among others CIITA, DEXI and SOCS1 in addition to CLEC16A. These genetic variants are located in intronic or intergenic regions and display strong linkage disequilibrium with each other, complicating the understanding of their functional contribution and the identification of the direct causal variant(s). Previous studies have shown that multiple sclerosis-associated risk variants in CLEC16A act as expression quantitative trait loci for CLEC16A itself in human pancreatic β-cells, for DEXI and SOCS1 in thymic tissue samples, and for DEXI in monocytes and lymphoblastoid cell lines. Since T cells are major players in multiple sclerosis pathogenesis, we have performed expression analyses of the CIITA-DEXI-CLEC16A-SOCS1 gene cluster in CD4+ and CD8+ T cells isolated from multiple sclerosis patients and healthy controls. We observed a higher expression of SOCS1 and CLEC16A in CD4+ T cells in samples homozygous for the risk allele of CLEC16A rs12927355. Pair-wise linear regression analysis revealed high correlation in gene expression in peripheral T cells of CIITA, DEXI, CLEC16A and SOCS1. Our data imply a possible regulatory role for the multiple sclerosis-associated rs12927355 in CLEC16A.

  20. Binding of /sup 125/I-labeled recombinant beta interferon (IFN-beta Ser17) to human cells

    SciTech Connect

    O'Rourke, E.C.; Drummond, R.J.; Creasey, A.A.

    1984-12-01

    The authors investigated the binding of /sup 125/I-labeled beta interferon (IFN-beta Ser17), a nonglycosylated recombinant human fibroblast interferon in which cysteine at position 17 is replaced by serine by site-specific mutagenesis. An optimized chloramine T radiolabeling method produced a highly labeled, fully active /sup 125/I-IFN suitable for these studies. Unlike the case with the chloramine T method, incorporation of a single mole of Bolton-Hunter reagent into a mole of IFN-beta Ser17 led to nearly complete loss of biological activity. /sup 125/I-IFN-beta Ser17, prepared by the chloramine T method, bound specifically to human lymphoblastoid cells (Daudi) with a dissociation constant of 0.24 nM. The number of binding sites per cell was 4,000. In competition assays, unlabeled beta interferons (native, recombinant IFN-beta Cys17, and various preparations of IFN-beta Ser17) equally displaced labeled IFN-beta Ser17 on Daudi cells. Recombinant IFN-alpha-1 displaced /sup 125/I-IFN-beta binding to Daudi cells less efficiently than did unlabeled native or recombinant beta interferon. However, at the concentrations tested, native gamma interferon showed no competition with /sup 125/I-IFN. The results indicate that IFN-beta Ser17 and native IFN-beta posses similar binding properties.

  1. SB subregion of the human major histocompatibility complex: gene organization, allelic polymorphism and expression in transformed cells.

    PubMed

    Okada, K; Prentice, H L; Boss, J M; Levy, D J; Kappes, D; Spies, T; Raghupathy, R; Mengler, R A; Auffray, C; Strominger, J L

    1985-03-01

    The SB region of the human major histocompatibility complex (MHC) has been cloned from cosmid and lambda phage libraries made from the human B-lymphoblastoid cell line Priess (DR4/4, DC4/4, SB3/4). Two alpha genes and two beta genes are encoded in the 100 kb long SB region in the order SB alpha-SB beta-SX alpha-SX beta. The SB alpha and SB beta genes encode the alpha and beta subunits of the SB subset of class II MHC molecules. Both the SX alpha and the SX beta genes are pseudogenes in the haplotype examined. From the isolated clones, the two haplotypes of the Priess cell line, SB3 and SB4, are distinguished by nucleotide sequencing and blot hybridization analyses. Restriction site polymorphisms between the SB3 and SB4 clones were observed only in relatively small regions of the SB beta and SX beta genes. A mouse macrophage cell line was transfected with one of the cosmid clones containing both SB alpha and SB beta genes. Expression of the alpha and beta genes was detected by fluorescene-activated cell sorting (FACS) and two-dimensional gel electrophoresis using SB-specific monoclonal antibodies.

  2. DNA Electrophoretic Migration Patterns Change after Exposure of Jurkat Cells to a Single Intense Nanosecond Electric Pulse

    PubMed Central

    Romeo, Stefania; Zeni, Luigi; Sarti, Maurizio; Sannino, Anna; Scarfì, Maria Rosaria; Vernier, P. Thomas; Zeni, Olga

    2011-01-01

    Intense nanosecond pulsed electric fields (nsPEFs) interact with cellular membranes and intracellular structures. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of nsPEFs, including cancer therapy, and b) better understanding of the mechanisms underlying such bioelectrical effects. In this work, we explored relatively mild exposure conditions to provide insight into weak, reversible effects, laying a foundation for a better understanding of the interaction mechanisms and kinetics underlying nsPEF bio-effects. In particular, we report changes in the nucleus of Jurkat cells (human lymphoblastoid T cells) exposed to single pulses of 60 ns duration and 1.0, 1.5 and 2.5 MV/m amplitudes, which do not affect cell growth and viability. A dose-dependent reduction in alkaline comet-assayed DNA migration is observed immediately after nsPEF exposure, accompanied by permeabilization of the plasma membrane (YO-PRO-1 uptake). Comet assay profiles return to normal within 60 minutes after pulse delivery at the highest pulse amplitude tested, indicating that our exposure protocol affects the nucleus, modifying DNA electrophoretic migration patterns. PMID:22164287

  3. Proteoglycan expression correlates with the phenotype of malignant and non-malignant EBV-positive B-cell lines

    PubMed Central

    Tsidulko, Alexandra Y.; Matskova, Liudmila; Astakhova, Lidiia A.; Ernberg, Ingemar; Grigorieva, Elvira V.

    2015-01-01

    The involvement of proteoglycans (PGs) in EBV-host interactions and lymphomagenesis remains poorly investigated. In this study, expression of major proteoglycans (syndecan-1, glypican-1, perlecan, versican, brevican, aggrecan, NG2, serglycin, decorin, biglycan, lumican, CD44), heparan sulphate (HS) metabolic system (EXT1/2, NDST1/2, GLCE, HS2ST1, HS3ST1/2, HS6ST1/2, SULF1/2, HPSE) and extracellular matrix (ECM) components (collagen 1A1, fibronectin, elastin) in primary B cells and EBV carrying cell lines with different phenotypes, patterns of EBV-host cell interaction and viral latency stages (type I-III) was investigated. Primary B cells expressed a wide repertoire of PGs (dominated by serglycin and CD44) and ECM components. Lymphoblastoid EBV+ B cell lines (LCLs) showed specific PG expression with down-regulation of CD44 and ECM components and up-regulation of serglycin and perlecan/HSPG2. For Burkitt's lymphoma cells (BL), serglycin was down-regulated in BL type III cells and perlecan in type I BL cells. The biosynthetic machinery for HS was active in all cell lines, with some tendency to be down-regulated in BL cells. 5′-aza-dC and/or Trichostatin A resulted in transcriptional upregulation of the genes, suggesting that low expression of ECM components, proteoglycan core proteins and HS biosynthetic system is due to epigenetic suppression in type I cells. Taken together, our data show that proteoglycans are expressed in primary B lymphocytes whereas they are not or only partly expressed in EBV-carrying cell lines, depending on their latency type program. PMID:26527314

  4. Chromosome damage evolution after low and high LET irradiation

    NASA Astrophysics Data System (ADS)

    Andreev, Sergey; Eidelman, Yuri

    Ionizing radiation induces DNA and chromatin lesions which are converted to chromosome lesions detected in the first post-irradiation mitosis by classic cytogenetic techniques as chromosomal aberrations (CAs). These techniques allow to monitor also delayed aberrations observed after many cell generations post-irradiation - the manifestation of chromosomal instability phenotype (CIN). The problem discussed is how to predict time evolution from initial to delayed DNA/chromosome damage. To address this question, in the present work a mechanistic model of CIN is elaborated which integrates pathways of (*) DNA damage induction and its conversion to chromosome lesions (aberrations), (**) lesion transmission and generation through cell cycles. Delayed aberrations in subsequent cycles are formed in the model owing to two pathways, DNA damage generation de novo as well as CA transmission from previous cycles. DNA damage generation rate is assumed to consist of bystander and non-bystander components. Bystander signals impact all cells roughly equally, whereas non-bystander DSB generation rate differs for the descendants of unirradiated and irradiated cells. Monte Carlo simulation of processes underlying CIN allows to predict the time evolution of initial radiation-induced damage - kinetics curve for delayed unstable aberrations (dicentrics) together with dose response and RBE as a function of time after high vs low LET irradiation. The experimental data for radiation-induced CIN in TK6 lymphoblastoid cells and human lymphocytes irradiated with low (gamma) and high (Fe, C) LET radiation are analyzed on the basis of the proposed model. One of the conclusions is that without bystander signaling, just taking into account the initial DNA damage and non-bystander DSB generation, it is impossible to describe the available experimental data for high-LET-induced CIN. The exact contribution of bystander effects for high vs low LET remains unknown, but the relative contribution may be

  5. CD8+ T-cell clones specific for the 5T4 antigen target renal cell carcinoma tumor-initiating cells in a murine xenograft model.

    PubMed

    Tykodi, Scott S; Satoh, Shoko; Deming, Janise D; Chou, Jeffrey; Harrop, Richard; Warren, Edus H

    2012-09-01

    The tumor antigen 5T4 is frequently expressed at high levels on renal cell carcinoma (RCC) and other epithelial carcinomas. Surveys of normal tissues demonstrate abundant 5T4 expression on placental trophoblast cells with limited expression elsewhere. 5T4 is the target for a therapeutic cancer vaccine (MVA-5T4) that elicits 5T4-specific serological, proliferative, and cytotoxic T lymphocyte (CTL) responses. However, the antitumor activity of 5T4-specific CTL has not been extensively characterized. CD8 T cells from HLA-A2 healthy donors (n=4) or RCC patients (n=2) were stimulated in vitro with the HLA-A2-binding nonamer peptides 5T417-25 or 5T497-105 and screened by flow cytometry with specific tetramers (TET). CD8/TET T-cell clones specific for 5T417-25 or 5T497-105 peptide were isolated from 4/6 and 1/4 donors, respectively. A subset of clones specific for 5T417-25 was cytolytic for MVA-5T4-infected HLA-A2 EBV-transformed lymphoblastoid cell line target cells and for constitutively HLA-A2-expressing and 5T4-expressing RCC tumor cell lines (including A498 RCC). In a xenoengraftment assay, the coinoculation of a representative 5T417-25-specific CTL clone with A498 RCC tumors cells into immune-deficient mice completely prevented growth of A498 tumors. Taken together, these data demonstrate high-avidity CD8 CTL able to recognize the naturally processed 5T417-25 epitope on RCC tumor cells including putative tumor-initiating cells are present in peripheral blood of both healthy donors and RCC patients. CD8T-cell immunity targeting 5T417-25 is therefore of substantial interest both as a potential target for further development of vaccination or adoptive cellular immunotherapy and for immune monitoring studies in association with nonspecific immunotherapies.

  6. Autocrine CCL3 and CCL4 Induced by the Oncoprotein LMP1 Promote Epstein-Barr Virus-Triggered B Cell Proliferation

    PubMed Central

    Tsai, Shu-Chun; Lin, Sue-Jane; Lin, Cheau-Jye; Chou, Ya-Ching; Lin, Jiun-Han; Yeh, Te-Huei; Chen, Mei-Ru; Huang, Li-Min; Lu, Meng-You; Huang, Ya-Chi; Chen, Huan-Yun

    2013-01-01

    Epstein-Barr virus (EBV) alters the regulation and expression of a variety of cytokines in its host cells to modulate host immune surveillance and facilitate viral persistence. Using cytokine antibody arrays, we found that, in addition to the cytokines reported previously, two chemotactic cytokines, CCL3 and CCL4, were induced in EBV-infected B cells and were expressed at high levels in all EBV-immortalized lymphoblastoid cell lines (LCLs). Furthermore, EBV latent membrane protein 1 (LMP1)-mediated Jun N-terminal protein kinase activation was responsible for upregulation of CCL3 and CCL4. Inhibition of CCL3 and CCL4 in LCLs using a short hairpin RNA approach or by neutralizing antibodies suppressed cell proliferation and caused apoptosis, indicating that autocrine CCL3 and CCL4 are required for LCL survival and growth. Importantly, significant amounts of CCL3 were detected in EBV-positive plasma from immunocompromised patients, suggesting that EBV modulates this chemokine in vivo. This study reveals the regulatory mechanism and a novel function of CCL3 and CCL4 in EBV-infected B cells. CCL3 might be useful as a therapeutic target in EBV-associated lymphoproliferative diseases and malignancies. PMID:23760235

  7. Rare Circulating Cells in Familial Waldenström Macroglobulinemia Displaying the MYD88 L265P Mutation Are Enriched by Epstein-Barr Virus Immortalization

    PubMed Central

    Pertesi, Maroulio; Galia, Perrine; Nazaret, Nicolas; Vallée, Maxime; Garderet, Laurent; Leleu, Xavier; Avet-Loiseau, Hervé; Foll, Matthieu; Byrnes, Graham; Lachuer, Joel; McKay, James D.; Dumontet, Charles

    2015-01-01

    The MYD88 L265P is a recurrent somatic mutation in neoplastic cells from patients with Waldenström Macroglobulinemia (WM). We identified the MYD88 L265P mutation in three individuals from unrelated families, but its presence did not explain the disease segregation within these WM pedigrees. We observed the mutation in these three individuals at high allele fractions in DNA extracted from EBV-immortalized Lymphoblastoid cell lines established from peripheral blood (LCL), but at much lower allele fractions in DNA extracted directly from peripheral blood, suggesting that this mutation is present in a clonal cell subpopulation rather than of germ-line origin. Furthermore, we observed that the MYD88 L265P mutation is enriched in WM families, detected in 40.5% of patients with familial WM or MGUS (10/22 WM, 5/15 MGUS), compared to 3.5% of patients with familial MM or MGUS (0/72 MM, 4/41 MGUS) (p = 10−7). The mutant allele frequency increased with passages in vitro after immortalization with Epstein-Barr virus (EBV) consistent with the MYD88 L265P described gain-of-function proposed for this mutation. The MYD88 L265P mutation appears to be frequently present in circulating cells in patients with WM, and MGUS, and these cells are amenable to immortalization by EBV. PMID:26352266

  8. Tracking the processing of damaged DNA double-strand break ends by ligation-mediated PCR: increased persistence of 3'-phosphoglycolate termini in SCAN1 cells.

    PubMed

    Akopiants, Konstantin; Mohapatra, Susovan; Menon, Vijay; Zhou, Tong; Valerie, Kristoffer; Povirk, Lawrence F

    2014-03-01

    To track the processing of damaged DNA double-strand break (DSB) ends in vivo, a method was devised for quantitative measurement of 3'-phosphoglycolate (PG) termini on DSBs induced by the non-protein chromophore of neocarzinostatin (NCS-C) in the human Alu repeat. Following exposure of cells to NCS-C, DNA was isolated, and labile lesions were chemically stabilized. All 3'-phosphate and 3'-hydroxyl ends were enzymatically capped with dideoxy termini, whereas 3'-PG ends were rendered ligatable, linked to an anchor, and quantified by real-time Taqman polymerase chain reaction. Using this assay and variations thereof, 3'-PG and 3'-phosphate termini on 1-base 3' overhangs of NCS-C-induced DSBs were readily detected in DNA from the treated lymphoblastoid cells, and both were largely eliminated from cellular DNA within 1 h. However, the 3'-PG termini were processed more slowly than 3'-phosphate termini, and were more persistent in tyrosyl-DNA phosphodiesterase 1-mutant SCAN1 than in normal cells, suggesting a significant role for tyrosyl-DNA phosphodiesterase 1 in removing 3'-PG blocking groups for DSB repair. DSBs with 3'-hydroxyl termini, which are not directly induced by NCS-C, were formed rapidly in cells, and largely eliminated by further processing within 1 h, both in Alu repeats and in heterochromatic α-satellite DNA. Moreover, absence of DNA-PK in M059J cells appeared to accelerate resolution of 3'-PG ends.

  9. Fluorinated 2-(4-amino-3-methylphenyl)benzothiazoles induce CYP1A1 expression, become metabolized, and bind to macromolecules in sensitive human cancer cells.

    PubMed

    Brantley, Eileen; Trapani, Valentina; Alley, Michael C; Hose, Curtis D; Bradshaw, Tracey D; Stevens, Malcolm F G; Sausville, Edward A; Stinson, Sherman F

    2004-12-01

    Fluorinated 2-(4-amino-3-methylphenyl)benzothiazoles possess potent antiproliferative activity against certain cancer cells, similar to the unfluorinated 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495). In "sensitive" cancer cells, DF 203 is metabolized by, can induce expression of, and binds covalently to CYP1A1. Metabolism appears to be essential for its antiproliferative activity through DNA adduct formation. However, a biphasic dose-response relationship compromises its straightforward development as a chemotherapeutic agent. We investigated whether fluorinated benzothiazoles inhibit cancer cell growth without the biphasic dose-response, and whether the fluorinated benzothiazoles are also metabolized into reactive species, with binding to macromolecules in sensitive cancer cells. One fluorinated benzothiazole, 2-(4-amino-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) did exhibit potent, antiproliferative activity without a biphasic dose-response. The fluorinated benzothiazoles were also metabolized only in cells, which subsequently showed evidence of cell death. We used microsomes from genetically engineered human B-lymphoblastoid cells expressing cytochromes P450 (CYP1A1, CYP1A2, or CYP1B1) to clarify the basis for fluorinated benzothiazole metabolism. 5F 203 induced CYP1A1 and CYP1B1 mRNA expression in sensitive breast and renal cancer cells, whereas 5F 203 induced CYP1A1 mRNA but not CYP1B1 mRNA expression in sensitive ovarian cancer cells. 5F 203 did not induce CYP1A1 or CYP1B1 mRNA expression in any "resistant" cancer cells. The fluorinated benzothiazoles induced CYP1A1 protein expression exclusively in sensitive cells. [14C]5F 203 bound substantially to subcellular fractions in sensitive cells but only minimally in resistant cells. These data are concordant with the antiproliferative activity of fluorinated benzothiazoles deriving from their ability to become metabolized and bind to macromolecules within sensitive cells.

  10. Bioenergetic and Antiapoptotic Properties of Mitochondria from Cultured Human Prostate Cancer Cell Lines PC-3, DU145 and LNCaP

    PubMed Central

    Panov, Alexander; Orynbayeva, Zulfiya

    2013-01-01

    The purpose of this work was to reveal the metabolic features of mitochondria that might be essential for inhibition of apoptotic potential in prostate cancer cells. We studied mitochondria isolated from normal prostate epithelial cells (PrEC), metastatic prostate cancer cell lines LNCaP, PC-3, DU145; and non-prostate cancer cells - human fibrosarcoma HT1080 cells; and normal human lymphoblastoid cells. PrEC cells contained 2 to 4 times less mitochondria per gram of cells than the three PC cell lines. Respiratory activities of PrEC cell mitochondria were 5-20-fold lower than PC mitochondria, depending on substrates and the metabolic state, due to lower content and lower activity of the respiratory enzyme complexes. Mitochondria from the three metastatic prostate cancer cell lines revealed several features that are distinctive only to these cells: low affinity of Complex I for NADH, 20-30 mV higher electrical membrane potential (ΔΨ). Unprotected with cyclosporine A (CsA) the PC-3 mitochondria required 4 times more Ca2+ to open the permeability transition pore (mPTP) when compared with the PrEC mitochondria, and they did not undergo swelling even in the presence of alamethicin, a large pore forming antibiotic. In the presence of CsA, the PC-3 mitochondria did not open spontaneously the mPTP. We conclude that the low apoptotic potential of the metastatic PC cells may arise from inhibition of the Ca2+-dependent permeability transition due to a very high ΔΨ and higher capacity to sequester Ca2+. We suggest that due to the high ΔΨ, mitochondrial metabolism of the metastatic prostate cancer cells is predominantly based on utilization of glutamate and glutamine, which may promote development of cachexia. PMID:23951286

  11. Antigenic Properties and Processing Requirements of 65-Kilodalton Mannoprotein, a Major Antigen Target of Anti-Candida Human T-Cell Response, as Disclosed by Specific Human T-Cell Clones

    PubMed Central

    Nisini, Roberto; Romagnoli, Giulia; Gomez, Maria Jesus; La Valle, Roberto; Torosantucci, Antonella; Mariotti, Sabrina; Teloni, Raffaela; Cassone, Antonio

    2001-01-01

    T-cell-mediated immunity is known to play a central role in the host response to Candida albicans. T-cell clones are useful tools for the exact identification of fungal T-cell epitopes and the processing requirements of C. albicans antigens. We isolated human T-cell clones from an HLA-DRB1*1101 healthy donor by using an antigenic extract (MP-F2) of the fungus. Specific clones were T-cell receptor α/β and CD4+/CD8− and showed a T-helper type 1 cytokine profile (production of gamma interferon and not interleukin-4). The large majority of these clones recognized both the natural (highly glycosylated) and the recombinant (nonglycosylated) 65-kDa mannoprotein (MP65), an MP-F2 minor constituent that was confirmed to be an immunodominant antigen of the human T-cell response. Surprisingly, most of the clones recognized two synthetic peptides of different MP65 regions. However, the peptides shared the amino acid motif IXSXIXXL, which may be envisaged as a motif sequence representing the minimal epitope recognized by these clones. Three clones recognized natural and pronase-treated MP65 but did not detect nonglycosylated, recombinant MP65 or the peptides, suggesting a possible role for polysaccharides in T-cell recognition of C. albicans. Finally, lymphoblastoid B-cell lines were efficient antigen-presenting cells (APC) for recombinant MP65 and peptides but failed to present natural, glycosylated antigens, suggesting that nonprofessional APC might be defective in processing highly glycosylated yeast proteins. In conclusion, this study provides the first characterization of C. albicans-specific human T-cell clones and provides new clues for the definition of the cellular immune response against C. albicans. PMID:11349037

  12. Growth inhibitive effect of betulinic acid combined with tripterine on MSB-1 cells and its mechanism.

    PubMed

    An, N; Li, H Y; Zhang, X M

    2015-12-01

    Marek's disease (MD), a highly infectious lymphoproliferative disease in chickens, is caused by a cell-associated oncogenic herpesvirus, Marek's disease virus (MDV). MSB-1 is a MD-derived lymphoblastoid cell line and can induce tumors when inoculated into susceptible chickens. Betulinic acid, which is present as one of the major effective components in many traditional Chinese medicines, has recently been reported to inhibit growth of cancer cells and employed as a potential anticancer agent. Tripterine, a major active compound extracted from the Chinese herb Tripterygium wilfordii Hook F, has now also shown anti-tumor activities in various cancers. The aim of this study was to investigate the synergistic growth-inhibitive effect of betulinic acid combined with tripterine on MSB-1 cells and its mechanism. Viability of MSB-1 cells was assessed by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) method. Cell apoptotic analysis was performed by fluorescence detection. NF-κB transcription activity was detected by measuring luciferase activity. Western blotting was used to analyze the expression of p65, IκB and Meq. Our results showed that the proliferation in the combination group was significantly decreased as compared with that of monotherapy using betulinic acid or tripterine, accompanied by an induction of apoptosis, inhibition of NF-κB transcriptional activity and its targeting oncogenic gene Meq. The results suggest that the combination of betulinic acid and tripterine at lower concentration may produce a synergistic inhibitive effect on MSB-1 cells that warrants further investigation for its potential clinical applications.

  13. EBV infection of B-CLL cells in vitro potentiates their allostimulatory capacity if accompanied by acquisition of the activated phenotype.

    PubMed

    Avila-Cariño, J; Lewin, N; Yamamoto, K; Tomita, Y; Mellstedt, H; Brodin, B; Rosén, A; Klein, E

    1994-09-01

    Epstein-Barr virus (EBV)-carrying immortalized lymphoblastoid cell lines (LCLs) stimulate autologous T lymphocytes in vitro. This T-cell response is independent of the EBV-specific cellular memory because it also occurs in experiments with cells of seronegative individuals. The question can be posed whether the T-cell-stimulatory potential of the LCL is coupled to its immortalized state. B-CLL cells were exploited to study this question because the majority of clones, represented by different patients, can be infected with EBV but they rarely become immortalized. We have investigated the phenotypic changes and the T-cell-stimulatory capacity of EBV-infected B-CLL cells. One aliquot of CLL cells was infected with EBV, another was activated with a mixture of Staphylococcus aureus (SAC), IL-2 and the supernatant from the T-cell hybridoma MP6 (activation mixture, AcMx) and the third aliquot received both treatments. In accordance with the individual features of the clonal populations represented by each patient, the immunophenotypic changes imposed by these treatments differed. With the samples of 3 patients the allo-stimulatory potential showed the following ranking order: EBV and AcMx-treated cells > AcMx-treated > EBV-infected. An analysis of several activation-related surface markers and adhesion molecules on the cells did not reveal any association between their expression and the EBV-imposed potentiation of allostimulatory capacity. These results may be extrapolated to EBV-genome-carrying normal B cells, suggesting that they can persist in vivo only as long as they have the resting phenotype. Once they are activated, these cells may be recognized and eliminated by T lymphocytes.

  14. Assessment of targeted and non-targeted responses in cells deficient in ATM function following exposure to low and high dose X-rays.

    PubMed

    Kiuru, Anne; Kämäräinen, Meerit; Heinävaara, Sirpa; Pylkäs, Katri; Chapman, Kim; Koivistoinen, Armi; Parviainen, Teuvo; Winqvist, Robert; Kadhim, Munira; Launonen, Virpi; Lindholm, Carita

    2014-01-01

    Radiation sensitivity at low and high dose exposure to X-rays was investigated by means of chromosomal aberration (CA) analysis in heterozygous ATM mutation carrier and A-T patient (biallelic ATM mutation) lymphoblastoid cell lines (LCLs). Targeted and non-targeted responses to acutely delivered irradiation were examined by applying a co-culture system that enables study of both directly irradiated cells and medium-mediated bystander effects in the same experimental setting. No indication of radiation hypersensitivity was observed at doses of 0.01 Gy or 0.1 Gy for the ATM mutation carrier LCL. The A-T patient cells also did not show low-dose response. There was significant increase in unstable CA yields for both ATM mutation carrier and A-T LCLs at 1 and 2 Gy, the A-T cells displaying more distinct dose dependency. Both chromosome and chromatid type aberrations were induced at an increased rate in the irradiated A-T cells, whereas for ATM carrier cells, only unstable chromosomal aberrations were increased above the level observed in the wild type cell line. No bystander effect could be demonstrated in any of the cell lines or doses applied. Characteristics typical for the A-T cell line were detected, i.e., high baseline frequency of CA that increased with dose. In addition, dose-dependent loss of cell viability was observed. In conclusion, CA analysis did not demonstrate low-dose (≤100 mGy) radiosensitivity in ATM mutation carrier cells or A-T patient cells. However, both cell lines showed increased radiosensitivity at high dose exposure.

  15. Assessment of Targeted and Non-Targeted Responses in Cells Deficient in ATM Function following Exposure to Low and High Dose X-Rays

    PubMed Central

    Heinävaara, Sirpa; Pylkäs, Katri; Chapman, Kim; Koivistoinen, Armi; Parviainen, Teuvo; Winqvist, Robert; Kadhim, Munira; Launonen, Virpi; Lindholm, Carita

    2014-01-01

    Radiation sensitivity at low and high dose exposure to X-rays was investigated by means of chromosomal aberration (CA) analysis in heterozygous ATM mutation carrier and A-T patient (biallelic ATM mutation) lymphoblastoid cell lines (LCLs). Targeted and non-targeted responses to acutely delivered irradiation were examined by applying a co-culture system that enables study of both directly irradiated cells and medium-mediated bystander effects in the same experimental setting. No indication of radiation hypersensitivity was observed at doses of 0.01 Gy or 0.1 Gy for the ATM mutation carrier LCL. The A-T patient cells also did not show low-dose response. There was significant increase in unstable CA yields for both ATM mutation carrier and A-T LCLs at 1 and 2 Gy, the A-T cells displaying more distinct dose dependency. Both chromosome and chromatid type aberrations were induced at an increased rate in the irradiated A-T cells, whereas for ATM carrier cells, only unstable chromosomal aberrations were increased above the level observed in the wild type cell line. No bystander effect could be demonstrated in any of the cell lines or doses applied. Characteristics typical for the A-T cell line were detected, i.e., high baseline frequency of CA that increased with dose. In addition, dose-dependent loss of cell viability was observed. In conclusion, CA analysis did not demonstrate low-dose (≤100 mGy) radiosensitivity in ATM mutation carrier cells or A-T patient cells. However, both cell lines showed increased radiosensitivity at high dose exposure. PMID:24681528

  16. Analysis of HLA-DR glycoproteins by DNA-mediated gene transfer. Definition of DR2 beta gene products and antigen presentation to T cell clones from leprosy patients

    PubMed Central

    1988-01-01

    We have used DNA-mediated gene transfer to express HLA class II molecules in mouse L cells for serological, biochemical, and functional analysis. cDNA clones encoding the DR2 beta a and DR2 beta b products of the DR2Dw2 haplotype were subcloned into a mouse Moloney leukemia virus-based expression vector (pJ4) and transfected separately into mouse L cells together with a HLA-DR alpha/pJ4 construct. These transfectants have allowed differential analysis of the two DR2 beta products in a manner normally prohibited by the concomitant expression seen in B cells. Two-dimensional SDS-PAGE analysis of the transfectants defines the more acidic beta chain as the product of the DR2 beta a sequence, and the more basic chain as the product of the DR2 beta b sequence. The LDR2a transfectants present antigen efficiently to M.leprae-specific T cell clones and are capable of presenting synthetic peptide, 65-kD recombinant mycobacterial antigen and M.leprae. Of the DR2Dw2-restricted T cell clones we have tested, all use the DR2 beta a chain as their restriction element. Inhibition studies with mAbs demonstrate the dependence of presentation by the transfectant on class II and CD4, while mAbs against LFA-1, which substantially inhibit presentation by B-lymphoblastoid cell lines, do not inhibit transfectant presentation. PMID:3128633

  17. Characterization of Epstein-Barr virus-induced lymphoproliferation derived from human peripheral blood mononuclear cells transferred to severe combined immunodeficient mice.

    PubMed Central

    Okano, M.; Taguchi, Y.; Nakamine, H.; Pirruccello, S. J.; Davis, J. R.; Beisel, K. W.; Kleveland, K. L.; Sanger, W. G.; Fordyce, R. R.; Purtilo, D. T.

    1990-01-01

    Mice with severe combined immunodeficiency (SCID) received 6 X 10(7) fresh human peripheral blood mononuclear cells (PBMC) intraperitoneally from Epstein-Barr virus (EBV)-seropositive and -seronegative donors. B95-8 EBV was inoculated intraperitoneally and intravenously to the mice 6 weeks after transfer of seronegative PBMC. Three of four mice transferred with PBMC from two EBV-seropositive donors and two of four mice from two EBV-seronegative donors inoculated with EBV developed fatal EBV-induced lymphoproliferative disease within 6 to 10 weeks. These tumors were oligoclonal or polyclonal by cytoplasmic immunoglobulin expression. Furthermore no consistent clonal chromosomal abnormalities were shown. Cell lines established from these tumors showed low cloning efficiency in soft agarose. In addition, latent membrane protein, B-lymphocyte activation antigen (CD23), and cell-adhesion molecules (ICAM-1, CD18) all were expressed in the EBV-positive infiltrating lymphoproliferative lesions in each mouse. These results suggest that lymphoid tumors are comparable to lymphoblastoid cell lines immortalized by EBV and are not malignant lymphomas such as Burkitt's lymphoma. This model may be useful for investigating mechanisms responsible for the growing numbers of lymphoproliferative diseases that are occurring in patients with inherited or acquired immunodeficiencies. Images Figure 1 PMID:1975985

  18. Endogenous DNA Damage and Risk of Testicular Germ Cell Tumors

    SciTech Connect

    Cook, M B; Sigurdson, A J; Jones, I M; Thomas, C B; Graubard, B I; Korde, L; Greene, M H; McGlynn, K A

    2008-01-18

    Testicular germ cell tumors (TGCT) are comprised of two histologic groups, seminomas and nonseminomas. We postulated that the possible divergent pathogeneses of these histologies may be partially explained by variable endogenous DNA damage. To assess our hypothesis, we conducted a case-case analysis of seminomas and nonseminomas using the alkaline comet assay to quantify single-strand DNA breaks and alkali-labile sites. The Familial Testicular Cancer study and the U.S. Radiologic Technologists cohort provided 112 TGCT cases (51 seminomas & 61 nonseminomas). A lymphoblastoid cell line was cultured for each patient and the alkaline comet assay was used to determine four parameters: tail DNA, tail length, comet distributed moment (CDM) and Olive tail moment (OTM). Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated using logistic regression. Values for tail length, tail DNA, CDM and OTM were modeled as categorical variables using the 50th and 75th percentiles of the seminoma group. Tail DNA was significantly associated with nonseminoma compared to seminoma (OR{sub 50th percentile} = 3.31, 95%CI: 1.00, 10.98; OR{sub 75th percentile} = 3.71, 95%CI: 1.04, 13.20; p for trend=0.039). OTM exhibited similar, albeit statistically non-significant, risk estimates (OR{sub 50th percentile} = 2.27, 95%CI: 0.75, 6.87; OR{sub 75th percentile} = 2.40, 95%CI: 0.75, 7.71; p for trend=0.12) whereas tail length and CDM showed no association. In conclusion, the results for tail DNA and OTM indicate that endogenous DNA damage levels are higher in patients who develop nonseminoma compared with seminoma. This may partly explain the more aggressive biology and younger age-of-onset of this histologic subgroup compared with the relatively less aggressive, later-onset seminoma.

  19. Cell Lines Models of Drug Response: Successes and Lessons from this Pharmacogenomic Model

    PubMed Central

    Jack, J.; Rotroff, D.; Motsinger-Reif, A.

    2015-01-01

    A new standard for medicine is emerging that aims to improve individual drug responses through studying associations with genetic variations. This field, pharmacogenomics, is undergoing a rapid expansion due to a variety of technological advancements that are enabling higher throughput with reductions in cost. Here we review the advantages, limitations, and opportunities for using lymphoblastoid cell lines (LCL) as a model system for human pharmacogenomic studies. There are a wide range of publicly available resources with genome-wide data available for LCLs from both related and unrelated populations, removing the cost of genotyping the data for drug response studies. Furthermore, in contrast to human clinical trials or in vivo model systems, with high-throughput in vitro screening technologies, pharmacogenomics studies can easily be scaled to accommodate large sample sizes. An important component to leveraging genome-wide data in LCL models is association mapping. Several methods are discussed herein, and include multivariate concentration response modeling, issues with multiple testing, and successful examples of the ‘triangle model’ to identify candidate variants. Once candidate gene variants have been determined, their biological roles can be elucidated using pathway analyses and functionally confirmed using siRNA knockdown experiments. The wealth of genomics data being produced using related and unrelated populations is creating many exciting opportunities leading to new insights into the genetic contribution and heritability of drug response. PMID:25109794

  20. Endocytosis of receptor-bound insulin-like growth factor II is enhanced by mannose-6-phosphate in IM9 cells.

    PubMed

    Polychronakos, C; Piscina, R

    1988-10-01

    The insulin-like growth factor II (IGF-II), and glycoproteins containing mannose 6-phosphate (M6P), bind to two different sites of the same receptor molecule (Morgan et al, Nature 329:301, 1987). To study the interactions between the two ligands on their common receptor in intact cells, we examined the effect of free M6P on IGF-II binding and endocytosis in the IM9 human lymphoblastoid cell line. M6P, up to a 3 mM concentration, had no effect on the binding of IGF-II to the cell surface receptor of intact IM9 cells at 4 degrees C. By contrast, when IM9 cells were incubated with 125I-IGF-II at 37 degrees C, 1mM M6P increased cell-associated radioactivity by twofold. The increase was resistant to acid wash at 4 degrees C, and therefore assumed to represent endocytosed IGF-II. Acid-washable radioactivity was no different, confirming that, in intact cells, M6P does not affect IGF-II surface binding. In addition, preincubation of cells with M6P at 37 degrees C for up to 3 hours did not change the abundance of receptor on the cell surface, as measured by a subsequent 4 degrees C binding assay. We conclude that M6P causes a shift of IGF-II-occupied receptors form the cell surface to intracellular locations without affecting surface binding of this ligand in IM9 cells. The effect could be produced by the binding of M6P itself, or by the displacement of endogenous phosphomannosylated ligands.

  1. Endocytosis of receptor-bound insulin-like growth factor II is enhanced by mannose-6-phosphate in IM9 cells.

    PubMed

    Polychronakos, C; Piscina, R

    1988-12-01

    The insulin-like growth factor II (IGF-II), and glycoproteins containing mannose 6-phosphate (M6P), bind to two different sites of the same receptor molecule (Morgan et al, Nature 329:301, 1987). To study the interactions between the two ligands on their common receptor in intact cells, we examined the effect of free M6P on IGF-II binding and endocytosis in the IM9 human lymphoblastoid cell line. M6P, up to a 3 mM concentration, had no effect on the binding of IGF-II to the cell surface receptor of intact IM9 cells at 4 degrees C. By contrast, when IM9 cells were incubated with 125I-IGF-II at 37 degrees C, 1 mM M6P increased cell-associated radioactivity by twofold. The increase was resistant to acid wash at 4 degrees C, and therefore assumed to represent endocytosed IGF-II. Acid-washable radioactivity was no different, confirming that, in intact cells, M6P does not affect IGF-II surface binding. In addition, preincubation of cells with M6P at 37 degrees C for up to 3 hours did not change the abundance of receptor on the cell surface, as measured by a subsequent 4 degrees C binding assay. We conclude that M6P causes a shift of IGF-II-occupied receptors form the cell surface to intracellular locations without affecting surface binding of this ligand in IM9 cells. The effect could be produced by the binding of M6P itself, or by the displacement of endogenous phosphomannosylated ligands.

  2. Comprehensive genetic analysis of cytarabine sensitivity in a cell-based model identifies polymorphisms associated with outcome in AML patients.

    PubMed

    Gamazon, Eric R; Lamba, Jatinder K; Pounds, Stanley; Stark, Amy L; Wheeler, Heather E; Cao, Xueyuan; Im, Hae K; Mitra, Amit K; Rubnitz, Jeffrey E; Ribeiro, Raul C; Raimondi, Susana; Campana, Dario; Crews, Kristine R; Wong, Shan S; Welsh, Marleen; Hulur, Imge; Gorsic, Lidija; Hartford, Christine M; Zhang, Wei; Cox, Nancy J; Dolan, M Eileen

    2013-05-23

    A whole-genome approach was used to investigate the genetic determinants of cytarabine-induced cytotoxicity. We performed a meta-analysis of genome-wide association studies involving 523 lymphoblastoid cell lines (LCLs) from individuals of European, African, Asian, and African American ancestry. Several of the highest-ranked single-nucleotide polymorphisms (SNPs) were within the mutated in colorectal cancers (MCC) gene. MCC expression was induced by cytarabine treatment from 1.7- to 26.6-fold in LCLs. A total of 33 SNPs ranked at the top of the meta-analysis (P < 10(-5)) were successfully tested in a clinical trial of patients randomized to receive low-dose or high-dose cytarabine plus daunorubicin and etoposide; of these, 18 showed association (P < .05) with either cytarabine 50% inhibitory concentration in leukemia cells or clinical response parameters (minimal residual disease, overall survival (OS), and treatment-related mortality). This count (n = 18) was significantly greater than expected by chance (P = .016). For rs1203633, LCLs with AA genotype were more sensitive to cytarabine-induced cytotoxicity (P = 1.31 × 10(-6)) and AA (vs GA or GG) genotype was associated with poorer OS (P = .015), likely as a result of greater treatment-related mortality (P = .0037) in patients with acute myeloid leukemia (AML). This multicenter AML02 study trial was registered at www.clinicaltrials.gov as #NCT00136084.

  3. Comprehensive genetic analysis of cytarabine sensitivity in a cell-based model identifies polymorphisms associated with outcome in AML patients

    PubMed Central

    Gamazon, Eric R.; Lamba, Jatinder K.; Pounds, Stanley; Stark, Amy L.; Wheeler, Heather E.; Cao, Xueyuan; Im, Hae K.; Mitra, Amit K.; Rubnitz, Jeffrey E.; Ribeiro, Raul C.; Raimondi, Susana; Campana, Dario; Crews, Kristine R.; Wong, Shan S.; Welsh, Marleen; Hulur, Imge; Gorsic, Lidija; Hartford, Christine M.; Zhang, Wei; Cox, Nancy J.; Dolan, M. Eileen

    2013-01-01

    A whole-genome approach was used to investigate the genetic determinants of cytarabine-induced cytotoxicity. We performed a meta-analysis of genome-wide association studies involving 523 lymphoblastoid cell lines (LCLs) from individuals of European, African, Asian, and African American ancestry. Several of the highest-ranked single-nucleotide polymorphisms (SNPs) were within the mutated in colorectal cancers (MCC) gene. MCC expression was induced by cytarabine treatment from 1.7- to 26.6-fold in LCLs. A total of 33 SNPs ranked at the top of the meta-analysis (P < 10−5) were successfully tested in a clinical trial of patients randomized to receive low-dose or high-dose cytarabine plus daunorubicin and etoposide; of these, 18 showed association (P < .05) with either cytarabine 50% inhibitory concentration in leukemia cells or clinical response parameters (minimal residual disease, overall survival (OS), and treatment-related mortality). This count (n = 18) was significantly greater than expected by chance (P = .016). For rs1203633, LCLs with AA genotype were more sensitive to cytarabine-induced cytotoxicity (P = 1.31 × 10−6) and AA (vs GA or GG) genotype was associated with poorer OS (P = .015), likely as a result of greater treatment-related mortality (P = .0037) in patients with acute myeloid leukemia (AML). This multicenter AML02 study trial was registered at www.clinicaltrials.gov as #NCT00136084. PMID:23538338

  4. Apoptotic effect of ethyl-4-isothiocyanatobutanoate is associated with DNA damage, proteasomal activity and induction of p53 and p21cip1/waf1.

    PubMed

    Bodo, Juraj; Jakubikova, Jana; Chalupa, Ivan; Bartosova, Zdena; Horakova, Katarina; Floch, Lubomir; Sedlak, Jan

    2006-08-01

    The effect of synthetic isothiocyanate ethyl-4-isothiocyanatobutanoate (E-4IB) on survival of mismatch repair-proficient TK6 and -deficient MT1 cell lines as well as the influence of proteasomal inhibitor MG132, caspase inhibitor Z-VAD-fmk, and ATM inhibitor caffeine on E-4IB modulation of cell cycle and apoptosis was evaluated. Flow cytometric analyses of DNA double strand breaks (gamma-H2AX), mitotic fraction (phospho-histone H3), cell cycle modulation, apoptosis induction (sub-G(0) fraction and fluorescein diacetate staining), and dissipation of transmembrane mitochondrial potential (JC-1 staining) were performed. Western blotting was used for the evaluation of ERK activation, expression of p53, p21(cip1/waf1) and GADD45alpha proteins, as well as PARP fragmentation. Analysis of mitotic nuclei was performed for chromosomal aberrations assessment. MT1 cells were more resistant to E-4IB treatment then TK6 cells (IC(50) 8 muM vs. 4 muM). In both cell lines E-4IB treatment induced phosphorylation of H2AX, increase of p53 protein level, phospho-histone H3 staining, and G(2)/M arrest. The sub-G(0) fragmentation was accompanied by PARP degradation, decreased mitochondrial transmembrane potential, and diminished p21(cip1/waf1) protein expression in TK6 cells. Caspase inhibitor Z-VAD-fmk decreased E-4IB induced sub-G(0) fragmentation and extent of apoptosis in TK6 cells, while proteasome inhibitor MG132 increased number of apoptotic cells in both cell lines tested. A number of aberrant metaphases and clastogenic effect of high E-4IB concentration was observed. The synthetic isothiocyanate E-4IB induced DNA strand breaks, increased mitotic fraction and apoptosis potentiated by MG132 inhibitor in both mismatch repair-proficient and -deficient cell lines.

  5. The landscape of histone modifications across 1% of the human genome in five human cell lines

    PubMed Central

    Koch, Christoph M.; Andrews, Robert M.; Flicek, Paul; Dillon, Shane C.; Karaöz, Ulaş; Clelland, Gayle K.; Wilcox, Sarah; Beare, David M.; Fowler, Joanna C.; Couttet, Phillippe; James, Keith D.; Lefebvre, Gregory C.; Bruce, Alexander W.; Dovey, Oliver M.; Ellis, Peter D.; Dhami, Pawandeep; Langford, Cordelia F.; Weng, Zhiping; Birney, Ewan; Carter, Nigel P.; Vetrie, David; Dunham, Ian

    2007-01-01

    We generated high-resolution maps of histone H3 lysine 9/14 acetylation (H3ac), histone H4 lysine 5/8/12/16 acetylation (H4ac), and histone H3 at lysine 4 mono-, di-, and trimethylation (H3K4me1, H3K4me2, H3K4me3, respectively) across the ENCODE regions. Studying each modification in five human cell lines including the ENCODE Consortium common cell lines GM06990 (lymphoblastoid) and HeLa-S3, as well as K562, HFL-1, and MOLT4, we identified clear patterns of histone modification profiles with respect to genomic features. H3K4me3, H3K4me2, and H3ac modifications are tightly associated with the transcriptional start sites (TSSs) of genes, while H3K4me1 and H4ac have more widespread distributions. TSSs reveal characteristic patterns of both types of modification present and the position relative to TSSs. These patterns differ between active and inactive genes and in particular the state of H3K4me3 and H3ac modifications is highly predictive of gene activity. Away from TSSs, modification sites are enriched in H3K4me1 and relatively depleted in H3K4me3 and H3ac. Comparison between cell lines identified differences in the histone modification profiles associated with transcriptional differences between the cell lines. These results provide an overview of the functional relationship among histone modifications and gene expression in human cells. PMID:17567990

  6. Study of the Cytotoxic Effects of the New Synthetic Isothiocyanate CM9 and Its Fullerene Derivative on Human T-Leukemia Cells

    PubMed Central

    De Gianni, Elena; Turrini, Eleonora; Milelli, Andrea; Maffei, Francesca; Carini, Marco; Minarini, Anna; Tumiatti, Vincenzo; Da Ros, Tatiana; Prato, Maurizio; Fimognari, Carmela

    2015-01-01

    One important strategy to develop effective anticancer agents is based on natural products. Many active phytochemicals are in human clinical trials and have been used for a long time, alone and in association with conventional anticancer drugs, for the treatment of various types of cancers. A great number of in vitro, in vivo and clinical reports document the multi-target anticancer activities of isothiocyanates and of compounds characterized by a naphthalenetetracarboxylic diimide scaffold. In order to search for new anticancer agents with a better pharmaco-toxicological profile, we investigated hybrid compounds obtained by inserting isothiocyanate group(s) on a naphthalenetetracarboxylic diimide scaffold. Moreover, since water-soluble fullerene derivatives can cross cell membranes thus favoring the delivery of anticancer therapeutics, we explored the cytostatic and cytotoxic activity of hybrid compounds conjugated with fullerene. We studied their cytostatic and cytotoxic effects on a human T-lymphoblastoid cell line by using different flow cytometric assays. In order to better understand their pharmaco-toxicological potential, we also analyzed their genotoxicity. Our global results show that the synthesized compounds reduced significantly the viability of leukemia cells. However, the conjugation with a non-toxic vector did not increase their anticancer potential. This opens an interesting research pattern for certain fullerene properties. PMID:25679371

  7. Effector properties and glycosylation patterns of recombinant human anti-D-IgG1 antibodies produced by human PER.C6(®) cells.

    PubMed

    Olovnikova, N I; Grigorieva, O V; Petrov, A V

    2012-12-01

    Creation of effective monoclonal anti-D immunoglobulin for prevention of hemolytic disease of the newborn remains an unsolved problem because there is still no producer cell strain providing stable production and adequate glycosylation of antibodies. Recombinant anti-D have been obtained on the basis of human PER.C6(®) cells and characterized. Anti-D antibodies expressed in PER.C6(®) exhibited lower hemolytic activity in antibody-dependent cytotoxicity (ADCC) reaction mediated by low-affinity Fcγ receptors in comparison with identical antibodies of lymphoblastoid origin. Monoclonal antibodies produced by PER.C6(®) are completely fucosylated and desialylated, i.e. are characterized by abnormal glycosylation. Addition of kifunensine (α-mannosidase I inhibitor) to the medium led to production of antibodies with high hemolytic activity. Reduced activity of monoclonal antibodies in PER.C6(®) cells and the effect of kifunensine (causing synthesis of defucosylated glycans) suggest that the absence of fucose is the key factor responsible for Fc affinity for low-affinity receptors.

  8. Molecular basis for paradoxical carriers of adenosine deaminase (ADA) deficiency that show extremely low levels of ADA activity in peripheral blood cells without immunodeficiency.

    PubMed

    Ariga, T; Oda, N; Sanstisteban, I; Arredondo-Vega, F X; Shioda, M; Ueno, H; Terada, K; Kobayashi, K; Hershfield, M S; Sakiyama, Y

    2001-02-01

    Adenosine deaminase (ADA) deficiency causes an autosomal recessive form of severe combined immunodeficiency and also less severe phenotypes, depending to a large degree on genotype. In general, ADA activity in cells of carriers is approximately half-normal. Unexpectedly, healthy first-degree relatives of two unrelated ADA-deficient severe combined immunodeficient patients (mother and brother in family I; mother in family II) had only 1-2% of normal ADA activity in PBMC, lower than has previously been found in PBMC of healthy individuals with so-called "partial ADA deficiency." The level of deoxyadenosine nucleotides in erythrocytes of these paradoxical carriers was slightly elevated, but much lower than levels found in immunodeficient patients with ADA deficiency. ADA activity in EBV-lymphoblastoid cell lines (LCL) and T cell lines established from these carriers was 10-20% of normal. Each of these carriers possessed two mutated ADA alleles. Expression of cloned mutant ADA cDNAs in an ADA-deletion strain of Escherichia coli indicated that the novel mutations G239S and M310T were responsible for the residual ADA activity. ADA activity in EBV-LCL extracts of the paradoxical carriers was much more labile than ADA from normal EBV-LCL. Immunoblotting suggested that this lability was due to denaturation rather than to degradation of the mutant protein. These results further define the threshold level of ADA activity necessary for sustaining immune function.

  9. Inferring a role for methylation of intergenic DNA in the regulation of genes aberrantly expressed in precursor B-cell acute lymphoblastic leukemia.

    PubMed

    Almamun, Md; Kholod, Olha; Stuckel, Alexei J; Levinson, Benjamin T; Johnson, Nathan T; Arthur, Gerald L; Davis, J Wade; Taylor, Kristen H

    2017-01-17

    A complete understanding of the mechanisms involved in the development of pre-B ALL is lacking. In this study, we integrated DNA methylation data and gene expression data to elucidate the impact of aberrant intergenic DNA methylation on gene expression in pre-B ALL. We found a subset of differentially methylated intergenic loci that were associated with altered gene expression in pre-B ALL patients. Notably, 84% of these regions were also bound by transcription factors (TF) known to play roles in differentiation and B-cell development in a lymphoblastoid cell line. Further, an overall downregulation of eRNA transcripts was observed in pre-B ALL patients and these transcripts were associated with the downregulation of putative target genes involved in B-cell migration, proliferation, and apoptosis. The identification of novel putative regulatory regions highlights the significance of intergenic DNA sequences and may contribute to the identification of new therapeutic targets for the treatment of pre-B ALL.

  10. Interferon-induced 56,000 Mr protein and its mRNA in human cells: molecular cloning and partial sequence of the cDNA.

    PubMed Central

    Chebath, J; Merlin, G; Metz, R; Benech, P; Revel, M

    1983-01-01

    Treatment of responsive cells by interferons (IFNs) induces within a few hours a rise in the concentration of several proteins and mRNAs. In order to characterize these IFN-induced mRNA species, we have cloned in E. coli the cDNA made from a 17-18S poly(A)+ RNA of human fibroblastoid cells (SV80) treated with IFN-beta. We describe here a pBR322 recombinant plasmid (C56) which contains a 400 bp cDNA insert corresponding to a 18S mRNA species newly induced by IFN. The C56 mRNA codes for a 56,000 dalton protein easily detectable by hybridization-translation experiments. The sequence of 66 of the carboxy-terminal amino-acids of the protein can be deduced from the cDNA sequence. IFNs-alpha, beta or gamma are able to activate the expression of this gene in human fibroblasts as well as lymphoblastoid cells. The mRNA is not detectable without IFN; it reaches maximum levels (0.1% of the total poly(A)+ RNA) within 4-8 hrs and decreases after 16 hrs. Images PMID:6186990

  11. Dendritic cells generated from blood precursors of chronic myelogenous leukemia patients carry the Philadelphia translocation and can induce a CML-specific primary cytotoxic T-cell response.

    PubMed

    Eibl, B; Ebner, S; Duba, C; Böck, G; Romani, N; Erdel, M; Gächter, A; Niederwieser, D; Schuler, G

    1997-11-01

    Dendritic cells (DC) are professional antigen-presenting cells specialized in the initiation of primary immune responses. We were interested to know whether mature DC can be grown in vitro from peripheral blood mononuclear cells (PBMC) of patients with chronic myelogenous leukemia (CML), and whether they carry the Philadelphia (Ph) translocation. Using a method recently described, DC were generated from PBMC precursors of 12 patients with CML using GM-CSF, IL-4, and monocyte-conditioned medium. DC exhibited the typical morphology with thin cytoplasmatic processes and expressed high levels of MHC class II, CD86, and CD83 typical for mature DC. After sorting with the monoclonal antibody CD83, a cell population of more than 95% CD83 positive cells was obtained. The presence of the Ph translocation was analyzed in these cells, in PBMC, lymphoblastoid cell lines (LCL), and in phytohemagglutinin (PHA)-induced T blasts from the same patients by fluorescence in situ hybridization (FISH). In contrast to all other cells analyzed, the vast majority of DC (95.9 +/- 0.7%) displayed the Ph translocation, irrespective of disease stage or therapy. PBMC were predominantly positive for the Ph chromosome (67.6 +/- 7.3%), whereas only 11.4 +/- 1% of the B cells and 4.4 +/- 1.1% of the PHA blasts carried the Ph translocation. Using such leukemic DC as antigen-presenting cells, a primary CML-directed cytotoxic immune response in vitro was obtained, as shown by the specific recognition of Ph chromosome positive cells. We conclude that DC can be generated from blood progenitors of CML patients in vitro and exhibit, to a large extent, the Ph translocation. Such DC, which in a preliminary experiment have been able to induce a primary CML-directed cytotoxic immune response in vitro, might be ideal candidates for adoptive immunotherapy either by direct transfer of DC for in vivo generation of a T-cell response or by in vitro generation of CML-specific cytotoxic autologous or HLA

  12. Short-term inhibition of TERT induces telomere length-independent cell cycle arrest and apoptotic response in EBV-immortalized and transformed B cells

    PubMed Central

    Celeghin, Andrea; Giunco, Silvia; Freguja, Riccardo; Zangrossi, Manuela; Nalio, Silvia; Dolcetti, Riccardo; De Rossi, Anita

    2016-01-01

    Besides its canonical role in stabilizing telomeres, telomerase reverse transcriptase (TERT) may promote tumorigenesis through extra-telomeric functions. The possible therapeutic effects of BIBR1532 (BIBR), a powerful TERT inhibitor, have been evaluated in different cellular backgrounds, but no data are currently available regarding Epstein–Barr virus (EBV)-driven B-cell malignancies. Our aim was to characterize the biological effects of TERT inhibition by BIBR on EBV-immortalized lymphoblastoid cell lines (LCLs) and fully transformed Burkitt's lymphoma (BL) cell lines. We found that BIBR selectively inhibits telomerase activity in TERT-positive 4134/Late and 4134/TERT+ LCLs and EBV-negative BL41 and EBV-positive BL41/B95.8 BL cell lines. TERT inhibition led to decreased cell proliferation, accumulation of cells in the S-phase and ultimately to increased apoptosis, compared with mock-treated control cells. All these effects occurred within 72 h and were not observed in BIBR-treated TERT-negative 4134/TERT- and U2OS cells. The cell cycle arrest and apoptosis, consequent upon short-term TERT inhibition, were associated with and likely dependent on the activation of the DNA damage response (DDR), highlighted by the increased levels of γH2AX and activation of ATM and ATR pathways. Analyses of the mean and range of telomere lengths and telomere dysfunction-induced foci indicated that DDR after short-term TERT inhibition was not related to telomere dysfunction, thus suggesting that TERT, besides stabilizing telomere, may protect DNA via telomere-independent mechanisms. Notably, TERT-positive LCLs treated with BIBR in combination with fludarabine or cyclophosphamide showed a significant increase in the number of apoptotic cells with respect to those treated with chemotherapeutic agents alone. In conclusion, TERT inhibition impairs cell cycle progression and enhances the pro-apoptotic effects of chemotherapeutic agents in TERT-positive cells. These results support new

  13. Neutron Exposures in Human Cells: Bystander Effect and Relative Biological Effectiveness

    PubMed Central

    Seth, Isheeta; Schwartz, Jeffrey L.; Stewart, Robert D.; Emery, Robert; Joiner, Michael C.; Tucker, James D.

    2014-01-01

    Bystander effects have been observed repeatedly in mammalian cells following photon and alpha particle irradiation. However, few studies have been performed to investigate bystander effects arising from neutron irradiation. Here we asked whether neutrons also induce a bystander effect in two normal human lymphoblastoid cell lines. These cells were exposed to fast neutrons produced by targeting a near-monoenergetic 50.5 MeV proton beam at a Be target (17 MeV average neutron energy), and irradiated-cell conditioned media (ICCM) was transferred to unirradiated cells. The cytokinesis-block micronucleus assay was used to quantify genetic damage in radiation-naïve cells exposed to ICCM from cultures that received 0 (control), 0.5, 1, 1.5, 2, 3 or 4 Gy neutrons. Cells grown in ICCM from irradiated cells showed no significant increase in the frequencies of micronuclei or nucleoplasmic bridges compared to cells grown in ICCM from sham irradiated cells for either cell line. However, the neutron beam has a photon dose-contamination of 5%, which may modulate a neutron-induced bystander effect. To determine whether these low doses of contaminating photons can induce a bystander effect, cells were irradiated with cobalt-60 at doses equivalent to the percent contamination for each neutron dose. No significant increase in the frequencies of micronuclei or bridges was observed at these doses of photons for either cell line when cultured in ICCM. As expected, high doses of photons induced a clear bystander effect in both cell lines for micronuclei and bridges (p<0.0001). These data indicate that neutrons do not induce a bystander effect in these cells. Finally, neutrons had a relative biological effectiveness of 2.0±0.13 for micronuclei and 5.8±2.9 for bridges compared to cobalt-60. These results may be relevant to radiation therapy with fast neutrons and for regulatory agencies setting standards for neutron radiation protection and safety. PMID:24896095

  14. Definition of a natural killer NKR-P1A+/CD56-/CD16- functionally immature human NK cell subset that differentiates in vitro in the presence of interleukin 12 [published erratum appears in J Exp Med 1997 Mar 17;185(6):1150-1

    PubMed Central

    1996-01-01

    Human natural killer (NK) cell differentiation from immature lineage negative (Lin-) umbilical cord blood cells was examined in vitro. Cells expressing differentiation antigens of mature NK cells (CD56, CD16, CD2, CD8, NKR-P1A) were generated from Lin- cells cultured with interleukin (IL)-2 and a murine bone marrow stromal cell line expressing the human membrane-bound form of stem cell factor. Two subsets of NK cells were identified in these cultures: one expressed both NKR-P1A and CD56 and, in variable proportions, all other NK cell differentiation antigens; the second subset expressed only NKR-P1A and, unlike the former, was not cytotoxic. Neither subset expressed interferon (IFN)-gamma mRNA even after stimulation with phorbol di- ester and Ca2+ ionophore, but both expressed tumor necrosis factor alpha mRNA and the cytotoxic granule-associated proteins TIA-1, perforin, and serine esterase-1. After 10-d culture with IL-2, IL-12, and irradiated B lymphoblastoid cells, approximately 45% of the NKR- P1A+/ CD56- cells became CD56+, and the same cultures contained cells capable of cytotoxicity and of IFN-gamma production. These results indicate that NKR-P1A expression in the absence of other NK cell markers defines an intermediate, functionally immature stage of NK cell differentiation, and that effector functions develop in these cells, concomitantly with CD56 expression, in the presence of IL-12. These cells likely represent the counterpart of a CD3-/NKR-P1A+/ CD56-/CD16- cell subset that, as shown here, is present both in adult and neonatal circulating lymphocytes. PMID:8920872

  15. Detection of cell surface and intracellular antigens by human monoclonal antibodies. Hybrid cell lines derived from lymphocytes of patients with malignant melanoma

    PubMed Central

    1983-01-01

    This study represents an initial attempt to analyze the humoral immune reactions of patients with malignant melanoma by hybridoma methodology. Using lymphocytes from regional lymph nodes, peripheral blood and tumor infiltrates, 158 fusions were performed with SKO-007 (human myeloma line), LICR-LON-HMy2 (LICR-2), GM 4672 (human lymphoblastoid lines), or NS-1 (mouse myeloma line). Fusion of lymph node lymphocytes with NS-1 resulted in a 3-4 times higher frequency of clones than fusion with LICR-2, and a 10 times higher frequency than fusion with SKO-007 or GM 4672. In the case of peripheral blood lymphocytes, fusion with NS-1 gave greater than 25 times higher frequency of clones than fusion with LICR-2 or SKO-007. Production of human mu, gamma, or alpha heavy chains was detected in 50-80% of wells containing growing clones, and the levels of immunoglobulin ranged from 0.3 micrograms to 40 micrograms/ml. NS-1-derived clones could be easily subcultured, while LICR-2 and SKO-007 clones grew more slowly on subculturing. In this study, Ig secretion appeared to be a more stable property of LICR-2- derived clones than NS-1-derived clones. A panel of 20 human cancer cell lines was used to screen 771 Ig-secreting cultures for antibody to cell surface or intracellular antigens. Reactivity with cell surface antigens was found infrequently (6 cultures), whereas reactivity with intracellular antigens was more common (27 cultures). A new cell surface antigen with properties of a glycolipid was defined with an IgM monoclonal antibody secreted by a tetraploid cell derived from a fusion of LICR-2 with lymphocytes from the axillary lymph node of a patient with melanoma. The hybrid cell line has been subcloned four times and secretes 5 micrograms IgM/ml. The antigen detected by this IgM antibody was found on 5 of 23 melanoma cell lines and 12 of 30 epithelial cancer cell lines. No reactions were found with 11 cultures derived from normal cells. Stable cell lines secreting human

  16. Radio-protective effects of manganese-containing superoxide dismutase mimics on ataxia telangiectasia cells

    PubMed Central

    Pollard, Julianne M.; Reboucas, Julio S.; Durazo, Armando; Kos, Ivan; Fike, Francesca; Panni, Moeen; Gralla, Edith Butler; Valentine, Joan Selverstone; Batinic-Haberle, Ines; Gatti, Richard A.

    2009-01-01

    We tested several classes of antioxidant manganese compounds for radioprotective effects using human lymphoblastoid cells: six porphyrins, three salens and two cyclic polyamines. Radioprotection was evaluated by seven assays: XTT; Annexin V and propidium iodide flow cytometry analysis; γ-H2AX immunofluorescence; the neutral comet assay; dichlorofluorescein and dihydroethidium staining; resazurin and colony survival assay. Two compounds were most effective in protecting wildtype, and A-T cells, against radiation-induced damage: MnMx-2-PyP-Calbio (a mixture of differently N-methylated MnT-2-PyP+ from Calbiochem) and MnTnHex-2-PyP. MnTnHex-2-PyP protected WT cells against radiation-induced apoptosis by 58% (p=0.04) in WT by XTT and 39% (p=0.01) in A-T by Annexin V and propidium iodide staining. MnTnHex-2-PyP protected WT cells against DNA damage by 57% (p=0.005) by Gamma H2AX immunofluorescence and by 30% (p<0.01) by neutral comet assay. MnTnHex-2-PyP is more lipophilic than MnMx-2-PyP-Calbio and is also >10-fold more SOD-active; consequently it is >50-fold more potent as a radioprotectant, as supported by six of the tests employed in this study. Thus, lipophilicity and antioxidant potency correlated with the magnitude of the beneficial radioprotectant effects observed. Our results identify a new class of porphyrinic radioprotectants for the general and radiosensitive populations and may also provide a new option for treating A-T patients. PMID:19389472

  17. Rapid Cell-Based Assay for Detection and Quantification of Active Staphylococcal Enterotoxin Type D.

    PubMed

    Rasooly, Reuven; Do, Paula M; Hernlem, Bradley J

    2017-03-01

    Food poisoning by Staphylococcus aureus is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by this bacterium and is a major source of foodborne illness. Staphylococcal enterotoxin D (SED) is one of the predominant enterotoxins recovered in Staphylococcal food poisoning incidences, including a recent outbreak in Guam affecting 300 children. Current immunology methods for SED detection cannot distinguish between the biologically active form of the toxin, which poses a threat, from the inactive form, which poses no threat. In vivo bioassays that measure emetic activity in kitten and monkeys have been used, but these methods rely upon expensive procedures using live animals and raising ethical concerns. A rapid (5 h) quantitative bioluminescence assay, using a genetically engineered T-cell Jurkat cell line expressing luciferase under regulation of nuclear factor of activated T cells response elements, in combination with the lymphoblastoid B-cell line Raji for antigen presentation, was developed. In this assay, the detection limit of biologically active SED is 100 ng/mL, which is 10 times more sensitive than the splenocyte proliferation assay, and 10(5) times more sensitive than monkey or kitten bioassay. Pasteurization or repeated freeze-thaw cycles had no effect on SED activity, but reduction in SED activity was shown with heat treatment at 100°C for 5 min. It was also shown that milk exhibits a protective effect on SED. This bioluminescence assay may also be used to rapidly evaluate antibodies to SED for potential therapeutic application as a measurement of neutralizing biological effects of SED.

  18. Identification of candidate genes involved in coronary artery calcification by transcriptome sequencing of cell lines

    PubMed Central

    2014-01-01

    Background Massively-parallel cDNA sequencing (RNA-Seq) is a new technique that holds great promise for cardiovascular genomics. Here, we used RNA-Seq to study the transcriptomes of matched coronary artery disease cases and controls in the ClinSeq® study, using cell lines as tissue surrogates. Results Lymphoblastoid cell lines (LCLs) from 16 cases and controls representing phenotypic extremes for coronary calcification were cultured and analyzed using RNA-Seq. All cell lines were then independently re-cultured and along with another set of 16 independent cases and controls, were profiled with Affymetrix microarrays to perform a technical validation of the RNA-Seq results. Statistically significant changes (p < 0.05) were detected in 186 transcripts, many of which are expressed at extremely low levels (5–10 copies/cell), which we confirmed through a separate spike-in control RNA-Seq experiment. Next, by fitting a linear model to exon-level RNA-Seq read counts, we detected signals of alternative splicing in 18 transcripts. Finally, we used the RNA-Seq data to identify differential expression (p < 0.0001) in eight previously unannotated regions that may represent novel transcripts. Overall, differentially expressed genes showed strong enrichment (p = 0.0002) for prior association with cardiovascular disease. At the network level, we found evidence for perturbation in pathways involving both cardiovascular system development and function as well as lipid metabolism. Conclusions We present a pilot study for transcriptome involvement in coronary artery calcification and demonstrate how RNA-Seq analyses using LCLs as a tissue surrogate may yield fruitful results in a clinical sequencing project. In addition to canonical gene expression, we present candidate variants from alternative splicing and novel transcript detection, which have been unexplored in the context of this disease. PMID:24628908

  19. Decreased expression of the mitochondrial matrix proteases Lon and ClpP in cells from a patient with hereditary spastic paraplegia (SPG13).

    PubMed

    Hansen, J; Corydon, T J; Palmfeldt, J; Dürr, A; Fontaine, B; Nielsen, M N; Christensen, J H; Gregersen, N; Bross, P

    2008-05-02

    The mitochondrial chaperonin heat shock protein 60 (Hsp60) assists the folding of a subset of proteins localized in mitochondria and is an essential component of the mitochondrial protein quality control system. Mutations in the HSPD1 gene that encodes Hsp60 have been identified in patients with an autosomal dominant form of hereditary spastic paraplegia (SPG13), a late-onset neurodegenerative disorder characterized by a progressive paraparesis of the lower limbs. The disease-associated Hsp60-(p.Val98Ile) protein, encoded by the c.292G>A HSPD1 allele, has reduced chaperonin activity, but how its expression affects mitochondrial functions has not been investigated. We have studied mitochondrial function and expression of genes encoding mitochondrial chaperones and proteases in a human lymphoblastoid cell line and fibroblast cells from a patient who is heterozygous for the c.292G>A HSPD1 allele. We found that both the c.292G>A RNA transcript and the corresponding Hsp60-(p.Val98Ile) protein were present at comparable levels to their wild-type counterparts in SPG13 patient cells. Compared with control cells, we found no significant cellular or mitochondrial dysfunctions in SPG13 patient cells by assessing the mitochondrial membrane potential, cell viability, and sensitivity toward oxidative stress. However, a decreased expression of the mitochondrial protein quality control proteases Lon and ClpP, both at the RNA and protein level, was demonstrated in SPG13 patient cells. We propose that decreased levels of mitochondrial proteases Lon and ClpP may allow Hsp60 substrate proteins to go through more folding attempts instead of being prematurely degraded, thereby supporting productive folding in cells with reduced Hsp60 chaperonin activity. In conclusion, our studies with SPG13 patient cells expressing the functionally impaired mutant Hsp60 chaperonin suggest that reduction of the degradative activity of the protein quality control system may represent a previously

  20. Interleukin-21 regulates expression of key Epstein-Barr virus oncoproteins, EBNA2 and LMP1, in infected human B cells

    SciTech Connect

    Konforte, Danijela Simard, Nathalie; Paige, Christopher J.

    2008-04-25

    Epstein-Barr virus (EBV) persists for the life of the host by accessing the long-lived memory B cell pool. It has been proposed that EBV uses different combinations of viral proteins, known as latency types, to drive infected B cells to make the transition from resting B cells to memory cells. This process is normally antigen-driven. A major unresolved question is what factors coordinate expression of EBV latency proteins. We have recently described novel type III latency EBV{sup +} B cell lines (OCI-BCLs) that were induced to differentiate into late plasmablasts/early plasma cells in culture with interleukin-21 (IL-21), mimicking normal B cell development. The objective of this study was to determine whether IL-21-mediated signals also regulate the expression of key EBV latent proteins during this window of development. Here we show that IL-21-reduced gene and protein expression of growth-transforming EBV nuclear antigen 2 (EBNA2) in OCI-BCLs. By contrast, the expression of CD40-like, latent membrane protein 1 (LMP1) strongly increased in these cells suggesting an EBNA2-independent mode of regulation. Same results were also observed in Burkitt's lymphoma line Jijoye and B95-8 transformed lymphoblastoid cell lines. The effect of IL-21 on EBNA2 and LMP1 expression was attenuated by a pharmacological JAK inhibitor indicating involvement of JAK/STAT signalling in this process. Our study also shows that IL-21 induced transcription of ebna1 from the viral Q promoter (Qp)

  1. Application of next generation sequencing to CEPH cell lines to discover variants associated with FDA approved chemotherapeutics

    PubMed Central

    2014-01-01

    Background The goal of this study was to perform candidate gene association with cytotoxicity of chemotherapeutics in cell line models through resequencing and discovery of rare and low frequency variants along with common variations. Here, an association study of cytotoxicity response to 30 FDA approved drugs was conducted and we applied next generation targeted sequencing technology to discover variants from 103 candidate genes in 95 lymphoblastoid cell lines from 14 CEPH pedigrees. In this article, we called variants across 95 cell lines and performed association analysis for cytotoxic response using the Family Based Association Testing method and software. Results We called 2281 variable SNP genotypes across the 103 genes for these cell lines and identified three genes of significant association within this marker set. Specifically, ATP-binding cassette, sub-family C, member 5 (ABCC5), metallothionein 1A (MT1A) and NAD(P)H dehydrogenase quinone1 (NQO1) were significantly associated with oxaliplatin drug response. The significant SNP on NQO1 (rs1800566) has been linked with poor survival rates in patients with non-small cell lung cancer treated with cisplatin (which belongs to the same class of drugs as oxaliplatin). A SNP (rs1846692) near the 5′ region of MT1A was associated with arsenic trioxide. Conclusions The results from this study are promising and this serves as a proof-of-principle demonstration of the use of sequencing data in the cytotoxicity models of human cell lines. With increased sample sizes, such studies will be a fast and powerful way to associate common and rare variants with drug response; while overcoming the cost and time limitations to recruit cohorts for association study. PMID:24924344

  2. Reprogramming erythroid cells for lysosomal enzyme production leads to visceral and CNS cross-correction in mice with Hurler syndrome.

    PubMed

    Wang, Daren; Zhang, Wei; Kalfa, Theodosia A; Grabowski, Gregory; Davies, Stella; Malik, Punam; Pan, Dao

    2009-11-24

    Restricting transgene expression to maturing erythroid cells can reduce the risk for activating oncogenes in hematopoietic stem cells (HSCs) and their progeny, yet take advantage of their robust protein synthesis machinery for high-level protein production. This study sought to evaluate the feasibility and efficacy of reprogramming erythroid cells for production of a lysosomal enzyme, alpha-L-iduronidase (IDUA). An erythroid-specific hybrid promoter provided inducible IDUA expression and release during in vitro erythroid differentiation in murine erythroleukemia cells, resulting in phenotypical cross-correction in an enzyme-deficient lymphoblastoid cell line derived from patients with mucopolysaccharidosis type I (MPS I). Stable and higher than normal plasma IDUA levels were achieved in vivo in primary and secondary MPS I chimeras for at least 9 months after transplantation of HSCs transduced with the erythroid-specific IDUA-containing lentiviral vector (LV). Moreover, long-term metabolic correction was demonstrated by normalized urinary glycosaminoglycan accumulation in all treated MPS I mice. Complete normalization of tissue pathology was observed in heart, liver, and spleen. Notably, neurological function and brain pathology were significantly improved in MPS I mice by erythroid-derived, higher than normal peripheral IDUA protein. These data demonstrate that late-stage erythroid cells, transduced with a tissue-specific LV, can deliver a lysosomal enzyme continuously at supraphysiological levels to the bloodstream and can correct the disease phenotype in both viscera and CNS of MPS I mice. This approach provides a paradigm for the utilization of RBC precursors as a depot for efficient and potentially safer systemic delivery of nonsecreted proteins by ex vivo HSC gene transfer.

  3. Inhibitory Effect of Individual or Combinations of Broadly Neutralizing Antibodies and Antiviral Reagents against Cell-Free and Cell-to-Cell HIV-1 Transmission

    PubMed Central

    Kolodkin-Gal, Dror; Eslamizar, Leila; Owuor, Joshua O.; Mazzola, Emanuele; Gonzalez, Ana M.; Korioth-Schmitz, Birgit; Gelman, Rebecca S.; Montefiori, David C.; Haynes, Barton F.; Schmitz, Joern E.

    2015-01-01

    ABSTRACT To date, most therapeutic and vaccine candidates for human immunodeficiency virus type 1 (HIV-1) are evaluated preclinically for efficacy against cell-free viral challenges. However, cell-associated HIV-1 is suggested to be a major contributor to sexual transmission by mucosal routes. To determine if neutralizing antibodies or inhibitors block cell-free and cell-associated virus transmission of diverse HIV-1 strains with different efficiencies, we tested 12 different antibodies and five inhibitors against four green fluorescent protein (GFP)-labeled HIV-1 envelope (Env) variants from transmitted/founder (T/F) or chronic infection isolates. We evaluated antibody/inhibitor-mediated virus neutralization using either TZM-bl target cells, in which infectivity was determined by virus-driven luciferase expression, or A3R5 lymphoblastoid target cells, in which infectivity was evaluated by GFP expression. In both the TZM-bl and A3R5 assays, cell-free virus or infected CD4+ lymphocytes were used as targets for neutralization. We further hypothesized that the combined use of specific neutralizing antibodies targeting HIV-1 Env would more effectively prevent cell-associated virus transmission than the use of individual antibodies. The tested antibody combinations included two gp120-directed antibodies, VRC01 and PG9, or VRC01 with the gp41-directed antibody 10E8. Our results demonstrated that cell-associated virus was less sensitive to neutralizing antibodies and inhibitors, particularly using the A3R5 neutralization assay, and the potencies of these neutralizing agents differed among Env variants. A combination of different neutralizing antibodies that target specific sites on gp120 led to a significant reduction in cell-associated virus transmission. These assays will help identify ideal combinations of broadly neutralizing antibodies to use for passive preventive antibody administration and further characterize targets for the most effective neutralizing antibodies

  4. Differential regulation of membrane and secretory mu chain synthesis in human beta cell lines. Regulation of membrane mu or secreted mu

    PubMed Central

    1982-01-01

    Regulation of membrane and secretory mu synthesis was examined in human lymphoblastoid cell lines representing various stages of differentiation. Immunoglobulin phenotype was determined by surface and cytoplasmic staining with fluorochrome-conjugated antibodies and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of anti-mu precipitable cellular products. The thymidine analogue, 5-bromo-2'-deoxyuridine (BUdR), which inhibits differentiation-specific proteins in a variety of systems, was used to examine regulation of immunoglobulin synthesis. We found that BUdR had a differential effect on membrane (mum) and secretory (mus) type mu heavy chains. Ig production in pre-B and plasma cell-like lines, which make mus, was unaffected by BUdR. However, surface expression of IgM (mum) in B cell lines was drastically inhibited at similar doses of BUdR without diminishing total Ig or protein synthesis. Examination of labeled mu chains from control and BUdR-treated B cell lines by SDS- PAGE revealed the production of two sizes of mu (mum and mus) in control cells and only the smaller size (mus) in BUdR-treated cells. This size difference could not be attributed to alterations in glycosylation of the molecules. These data show that BUdR inhibits the production of membrane mu chains without diminishing secretory mu chain synthesis in the same cell. Our findings suggest that thymidine-rich regions of the genome are involved in the regulation of mum vs. mus during B cell differentiation. PMID:6816895

  5. Cloning and partial nucleotide sequence of human immunoglobulin mu chain cDNA from B cells and mouse-human hybridomas.

    PubMed Central

    Dolby, T W; Devuono, J; Croce, C M

    1980-01-01

    Purified mRNAs coding for mu and kappa human immunoglobulin polypeptides were translated in vitro and their products were characterized. The mu-specific mRNAs, derived from both human lymphoblastoid cells (GM607) and from a mouse-human somatic cell hybrid secreting human mu chains (alpha D5-H11-BC11), were copied into cDNAs and inserted into the plasmid pBR322. Several recombinant cDNAs that were obtained were identified by a combination of colony hybridization with labeled probes, in vitro translation of plasmid-selected mu mRNAs, and DNA nucleotide sequence determination. One recombinant DNA, for which the sequence has been partially determined, contains the codons for part of the C3 constant region domain through the carboxy-terminal piece (155 amino acids total) as well as the entire 3' noncoding sequence up to the poly(A) site of the human mu mRNA. The sequence A-A-U-A-A occurs 12 nucleotides prior to the poly(A) addition site in the human mu mRNA. Considerable sequence homology is observed in the mouse and human mu mRNA 3' coding and noncoding sequences. Images PMID:6777778

  6. Suberoylanilide Hydroxyamic Acid Modification of Chromatin Architecture Affects DNA Break Formation and Repair

    SciTech Connect

    Singh, Sheetal; Le Hongan; Shih, S.-J.; Ho, Bay; Vaughan, Andrew T.

    2010-02-01

    Purpose: Chromatin-modifying compounds that inhibit the activity of histone deacetylases have shown potency as radiosensitizers, but the action of these drugs at a molecular level is not clear. Here we investigated the effect of suberoylanilide hydroxyamic acid (SAHA) on DNA breaks and their repair and induction of rearrangements. Methods and Materials: The effect of SAHA on both clonogenic survival and repair was assessed using cell lines SCC-25, MCF7, and TK6. In order to study unique DNA double-strand breaks, anti-CD95 antibody was employed to introduce a DNA double-strand break at a known location within the 11q23 region. The effects of SAHA on DNA cleavage and rearrangements were analyzed by ligation-mediated PCR and inverse PCR, respectively. Results: SAHA acts as radiosensitizer at 1 {mu}M, with dose enhancement factors (DEFs) at 10% survival of: SCC-25 - 1.24 +- 0.05; MCF7 - 1.16 +- 0.09 and TK6 - 1.17 +- 0.05, and it reduced the capacity of SCC-25 cells to repair radiation induced lesions. Additionally, SAHA treatment diffused site-specific fragmentation over at least 1 kbp in TK6 cells. Chromosomal rearrangements produced in TK6 cells exposed to SAHA showed a reduction in microhomology at the breakpoint between 11q23 and partner chromosomes. Conclusions: SAHA shows efficacy as a radiosensitizer at clinically obtainable levels. In its presence, targeted DNA strand breaks occur over an expanded region, indicating increased chromatin access. The rejoining of such breaks is degraded by SAHA when measured as rearrangements at the molecular level and rejoining that contributes to cell survival.

  7. Physical mapping of new DNA probes near the fragile X mutation (FRAXA) by using a panel of cell lines

    PubMed Central

    Suthers, G. K.; Hyland, V. J.; Callen, D. F.; Oberle, I.; Rocchi, M.; Thomas, N. S.; Morris, C. P.; Schwartz, C. E.; Schmidt, M.; Ropers, H. H.; Baker, E.; Oostra, B. A.; Dahl, N.; Wilson, P. J.; Hopwood, J. J.; Sutherland, G. R.

    1990-01-01

    The fragile X syndrome is a very common disorder, but there has been little progress toward isolating the fragile X mutation (FRAXA). We describe a panel of 14 somatic cell hybrid lines, lymphoblastoid cell lines, and peripheral lymphocytes with X-chromosome translocation or deletion breakpoints near FRAXA. The locations of the breakpoints were defined with 16 established probes between pX45d (DXS100) and St14–1 (DXS52). Seven of the cell lines had breakpoints between the probes RN1 (DXS369) and U6.2 (DXS304), which flank FRAXA at distances of 3–5 centimorgans. The panel of cell lines was used to localize 16 new DNA probes in this region. Six of the probes–VK16, VK18, VK23, VK24, VK37, and VK47–detected loci near FRAXA, and it was possible to order both the X-chromosome breakpoints and the probes in relation to FRAXA. The order of probes and loci near FRAXA is cen–RN1,VK24–VK47–VK23–VK16,FRAXA–VK21A–VK18–IDS–VK37–U6.2-qter. The breakpoints near FRAXA are sufficiently close together that probes localized with this panel can be linked on a large-scale restriction map by pulsed-field gel electrophoresis. This panel of cell lines will be valuable in rapidly localizing other probes near FRAXA. ImagesFigure 2 PMID:2378346

  8. Hydroxyurea induces chromosomal damage in G2 and enhances the clastogenic effect of mitomycin C in Fanconi anemia cells.

    PubMed

    Molina, Bertha; Marchetti, Francesco; Gómez, Laura; Ramos, Sandra; Torres, Leda; Ortiz, Rocio; Altamirano-Lozano, Mario; Carnevale, Alessandra; Frias, Sara

    2015-06-01

    Fanconi's anemia (FA) is a recessive disease; 16 genes are currently recognized in FA. FA proteins participate in the FA/BRCA pathway that plays a crucial role in the repair of DNA damage induced by crosslinking compounds. Hydroxyurea (HU) is an agent that induces replicative stress by inhibiting ribonucleotide reductase (RNR), which synthesizes deoxyribonucleotide triphosphates (dNTPs) necessary for DNA replication and repair. HU is known to activate the FA pathway; however, its clastogenic effects are not well characterized. We have investigated the effects of HU treatment alone or in sequential combination with mitomycin-C (MMC) on FA patient-derived lymphoblastoid cell lines from groups FA-A, B, C, D1/BRCA2, and E and on lymphocytes from two unclassified FA patients. All FA cells showed a significant increase (P < 0.05) in chromosomal aberrations following treatment with HU during the last 3 h before mitosis. Furthermore, when FA cells previously exposed to MMC were treated with HU, we observed an increase of MMC-induced DNA damage that was characterized by high occurrence of DNA breaks and a reduction in rejoined chromosomal aberrations. These findings show that exposure to HU during G2 induces chromosomal aberrations by a mechanism that is independent of its well-known role in replication fork stalling during S-phase and that HU interfered mainly with the rejoining process of DNA damage. We suggest that impaired oxidative stress response, lack of an adequate amount of dNTPs for DNA repair due to RNR inhibition, and interference with cell cycle control checkpoints underlie the clastogenic activity of HU in FA cells. Environ. Mol. Mutagen. 56:457-467, 2015. © 2015 Wiley Periodicals, Inc.

  9. A 1,4-dihydropyridine derivative reduces DNA damage and stimulates DNA repair in human cells in vitro.

    PubMed

    Ryabokon, Nadezhda I; Goncharova, Rose I; Duburs, Gunars; Rzeszowska-Wolny, Joanna

    2005-11-10

    Compounds of the 1,4-dihydropyridine (1,4-DHP) series have been shown to reduce spontaneous, alkylation- and radiation-induced mutation rates in animal test systems. Here we report studies using AV-153, the 1,4-DHP derivative that showed the highest antimutagenic activity in those tests, to examine if it modulates DNA repair in human peripheral blood lymphocytes and in two human lymphoblastoid cell lines, Raji and HL-60. AV-153 caused a 50% inhibition of growth (IC50) of Raji and HL-60 cells at 14.9+/-1.2 and 10.3+/-0.8mM, respectively, but did not show a cytotoxic effect at concentrations <100 microM. Alkaline single-cell gel electrophoresis (comet) assays showed that AV-153 reduced the number of DNA strand breaks in untreated cells and also in cells exposed to 2 Gy of gamma-radiation, 100 microM ethylmethane sulfonate (EMS), or 100 microM H2O2. DNA damage was reduced by up to 87% at AV-153 concentrations between 1 and 10nM, and a positive dose-effect relationship was seen between 0.01 and 1 nM. Comparison of the kinetics of DNA strand-break rejoining in the presence and absence of AV-153 revealed a considerable influence on the rate of repair. In view of the resemblance of this compound's structure to that of dihydronicotinamide, a substrate for poly(ADP-rybose)polymerase, the modulation of DNA repair by AV-153 could involve an influence on poly(ADP)ribosylation.

  10. Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells

    SciTech Connect

    Iordanskiy, Sergey; Van Duyne, Rachel; Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao; Romerio, Fabio; Kashanchi, Fatah

    2015-11-15

    The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4{sup +} T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4{sup +} T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4{sup +} T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the “Shock and Kill” strategy for latently HIV-1 infected cells. - Highlights: • X-ray irradiation

  11. MicroRNAs 221 and 222 target p27Kip1 in Marek's disease virus-transformed tumour cell line MSB-1.

    PubMed

    Lambeth, Luke S; Yao, Yongxiu; Smith, Lorraine P; Zhao, Yuguang; Nair, Venugopal

    2009-05-01

    MicroRNAs (miRNAs) are a class of short RNAs that function as post-transcriptional suppressors of protein expression and are involved in a variety of biological processes, including oncogenesis. Several recent studies have implicated the involvement of miR-221 and miR-222 in tumorigenesis as these miRNAs are upregulated in a number of cancers and affect the expression of cell cycle regulatory proteins such as the cyclin-dependent kinase (cdk) inhibitor p27(Kip1). Marek's disease virus (MDV) is a highly oncogenic herpesvirus that affects poultry, causing acute neoplastic disease with lymphomatous lesions in several organs. MDV-encoded oncogenes such as Meq are directly implicated in the neoplastic transformation of T cells and have been well studied. More recently, however, the involvement of both host and virus-encoded miRNAs in the induction of MD lymphomas is being increasingly recognized. We analysed the miRNA expression profiles in the MDV-transformed lymphoblastoid cell line MSB-1 and found that endogenous miRNAs miR-221 and miR-222 were significantly upregulated. Demonstration of the conserved binding sites for these miRNAs in the chicken p27(Kip1) 3'-untranslated region sequence and the repression of luciferase activity of reporter constructs indicated that miR-221 and miR-222 target p27(Kip1) in these cells. We also found that overexpression of miR-221 and miR-222 decreased p27(Kip1) levels and that treatment with retrovirally expressed antagomiRs partially alleviated this suppression. These data show that an oncogenic herpesvirus, as in the case of many cancers, can exploit the miRNA machinery for suppressing cell cycle regulatory molecules such as p27(Kip1) in the induction and progression of T-cell lymphomas.

  12. Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells

    PubMed Central

    Iordanskiy, Sergey; Van Duyne, Rachel; Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao; Romerio, Fabio; Kashanchi, Fatah

    2015-01-01

    The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4+ T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4+ T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4+ T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the “Shock and Kill” strategy for latently HIV-1 infected cells. PMID:26184775

  13. LMP1 and LMP2A collaborate to promote Epstein-Barr virus (EBV)-induced B cell lymphomas in a cord blood-humanized mouse model but are not essential.

    PubMed

    Ma, Shi-Dong; Tsai, Ming-Han; Romero-Masters, James C; Ranheim, Erik A; Huebner, Shane M; Bristol, Jillian; Delecluse, Henri-Jacques; Kenney, Shannon C

    2017-01-11

    Epstein-Barr virus (EBV) infection is associated with B cell lymphomas in humans. The ability of EBV to convert human B cells into long-lived lymphoblastoid cell lines (LCLs) in vitro requires the collaborative effects of EBNA2 (which hijacks notch signaling), LMP1 (which mimics CD40 signaling), and EBNA 3A/3C (which inhibit oncogene-induced senescence and apoptosis). However, we recently showed that an LMP1-deleted EBV mutant induces B cell lymphomas in a newly developed cord blood-humanized mouse model that allows EBV-infected B cells to interact with CD4 T cells (the major source of CD40 ligand). Here we examined whether the EBV LMP2A protein, which mimics constitutively active B cell receptor signaling, is required for EBV-induced lymphomas in this model. We find that deletion of LMP2A delays the onset of EBV-induced lymphomas, but does not affect the tumor phenotype or the number of tumors. Simultaneous deletion of both LMP1 and LMP2A results in fewer tumors, and a further delay in tumor onset. Nevertheless, the double LMP1/LMP2A mutant induces lymphomas in approximately half of the infected animals. These results indicate that neither LMP1 nor LMP2A is absolutely essential for the ability of EBV to induce B cell lymphomas in the cord blood-humanized mouse model, although simultaneous loss of both LMP1/LMP2A decreases the proportion of animals developing tumors and increases the time to tumor onset. Thus, either LMP1 or LMP2A expression may be sufficient to promote early-onset EBV-induced tumors in this model.

  14. Stem Cells

    MedlinePlus

    Stem cells are cells with the potential to develop into many different types of cells in the body. ... the body. There are two main types of stem cells: embryonic stem cells and adult stem cells. Stem ...

  15. MicroRNA profile of Marek's disease virus-transformed T-cell line MSB-1: predominance of virus-encoded microRNAs.

    PubMed

    Yao, Yongxiu; Zhao, Yuguang; Xu, Hongtao; Smith, Lorraine P; Lawrie, Charles H; Watson, Michael; Nair, Venugopal

    2008-04-01

    Research over the last few years has demonstrated the increasing role of microRNAs (miRNAs) as major regulators of gene expression in diverse cellular processes and diseases. Several viruses, particularly herpesviruses, also use the miRNA pathway of gene regulation by encoding their own miRNAs. Marek's disease (MD) is a widespread lymphomatous neoplastic disease of poultry caused by the highly contagious Marek's disease virus type 1 (MDV-1). Recent studies using virus-infected chicken embryo fibroblasts have identified at least eight miRNAs that map to the R(L)/R(S) region of the MDV genome. Since MDV is a lymphotropic virus that induces T-cell lymphomas, analysis of the miRNA profile in T-cell lymphoma would be more relevant for examining their role in oncogenesis. We determined the viral and host miRNAs expressed in MSB-1, a lymphoblastoid cell line established from an MDV-induced lymphoma of the spleen. In this paper, we report the identification of 13 MDV-1-encoded miRNAs (12 by direct cloning and 1 by Northern blotting) from MSB-1 cells. These miRNAs, five of which are novel MDV-1 miRNAs, map to the Meq and latency-associated transcript regions of the MDV genome. Furthermore, we show that miRNAs encoded by MDV-1 and the coinfected MDV-2 accounted for >60% of the 5,099 sequences of the MSB-1 "miRNAome." Several chicken miRNAs, some of which are known to be associated with cancer, were also cloned from MSB-1 cells. High levels of expression of MDV-1-encoded miRNAs and potentially oncogenic host miRNAs suggest that miRNAs may have major roles in MDV pathogenesis and neoplastic transformation.

  16. Modulation of mitochondrial function by the microbiome metabolite propionic acid in autism and control cell lines

    PubMed Central

    Frye, R E; Rose, S; Chacko, J; Wynne, R; Bennuri, S C; Slattery, J C; Tippett, M; Delhey, L; Melnyk, S; Kahler, S G; MacFabe, D F

    2016-01-01

    Propionic acid (PPA) is a ubiquitous short-chain fatty acid, which is a major fermentation product of the enteric microbiome. PPA is a normal intermediate of metabolism and is found in foods, either naturally or as a preservative. PPA and its derivatives have been implicated in both health and disease. Whereas PPA is an energy substrate and has many proposed beneficial effects, it is also associated with human disorders involving mitochondrial dysfunction, including propionic acidemia and autism spectrum disorders (ASDs). We aimed to investigate the dichotomy between the health and disease effects of PPA by measuring mitochondrial function in ASD and age- and gender-matched control lymphoblastoid cell lines (LCLs) following incubation with PPA at several concentrations and durations both with and without an in vitro increase in reactive oxygen species (ROS). Mitochondrial function was optimally increased at particular exposure durations and concentrations of PPA with ASD LCLs, demonstrating a greater enhancement. In contrast, increasing ROS negated the positive PPA effect with the ASD LCLs, showing a greater detriment. These data demonstrate that enteric microbiome metabolites such as PPA can have both beneficial and toxic effects on mitochondrial function, depending on concentration, exposure duration and microenvironment redox state with these effects amplified in LCLs derived from individuals with ASD. As PPA, as well as enteric bacteria, which produce PPA, have been implicated in a wide variety of diseases, including ASD, diabetes, obesity and inflammatory diseases, insight into this metabolic modulator from the host microbiome may have wide applications for both health and disease. PMID:27779624

  17. Autologous Epstein-Barr virus (EBV)-specific cytotoxic T cells for the treatment of persistent active EBV infection.

    PubMed

    Savoldo, Barbara; Huls, M Helen; Liu, Zhensheng; Okamura, Takayuki; Volk, Hans-Dieter; Reinke, Petra; Sabat, Robert; Babel, Nina; Jones, James F; Webster-Cyriaque, Jennifer; Gee, Adrian P; Brenner, Malcolm K; Heslop, Helen E; Rooney, Cliona M

    2002-12-01

    Chronic active Epstein-Barr virus (CAEBV) infection syndrome is a heterogeneous EBV-related disorder characterized by chronic fatigue, fever, lymphadenopathy, and/or hepatosplenomegaly, associated with abnormal patterns of antibody to EBV. CAEBV can range from disabling mild/moderate forms to rapidly lethal disorders. Even patients with mild/moderate disease frequently suffer adverse effects from long-term anti-inflammatory agents and have a quality of life that progressively deteriorates. It is still unknown why these individuals are unable to produce an effective immune response to control EBV, and no effective treatment is currently available. Since ex vivo-expanded EBV-specific cytotoxic T lymphocytes (EBV-CTLs) can safely restore EBV-specific cellular immune responses in immunodeficient patients, we assessed the possibility that adoptive immunotherapy might also effectively treat CAEBV infection. Following stimulation with irradiated EBV-transformed lymphoblastoid cell lines (LCLs), EBV-CTLs were successfully generated from 8 of 8 patients with the mild/moderate form of CAEBV infection. These CTLs were predominantly CD3(+) CD8(+) cells and produced specific killing of the autologous LCLs. There were 5 patients with 1- to 12-year histories of disease who were treated with 1 to 4 injections of EBV-CTLs. Following infusion, there was resolution of fatigue and malaise, disappearance of fever, and regression of lymphadenopathy and splenomegaly. The pattern and titers of anti-EBV antibodies also normalized. No toxicity was observed. There were 4 patients who did not show any relapse of disease within 6 to 36 months follow-up; one patient had recurrence of fatigue and myalgia one year after CTL infusion. We suggest that adoptive immunotherapy with autologous EBV-CTLs may represent a safe and feasible alternative treatment for patients affected with mild/moderate CAEBV infection and that this approach should be evaluated in the more severe forms of the disease.

  18. Integrating pathway analysis and genetics of gene expression for genome-wide association study of basal cell carcinoma.

    PubMed

    Zhang, Mingfeng; Liang, Liming; Morar, Nilesh; Dixon, Anna L; Lathrop, G Mark; Ding, Jun; Moffatt, Miriam F; Cookson, William O C; Kraft, Peter; Qureshi, Abrar A; Han, Jiali

    2012-04-01

    Genome-wide association studies (GWASs) have primarily focused on marginal effects for individual markers and have incorporated external functional information only after identifying robust statistical associations. We applied a new approach combining the genetics of gene expression and functional classification of genes to the GWAS of basal cell carcinoma (BCC) to identify potential biological pathways associated with BCC. We first identified 322,324 expression-associated single-nucleotide polymorphisms (eSNPs) from two existing GWASs of global gene expression in lymphoblastoid cell lines (n = 955), and evaluated the association of these functionally annotated SNPs with BCC among 2,045 BCC cases and 6,013 controls in Caucasians. We then grouped them into 99 KEGG pathways for pathway analysis and identified two pathways associated with BCC with p value <0.05 and false discovery rate (FDR) <0.5: the autoimmune thyroid disease pathway (mainly HLA class I and II antigens, p < 0.001, FDR = 0.24) and Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway (p = 0.02, FDR = 0.49). Seventy-nine (25.7%) out of 307 significant eSNPs in the JAK-STAT pathway were associated with BCC risk (p < 0.05) in an independent replication set of 278 BCC cases and 1,262 controls. In addition, the association of JAK-STAT signaling pathway was marginally validated using 16,691 eSNPs identified from 110 normal skin samples (p = 0.08). Based on the evidence of biological functions of the JAK-STAT pathway on oncogenesis, it is plausible that this pathway is involved in BCC pathogenesis.

  19. Polymerase ε (POLE) ultra-mutated tumors induce robust tumor-specific CD4+ T cell responses in endometrial cancer patients

    PubMed Central

    Bellone, Stefania; Centritto, Floriana; Black, Jonathan; Schwab, Carlton; English, Diana; Cocco, Emiliano; Lopez, Salvatore; Bonazzoli, Elena; Predolini, Federica; Ferrari, Francesca; Silasi, Dan-Arin; Ratner, Elena; Azodi, Masoud; Schwartz, Peter E.; Santin, Alessandro D.

    2015-01-01

    Objective Around 7–10% of endometrial carcinomas are characterized by Polymerase-ε-(POLE) exonuclease-domain-mutations, an ultra-mutated-phenotype and a favorable prognosis. It is currently unknown whether POLE ultra-mutated-tumors are more immunogenic when compared to the other groups of endometrial cancers. Methods We used autologous-dendritic-cells (DC) pulsed with whole-tumor-extracts to assess the level of CD8+ and CD4+ T-cell-activation induced by POLE-ultramutated (+) and POLE wild-type (−) endometrial cancer cells in vitro. T-lymphocyte-proliferations were evaluated using CFSE and/or [3H]thymidine-incorporation-assays while the ability to specifically kill autologous-tumor-cells by cytotoxic-T-lymphocyte (CTL) was tested in standard 4-hr-51Cr-cytotoxicity-assays. In order to correlate cytotoxic activity and proliferation by CD4+ and CD8+ T-lymphocytes, respectively, with a particular lymphoid subset, two-color-flow-cytometric analysis of intracellular-cytokine-expression (IFN-γ vs IL-4) at the single cell level, was also performed. Results DC-pulsed with tumor extracts were able to induce CTL-responses against autologous-tumor-cells in both POLE (+) and POLE (−) cancer patients (P=0.305). These CD8+ T-cell-populations were cytotoxic against tumor-cells but they did not lyse PHA-stimulated-autologous-lymphocytes or autologous-EBV-transformed-lymphoblastoid-control-cell-lines. In contrast, only POLE (+) tumor-lysate-pulsed-DC were able to induce significant proliferation and high IFN-γ expression (i.e., Th1-cytokine-bias) in autologous in vitro DC-stimulated CD4+ T-cells as well as naïve CD4+ and CD8+ T-cells from patients-peripheral-blood (P < 0.05). Conclusions POLE ultra-mutated-tumors are significantly more immunogenic when compared to POLE (−) tumors, in particular to the helper arm of the immune system. These data lend support to the hypothesis that the better prognosis of patients with POLE (+) tumors may at least in part be linked to their

  20. Correction of both spontaneous and DEB-induced chromosome instability in Fanconi anemia FA-C cells by FACC cDNA

    SciTech Connect

    Stavropoulos, D.J.; Tomkins, D.J.; Allingham-Hawkins, D.J.; Buchwald, M.

    1994-09-01

    Cells from all four Fanconi anemia complementation groups show hypersensitivity to cell-killing by mitomycin C (MMC), diepoxybutane (DEB) and other DNA cross-linking agents, and increased spontaneous and DEB-induced chromosome aberrations (CA). The extent of these phenotypes varies between lymphoblastoid cell lines from different complementation groups. Our data showed that the difference in MMC hypersensitivity and DEB-CA was not always coupled. While 230N (FA-B) had higher DEB-induced CA/cell than 536N (FA-C) (7.42 vs. 4.46 respectively), that latter was much more sensitive to cell-killing by MMC (dose at 10% survival, D{sub 10}: 5.2 vs. 1.2 ng/ml respectively). Strathdes et al. (1992) cloned a cDNA Fanconi anemia complementation group C (FACC) which complemented the hypersensitivity to MMC and DEB cell-killing of FA-C cells (536N) but not cells from the other three complementation groups. The present study was initiated to determine whether chromosome instability in 536N is also complemented by the FACC (FAC3) cDNA. The pREP4-FAC3 vector was transfected into 536N and transfectants selected with hygromycin B. The DEB D{sub 10} of 536N (1.0 {mu}M) was corrected to the control level (16.2 {mu}M for 3TO) by FACC (15.1 {mu}M for 536N-FACC), as previously demonstrated. Chromosome instability (cab, cse, ctb, cte) was determined without and with 0.1 {mu}g/ml DEB treatment. Spontaneous CA of 536N (0.30 aberrations/cell) was corrected to the control level (0.04 for 3TO) by FACC (0.06 for 536N-FACC). Similarly, the DEB-induced CA was corrected (2.74 for 536N vs. 0.06 and 0.02 for 3TO and 536N-FACC respectively). Thus, at least for FA complementation group C, hypersensitivity to cell-killing and chromosome instability are not dissociated and are most likely caused by the same gene defect.

  1. Heterogeneity of Multifunctional IL-17A Producing S. Typhi-Specific CD8+ T Cells in Volunteers following Ty21a Typhoid Immunization

    PubMed Central

    McArthur, Monica A.; Sztein, Marcelo B.

    2012-01-01

    Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, continues to cause significant morbidity and mortality world-wide. CD8+ T cells are an important component of the cell mediated immune (CMI) response against S. Typhi. Recently, interleukin (IL)-17A has been shown to contribute to mucosal immunity and protection against intracellular pathogens. To investigate multifunctional IL-17A responses against S. Typhi antigens in T memory subsets, we developed multiparametric flow cytometry methods to detect up to 6 cytokines/chemokines (IL-10, IL-17A, IL-2, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1β (MIP-1β)) simultaneously. Five volunteers were immunized with a 4 dose regimen of live-attenuated S. Typhi vaccine (Ty21a), peripheral blood mononuclear cells (PBMC) were isolated before and at 11 time points after immunization, and CMI responses were evaluated. Of the 5 immunized volunteers studied, 3 produced detectable CD8+ T cell responses following stimulation with S. Typhi-infected autologous B lymphoblastoid cell lines (B-LCL). Additionally, 2 volunteers had detectable levels of intracellular cytokines in response to stimulation with S. Typhi-infected HLA-E restricted cells. Although the kinetics of the responses differed among volunteers, all of the responses were bi- or tri-phasic and included multifunctional CD8+ T cells. Virtually all of the IL-17A detected was derived from multifunctional CD8+ T cells. The presence of these multifunctional IL-17A+ CD8+ T cells was confirmed using an unsupervised analysis program, flow cytometry clustering without K (FLOCK). This is the first report of IL-17A production in response to S. Typhi in humans, indicating the presence of a Tc17 response which may be important in protection. The presence of IL-17A in multifunctional cells co-producing Tc1 cytokines (IL-2, IFN-γ and TNF-α) may also indicate that the distinction between Tc17 and Tc1

  2. Reduced DNA double-strand break repair capacity and risk of squamous cell carcinoma of the head and neck--A case-control study.

    PubMed

    Liu, Zhensheng; Liu, Hongliang; Gao, Fengqin; Dahlstrom, Kristina R; Sturgis, Erich M; Wei, Qingyi

    2016-04-01

    Tobacco smoke and alcohol use play important roles in the etiology of squamous cell carcinoma of the head and neck (SCCHN). Smoking causes DNA damage, including double-strand DNA breaks (DSBs), that leads to carcinogenesis. To test the hypothesis that suboptimal DSB repair capacity is associated with risk of SCCHN, we applied a flow cytometry-based method to detect the DSB repair phenotype first in four EBV-immortalized human lymphoblastoid cell lines and then in human peripheral blood T-lymphocytes (PBTLs). With this blood-based laboratory assay, we conducted a pilot case-control study of 100 patients with newly diagnosed, previously untreated SCCHN and 124 cancer-free controls of non-Hispanic whites. We found that the mean DSB repair capacity level was significantly lower in cases (42.1%) than that in controls (54.4%) (P<0.001). When we used the median DSB repair capacity level in the controls as the cutoff value for calculating the odds ratios (ORs) with adjustment for age, sex, smoking and drinking status, the cases were more likely than the controls to have a reduced DSB repair capacity (adjusted OR=1.93; 95% confidence interval, CI=1.04-3.56, P=0.037), especially for those subjects who were ever drinkers (adjusted OR=2.73; 95% CI=1.17-6.35, P=0.020) and had oropharyngeal tumors (adjusted OR=2.17; 95% CI=1.06-4.45, P=0.035). In conclusion, these findings suggest that individuals with a reduced DSB repair capacity may be at an increased risk of developing SCCHN. Larger studies are warranted to confirm these preliminary findings.

  3. Signaling through P2X7 receptor in human T cells involves p56lck, MAP kinases, and transcription factors AP-1 and NF-kappa B.

    PubMed

    Budagian, Vadim; Bulanova, Elena; Brovko, Luba; Orinska, Zane; Fayad, Raja; Paus, Ralf; Bulfone-Paus, Silvia

    2003-01-17

    multiple downstream signaling events in a human T-lymphoblastoid cell line.

  4. Cell division

    MedlinePlus Videos and Cool Tools

    ... the first 12 hours after conception, the fertilized egg cell remains a single cell. After approximately 30 ... at the end of 3 days, the fertilized egg cell has become a berry-like structure made ...

  5. Stem cells.

    PubMed

    Behr, Björn; Ko, Sae Hee; Wong, Victor W; Gurtner, Geoffrey C; Longaker, Michael T

    2010-10-01

    Stem cells are self-renewing cells capable of differentiating into multiple cell lines and are classified according to their origin and their ability to differentiate. Enormous potential exists in use of stem cells for regenerative medicine. To produce effective stem cell-based treatments for a range of diseases, an improved understanding of stem cell biology and better control over stem cell fate are necessary. In addition, the barriers to clinical translation, such as potential oncologic properties of stem cells, need to be addressed. With renewed government support and continued refinement of current stem cell methodologies, the future of stem cell research is exciting and promises to provide novel reconstructive options for patients and surgeons limited by traditional paradigms.

  6. Stem Cell Information: Glossary

    MedlinePlus

    ... cells (skeletal stem cells) Cell-based therapies Cell culture Cell division Chromosome Clone Cloning Cord blood stem cells Culture medium Differentiation Directed differentiation DNA Ectoderm Embryo Embryoid ...

  7. Engineering Cell-Cell Signaling

    PubMed Central

    Milano, Daniel F.; Natividad, Robert J.; Asthagiri, Anand R.

    2014-01-01

    Juxtacrine cell-cell signaling mediated by the direct interaction of adjoining mammalian cells is arguably the mode of cell communication that is most recalcitrant to engineering. Overcoming this challenge is crucial for progress in biomedical applications, such as tissue engineering, regenerative medicine, immune system engineering and therapeutic design. Here, we describe the significant advances that have been made in developing synthetic platforms (materials and devices) and synthetic cells (cell surface engineering and synthetic gene circuits) to modulate juxtacrine cell-cell signaling. In addition, significant progress has been made in elucidating design rules and strategies to modulate juxtacrine signaling based on quantitative, engineering analysis of the mechanical and regulatory role of juxtacrine signals in the context of other cues and physical constraints in the microenvironment. These advances in engineering juxtacrine signaling lay a strong foundation for an integrative approach to utilizing synthetic cells, advanced ‘chassis’ and predictive modeling to engineer the form and function of living tissues. PMID:23856592

  8. Engineering cell-cell signaling.

    PubMed

    Blagovic, Katarina; Gong, Emily S; Milano, Daniel F; Natividad, Robert J; Asthagiri, Anand R

    2013-10-01

    Juxtacrine cell-cell signaling mediated by the direct interaction of adjoining mammalian cells is arguably the mode of cell communication that is most recalcitrant to engineering. Overcoming this challenge is crucial for progress in biomedical applications, such as tissue engineering, regenerative medicine, immune system engineering and therapeutic design. Here, we describe the significant advances that have been made in developing synthetic platforms (materials and devices) and synthetic cells (cell surface engineering and synthetic gene circuits) to modulate juxtacrine cell-cell signaling. In addition, significant progress has been made in elucidating design rules and strategies to modulate juxtacrine signaling on the basis of quantitative, engineering analysis of the mechanical and regulatory role of juxtacrine signals in the context of other cues and physical constraints in the microenvironment. These advances in engineering juxtacrine signaling lay a strong foundation for an integrative approach to utilize synthetic cells, advanced 'chassis' and predictive modeling to engineer the form and function of living tissues.

  9. Fuel cells

    NASA Astrophysics Data System (ADS)

    1984-12-01

    The US Department of Energy (DOE), Office of Fossil Energy, has supported and managed a fuel cell research and development (R and D) program since 1976. Responsibility for implementing DOE's fuel cell program, which includes activities related to both fuel cells and fuel cell systems, has been assigned to the Morgantown Energy Technology Center (METC) in Morgantown, West Virginia. The total United States effort of the private and public sectors in developing fuel cell technology is referred to as the National Fuel Cell Program (NFCP). The goal of the NFCP is to develop fuel cell power plants for base-load and dispersed electric utility systems, industrial cogeneration, and on-site applications. To achieve this goal, the fuel cell developers, electric and gas utilities, research institutes, and Government agencies are working together. Four organized groups are coordinating the diversified activities of the NFCP. The status of the overall program is reviewed in detail.

  10. Autologous hematopoietic stem cell transplantation in lymphoma patients is associated with a decrease in the double strand break repair capacity of peripheral blood lymphocytes

    PubMed Central

    Lacoste, Sandrine; Bhatia, Smita; Chen, Yanjun; Bhatia, Ravi; O’Connor, Timothy R.

    2017-01-01

    Patients who undergo autologous hematopoietic stem cell transplantation (aHCT) for treatment of a relapsed or refractory lymphoma are at risk of developing therapy related- myelodysplasia/acute myeloid leukemia (t-MDS/AML). Part of the risk likely resides in inherent interindividual differences in their DNA repair capacity (DRC), which is thought to influence the effect chemotherapeutic treatments have on the patient’s stem cells prior to aHCT. Measuring DRC involves identifying small differences in repair proficiency among individuals. Initially, we investigated the cell model in healthy individuals (primary lymphocytes and/or lymphoblastoid cell lines) that would be appropriate to measure genetically determined DRC using host-cell reactivation assays. We present evidence that interindividual differences in DRC double-strand break repair (by non-homologous end-joining [NHEJ] or single-strand annealing [SSA]) are better preserved in non-induced primary lymphocytes. In contrast, lymphocytes induced to proliferate are required to assay base excision (BER) or nucleotide excision repair (NER). We established that both NHEJ and SSA DRCs in lymphocytes of healthy individuals were inversely correlated with the age of the donor, indicating that DSB repair in lymphocytes is likely not a constant feature but rather something that decreases with age (~0.37% NHEJ DRC/year). To investigate the predictive value of pre-aHCT DRC on outcome in patients, we then applied the optimized assays to the analysis of primary lymphocytes from lymphoma patients and found that individuals who later developed t-MDS/AML (cases) were indistinguishable in their DRC from controls who never developed t-MDS/AML. However, when DRC was investigated shortly after aHCT in the same individuals (21.6 months later on average), aHCT patients (both cases and controls) showed a significant decrease in DSB repair measurements. The average decrease of 6.9% in NHEJ DRC observed among aHCT patients was much

  11. Molecular cloning of a new secreted sulfated mucin-like protein with a C-type lectin domain that is expressed in lymphoblastic cells.

    PubMed

    Bannwarth, S; Giordanengo, V; Lesimple, J; Lefebvre, J C

    1998-01-23

    We have previously demonstrated hyposialylation of the two major CD45 and leukosialin (CD43) molecules at the surface of latently human immunodeficiency virus type 1-infected CEM T cells (CEMLAI/NP), (Lefebvre, J. C., Giordanengo, V., Doglio, A., Cagnon, L., Breittmayer, J. P., Peyron, J. F., and Lesimple, J. (1994) Virology 199, 265-274; Lefebvre, J. C., Giordanengo, V., Limouse, M., Doglio, A., Cucchiarini, M., Monpoux, F., Mariani, R., and Peyron, J. F. (1994) J. Exp. Med. 180, 1609-1617). Searching to clarify mechanism(s) of hyposialylation, we observed two sulfated secreted glycoproteins (molecular mass approximately 47 and approximately 40 kDa) (P47 and P40), which were differentially sulfated and/or differentially secreted in the culture supernatants of CEMLAI/NP cells when compared with parental CEM cells. A hybridoma clone (7H1) resulting from the fusion between CEMLAI/NP and human embryonic fibroblasts MRC5 cells produced very large amounts of P47 that was purified using Jacalin lectin (specific for O-glycans) and microsequenced. Cloning of P47 was achieved using a CEMLAI/NP cDNA library screened with a degenerate oligonucleotide probe based on its NH2-terminal amino acid sequence. A single open reading frame encoding a protein of 323 amino acids was deduced from the longest isolated recombinant (1.4 kilobase). P47 is a secreted sulfated protein. It carries an NH2-terminal RGD (Arg-Gly-Asp) triplet, a striking alpha-helical leucine zipper composed of six heptads, and a C-terminal C-type lectin domain. The NH2-terminal portion is rich in glutamic acids with a predicted pI of 3.9. In addition, a hinge region with numerous condensed potential sites for O-glycan side chains, which are also the most likely sulfation sites, is located between the RGD and leucine zipper domains. Transcripts were detected in lymphoid tissues (notably bone marrow) and abundantly in T and B lymphoblastoid but very faintly in monocytoid cell lines.

  12. Photoelectrochemical cells

    NASA Astrophysics Data System (ADS)

    Nozik, A. J.

    1980-02-01

    The application of photoelectrochemical systems based on photoactive semiconducting electrodes to the problem of solar energy conversion and chemical synthesis is discussed. Three types of cells are described: electrochemical photovoltaic cells (wherein optical energy is converted into electrical energy); photoelectrolysis cells (wherein optical energy is converted into chemical free energy); and photocatalytic cells (wherein optical energy provides the activation energy for exoergic chemical reactions). The critical semiconductor electrode properties for these cells are the band gap, the flat-band potential, and the photoelectrochemical stability. No semiconductor electrode material is yet known for which all three parameters are simultaneously optimized. An interesting configurational variation of photoelectrolysis cells, labelled 'photochemical diodes', is described. These diodes comprise cells that have been collapsed into monolithic particles containing no external wires. Recent advances in several areas of photoelectrochemical systems are also described.

  13. Types of Stem Cells

    MedlinePlus

    ... Stem Cell Glossary Search Toggle Nav Types of Stem Cells Stem cells are the foundation from which all ... Learn About Stem Cells > Types of Stem Cells Stem cells Stem cells are the foundation for every organ ...

  14. Electrolytic cell

    NASA Astrophysics Data System (ADS)

    Bullock, J. S.; Hale, B. D.

    1984-09-01

    An apparatus is described for the separation of the anolyte and the catholyte during electrolysis. The electrolyte flows through an electrolytic cell between the oppositely charged electrodes. The cell is equipped with a wedge-shaped device, the tapered end is located between the electrodes on the effluent side of the cell. The wedge diverts the flow of the electrolyte to either side of the wedge, substantially separating the anolyte and the catholyte.

  15. Cell Migration

    PubMed Central

    Trepat, Xavier; Chen, Zaozao; Jacobson, Ken

    2015-01-01

    Cell migration is fundamental to establishing and maintaining the proper organization of multicellular organisms. Morphogenesis can be viewed as a consequence, in part, of cell locomotion, from large-scale migrations of epithelial sheets during gastrulation, to the movement of individual cells during development of the nervous system. In an adult organism, cell migration is essential for proper immune response, wound repair, and tissue homeostasis, while aberrant cell migration is found in various pathologies. Indeed, as our knowledge of migration increases, we can look forward to, for example, abating the spread of highly malignant cancer cells, retarding the invasion of white cells in the inflammatory process, or enhancing the healing of wounds. This article is organized in two main sections. The first section is devoted to the single-cell migrating in isolation such as occurs when leukocytes migrate during the immune response or when fibroblasts squeeze through connective tissue. The second section is devoted to cells collectively migrating as part of multicellular clusters or sheets. This second type of migration is prevalent in development, wound healing, and in some forms of cancer metastasis. PMID:23720251

  16. Cell Chauvinism

    ERIC Educational Resources Information Center

    Keller, Dolores Elaine

    1972-01-01

    Indicates that biological terminology, such as mother cell'' and labels of sex factors in bacteria, reflect discrimination against females by reinforcing perpetuation of stereotyped gender roles. (AL)

  17. Unit Cells

    ERIC Educational Resources Information Center

    Olsen, Robert C.; Tobiason, Fred L.

    1975-01-01

    Describes the construction of unit cells using clear plastic cubes which can be disassembled, and one inch cork balls of various colors, which can be cut in halves, quarters, or eighths, and glued on the inside face of the cube, thus simulating a unit cell. (MLH)

  18. T Cells

    MedlinePlus

    ... Definition of MS Myelin Immune-Mediated Disease T Cells d What Causes MS? Disproved Theories Viruses Clusters d Who Gets MS? Pediatric MS ... the progression of MS, without harming any immune cells that are not involved in the process of myelin destruction. Share Smaller ... More Immune-Mediated Disease Learn More Myelin ...

  19. Fuel Cells

    ERIC Educational Resources Information Center

    Hawkins, M. D.

    1973-01-01

    Discusses the theories, construction, operation, types, and advantages of fuel cells developed by the American space programs. Indicates that the cell is an ideal small-scale power source characterized by its compactness, high efficiency, reliability, and freedom from polluting fumes. (CC)

  20. Cell Phones

    MedlinePlus

    ... Emitting Products Radiation-Emitting Products and Procedures Home, Business, and Entertainment Products Cell Phones Cell Phones Share Tweet Linkedin Pin it More sharing options Linkedin Pin it Email Print Under the law, FDA does not review the safety of radiation- ...

  1. Cell polarity

    PubMed Central

    Romereim, Sarah M

    2011-01-01

    Despite extensive genetic analysis of the dynamic multi-phase process that transforms a small population of lateral plate mesoderm into the mature limb skeleton, the mechanisms by which signaling pathways regulate cellular behaviors to generate morphogenetic forces are not known. Recently, a series of papers have offered the intriguing possibility that regulated cell polarity fine-tunes the morphogenetic process via orienting cell axes, division planes and cell movements. Wnt5a-mediated non-canonical signaling, which may include planar cell polarity, has emerged as a common thread in the otherwise distinct signaling networks that regulate morphogenesis in each phase of limb development. These findings position the limb as a key model to elucidate how global tissue patterning pathways direct local differences in cell behavior that, in turn, generate growth and form. PMID:22064549

  2. Fuel cells 101

    SciTech Connect

    Hirschenhofer, J.H.

    1999-07-01

    This paper discusses the various types of fuel cells, the importance of cell voltage, fuel processing for natural gas, cell stacking, fuel cell plant description, advantages and disadvantages of the types of fuel cells, and applications. The types covered include: polymer electrolyte fuel cell, alkaline fuel cell, phosphoric acid fuel cell; molten carbonate fuel cell, and solid oxide fuel cell.

  3. 9. ENGINE TEST CELL BUILDING INTERIOR. CELL ACCESS ELEVATOR, CELLS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    9. ENGINE TEST CELL BUILDING INTERIOR. CELL ACCESS ELEVATOR, CELLS 2 AND 4, BASEMENT LEVEL. LOOKING SOUTHEAST. - Fairchild Air Force Base, Engine Test Cell Building, Near intersection of Arnold Street & George Avenue, Spokane, Spokane County, WA

  4. Bi-Cell Unit for Fuel Cell.

    DTIC Science & Technology

    The patent concerns a bi-cell unit for a fuel cell . The bi-cell unit is comprised of two electrode packs. Each of the electrode packs includes an...invention relates in general to a bi-cell unit for a fuel cell and in particular, to a bi-cell unit for a hydrazine-air fuel cell .

  5. Electrochemical cell

    DOEpatents

    Redey, Laszlo I.; Vissers, Donald R.; Prakash, Jai

    1994-01-01

    An electrochemical cell having a bimodal positive electrode, a negative electrode of an alkali metal, and a compatible electrolyte including an alkali metal salt molten at the cell operating temperature. The positive electrode has an electrochemically active layer of at least one transition metal chloride at least partially present as a charging product, and additives of bromide and/or iodide and sulfur in the positive electrode or the electrolyte. Electrode volumetric capacity is in excess of 400 Ah/cm.sup.3 ; the cell can be 90% recharged in three hours and can operate at temperatures below 160.degree. C. There is also disclosed a method of reducing the operating temperature and improving the overall volumetric capacity of an electrochemical cell and for producing a positive electrode having a BET area greater than 6.times.10.sup.4 cm.sup.2 /g of Ni.

  6. Electrochemical cell

    DOEpatents

    Redey, Laszlo I.; Vissers, Donald R.; Prakash, Jai

    1996-01-01

    An electrochemical cell having a bimodal positive electrode, a negative electrode of an alkali metal, and a compatible electrolyte including an alkali metal salt molten at the cell operating temperature. The positive electrode has an electrochemically active layer of at least one transition metal chloride at least partially present as a charging product, and additives of bromide and/or iodide and sulfur in the positive electrode or the electrolyte. Electrode volumetric capacity is in excess of 400 Ah/cm.sup.3 ; the cell can be 90% recharged in three hours and can operate at temperatures below 160.degree. C. There is also disclosed a method of reducing the operating temperature and improving the overall volumetric capacity of an electrochemical cell and for producing a positive electrode having a BET area greater than 6.times.10.sup.4 cm.sup.2 /g of Ni.

  7. Electrochemical cell

    DOEpatents

    Redey, L.I.; Vissers, D.R.; Prakash, J.

    1996-07-16

    An electrochemical cell is described having a bimodal positive electrode, a negative electrode of an alkali metal, and a compatible electrolyte including an alkali metal salt molten at the cell operating temperature. The positive electrode has an electrochemically active layer of at least one transition metal chloride at least partially present as a charging product, and additives of bromide and/or iodide and sulfur in the positive electrode or the electrolyte. Electrode volumetric capacity is in excess of 400 Ah/cm{sup 3}; the cell can be 90% recharged in three hours and can operate at temperatures below 160 C. There is also disclosed a method of r