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

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 suppressive effect was reversed by exogenous LT. Both p80 and p60 forms of soluble TNF receptors suppressed the lymphoblastoid cell line proliferation and their inhibitory effect was partially reversed by LT. Our data suggest that (a) LT is an autocrine growth factor for EBV-transformed lymphoblastoid B cell lines; and (b) anti-LT antibodies, soluble TNF/LT receptors, and TNF itself can suppress the growth of lymphoblastoid cells, probably by modulating or competing with LT.(ABSTRACT TRUNCATED AT 400 WORDS

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

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 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 the diabetic state. Lymphoblastoid cells may represent a useful tool to guide an individualized understanding of the development and potential treatment of diabetic complications like retinopathy.</p

Genetic Variation Is the Major Determinant of Individual Differences in Leukocyte Endothelial Adhesion

Objective: To determine the genetic contribution to leukocyte endothelial adhesion.Methods: Leukocyte endothelial adhesion was assessed through a novel cell-based assay using human lymphoblastoid cell lines. A high-throughput screening method was developed to evaluate the inter-individual variability in leukocyte endothelial adhesion using lymphoblastoid cell lines derived from different donors. To assess heritability, ninety-two lymphoblastoid cell lines derived from twenty-three monozygotic twin pairs and twenty-three sibling pairs were compared. These lymphoblastoid cell lines were plated with the endothelial cell line EA.hy926 and labeled with Calcein AM dye. Fluorescence was assessed to determine endothelial cell adhesion to each lymphoblastoid cell line. Intra-pair similarity was determined for monozygotic twins and siblings using Pearson pairwise correlation coefficients.Results: A leukocyte endothelial adhesion assay for lymphoblastoid cell lines was developed and optimized (CV = 8.68, Z′-factor = 0.67, SNR = 18.41). A higher adhesion correlation was found between the twins than that between the siblings. Intra-pair similarity for leukocyte endothelial adhesion in monozygotic twins was 0.60 compared to 0.25 in the siblings. The extent to which these differences are attributable to underlying genetic factors was quantified and the heritability of leukocyte endothelial adhesion was calculated to be 69.66% (p-valueConclusions: There is a heritable component to leukocyte endothelial adhesion. Underlying genetic predisposition plays a significant role in inter-individual variability of leukocyte endothelial adhesion.</p

Hypersensitivity of BRCA1 heterozygote lymphoblastoid cells to gamma radiation and PARP inhibitors

This article is made available through the Brunel Open Access Publishing Fund. Copyright @ 2013 Bourton EC, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.PARP inhibitors can be used to induce synthetic lethality in cells with bi-allelic BRCA1 and BRCA2 mutations. However the effect of PARP inhibitors in combination with radiation on cells with mono-allelic mutations of BRCA1 and BRCA2 is unknown. We have examined the cell survival response of lymphoblastoid cells derived from normal individuals and those derived from carriers of BRCA1 and BRCA2 mutations, following exposure to ionising radiation and the PARP inhibitor Olaparib. Two lymphoblastoid cell lines from normal individuals and three with mono-allelic mutations in BRCA1 and BRCA2 were exposed to increasing doses of gamma radiation either alone or in combination with 5 μM Olaparib. Cell survival was measured using the MTT assay. Exposure to increasing doses of gamma radiation caused a reduction in cell survival of all cell types. The combined exposure to gamma radiation and 5 μM Olaparib did not enhance cell kill in normal or BRCA2 heterozygote lymphoblastoid cells but significantly enhanced cell kill in cells derived from BRCA1 carriers (P = 0.02). The treatment of cancer patients carrying mutations in the BRCA1 gene with radiotherapy and the PARP inhibitor Olaparib may significantly enhance radiation induced normal tissue toxicity in these patients.Vidal Sassoon Foundation of America and “The Balls to Cancer” Charity, Coventry, U

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

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

Chromosome positioning is largely unaffected in lymphoblastoid cell lines containing emerin or A-type lamin mutations

Gene-poor human chromosomes are reproducibly found at the nuclear periphery in proliferating cells. There are a number of inner nuclear envelope proteins that may have roles in chromosome location and anchorage, e.g. emerin and A-type lamins. In the last decade, a number of diseases associated with tissue degeneration and premature aging have been linked with mutations in lamin A or emerin. These are termed laminopathies, withmutations in emerin causing Emery–Dreifuss muscular dystrophy. Despite highly aberrant nuclear distributions of A-type lamins and emerin in lymphoblastoid cell lines derived from patients with emerin or lamin A mutations, little or no change in chromosome location was detected

The Deafness-Associated Mitochondrial DNA Mutation at Position 7445, Which Affects tRNASer(UCN) Precursor Processing, Has Long-Range Effects on NADH Dehydrogenase Subunit ND6 Gene Expression

The pathogenetic mechanism of the deafness-associated mitochondrial DNA (mtDNA) T7445C mutation has been investigated in several lymphoblastoid cell lines from members of a New Zealand pedigree exhibiting the mutation in homoplasmic form and from control individuals. We show here that the mutation flanks the 3' end of the tRNASer(UCN) gene sequence and affects the rate but not the sites of processing of the tRNA precursor. This causes an average reduction of ~70% in the tRNASer(UCN) level and a decrease of ~45% in protein synthesis rate in the cell lines analyzed. The data show a sharp threshold in the capacity of tRNASer(UCN) to support the wild-type protein synthesis rate, which corresponds to ~40% of the control level of this tRNA. Strikingly, a 7445 mutation-associated marked reduction has been observed in the level of the mRNA for the NADH dehydrogenase (complex I) ND6 subunit gene, which is located ~7 kbp upstream and is cotranscribed with the tRNASer(UCN) gene, with strong evidence pointing to a mechanistic link with the tRNA precursor processing defect. Such reduction significantly affects the rate of synthesis of the ND6 subunit and plays a determinant role in the deafness-associated respiratory phenotype of the mutant cell lines. In particular, it accounts for their specific, very significant decrease in glutamate- or malate-dependent O2 consumption. Furthermore, several homoplasmic mtDNA mutations affecting subunits of NADH dehydrogenase may play a synergistic role in the establishment of the respiratory phenotype of the mutant cells

The PARP-1 inhibitor Olaparib causes retention of γ-H2AX foci in BRCA1 heterozygote cells following exposure to gamma radiation

This article is made available through the Brunel Open Access Publishing Fund. Copyright © 2013 Emma C. Bourton et al. This is an open access article distributed under the Creative Commons Attribution Li-cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.A novel treatment for cancer patients with homozygous deletions of BRCA1 and BRCA2 is to use drugs that inhibit the enzyme poly(ADP-ribose) polymerase (PARP). Specific inhibition of PARP-1 can induce synthetic lethality in irradi- ated cancer cells while theoretically leaving normal tissue unaffected. We recently demonstrated in a cell survival assay that lymphoblastoid cells with mono-allelic mutations of BRCA1 were hypersensitive to gamma radiation in the pres- ence of the PARP-1 inhibitor Olaparib compared to normal cells and mono-allelic BRCA2 cells. To determine if the enhanced radiation sensitivity was due to a persistence of DNA strand breaks, we performed γ-H2AX foci analysis in cells derived from two normal individuals, three heterozygous BRCA1 and three heterozygous BRCA2 cell lines. Cells were exposed to 2 Gy gamma radiation in the presence or absence of 5 μM Olaparib. Using immunofluorescence and imaging flow cytometry, foci were measured in untreated cells and at 0.5, 3, 5 and 24 hours post-irradiation. In all lymphoblastoid cells treated with 2 Gy gamma radiation, there was a predictable induction of DNA strand breaks, with a modest but significant retention of foci over 24 hours in irradiated cells treated with Olaparib (ANOVA P < 0.05). However, in mono-allelic BRCA1 cells, there was a failure to fully repair DNA double-strand breaks (DSB) in the pres- ence of Olaparib, evidenced by a significant retention of foci at 24 hours’ post irradiation (t-Test P < 0.05). These data show that the cellular hypersensitivity of mono-allelic BRCA1 lymphoblastoid cells to gamma radiation in the presence of the Olaparib is due to the retention of DNA DSB. These data may indicate that patients with inherited mutations in the BRCA1 gene treated with radiotherapy and PARP-1 inhibitors may experience elevated radiation-associated normal tissue toxicity.Vidal Sassoon Foundation of America

Inter- and intra-specific gene-density-correlated radial chromosome territory arrangements are conserved in Old World monkeys

Recently it has been shown that the gene-density correlated radial distribution of human 18 and 19 homologous chromosome territories (CTs) is conserved in higher primates in spite of chromosomal rearrangements that occurred during evolution. However, these observations were limited to apes and New World monkey species. In order to provide further evidence for the evolutionary conservation of gene-density-correlated CT arrangements, we extended our previous study to Old World monkeys. They comprise the remaining species group to be analyzed in order to obtain a comprehensive overview of the nuclear topology of human 18 and 19 homologous CTs in higher primates. In the present study we investigated four lymphoblastoid cell lines from three species of Old World monkeys by three-dimensional fluorescence in situ hybridization (3D-FISH): two individuals of Japanese macaque ( Macaca fuscata), crab-eating macaque ( Macaca fascicularis), and an interspecies hybrid individual between African green monkey (Cercopithecus aethiops) and Patas monkey ( Erythrocebus patas). Our data demonstrate that gene-poor human 18 homologous CTs are located preferentially close to the nuclear periphery, whereas gene-dense human 19 homologous CTs are oriented towards the nuclear center in all cell lines analyzed. The gene-density-correlated positioning of human 18 and 19 homologous CTs is evolutionarily conserved throughout all major higher primate lineages, despite chromosomal inversions, fusions, fissions or reciprocal translocations that occurred in the course of evolution in these species. This remarkable preservation of a gene-density-correlated chromatin arrangement gives further support for a functionally relevant higher-order chromatin architecture. Copyright (C) 2005 S. Karger AG, Basel