sq-23377 and Cell-Transformation--Viral

Cytolytic CD8+ T lymphocytes are the main cell type involved in the fatal lymphoproliferative-accelerated phase of the Chediak-Higashi syndrome (CHS). To generate a cellular tool to study the defects of this T cell subset in vitro, we have used Herpesvirus saimiri, a lymphotropic virus that transforms human T lymphocytes into extended growth and in addition, endows them with natural killer (NK) features. Transformed CHS CD8+ T cells were generated and characterized in comparison with healthy controls. The results showed that transformed CHS T cells maintained the defects described in primary CHS lymphocytes, such as giant secretory lysosomes and impaired NK and T cell receptor/CD3-induced, perforin-mediated cytolytic activity [which, however, could be restored after extended culture in the presence of interleukin-2 (IL-2)]. Upon activation with phorbol ester plus calcium ionophore or upon extended culture with IL-2, transformed CHS T cells showed normal, perforin-independent plasma membrane CD178/CD95L/FasL-mediated cytolytic activity but negligible secretion of microvesicle-bound CD95L. Transformed (and primary) CHS T cells were otherwise normal for cytolysis-independent activation functions, such as proliferation, surface expression of several activation markers including major histocompatibility complex class II, and cytokine or surface activation-marker induction. Therefore, the CHS protein [CHS1/LYST (for lysosomal traffic regulator)] can be dispensable for certain NK and T cell cytolytic activities of activated CHS CD8+ T lymphocytes, but it seems to be required for microvesicle secretion of CD95L. We conclude that transformed CHS T cells may be useful as a tool to study in vitro the relative role of CHS1/LYST in NK and T lymphocyte cytolysis and antigen presentation.

Topics: Antigen Presentation; CD8-Positive T-Lymphocytes; Cell Line; Cell Membrane; Cell Transformation, Viral; Chediak-Higashi Syndrome; Fas Ligand Protein; Female; Genes, MHC Class II; Humans; Interleukin-2; Ionomycin; Killer Cells, Natural; Lymphocyte Activation; Lysosomes; Membrane Glycoproteins; Proteins; Simplexvirus; Vesicular Transport Proteins

Apoptosis provoked by DNA damage requires the p53 tumor suppressor, but which of the many p53-regulated genes are required has remained unknown. Two genes induced by this transcription factor, noxa and puma (bbc3), stand out, because they encode BH3-only proteins, proapoptotic members of the Bcl-2 family required to initiate apoptosis. In mice with either noxa or puma disrupted, we observed decreased DNA damage-induced apoptosis in fibroblasts, although only loss of Puma protected lymphocytes from cell death. Puma deficiency also protected cells against diverse p53-independent cytotoxic insults, including cytokine deprivation and exposure to glucocorticoids, the kinase inhibitor staurosporine, or phorbol ester. Hence, Puma and Noxa are critical mediators of the apoptotic responses induced by p53 and other agents.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; B-Lymphocytes; Cell Transformation, Viral; Cytokines; Dexamethasone; DNA Damage; Etoposide; Fibroblasts; Gamma Rays; Gene Targeting; Ionomycin; Lymphocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Biological; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Suppressor Protein p53

Common variable immunodeficiency (CVID) is a very frequent but heterogeneous syndrome of antibody formation. The primary defect remains unknown, but many reports describe peripheral blood T lymphocyte dysfunctions in a substantial proportion of CVID patients, which may impair T--B cell collaboration. In order to investigate whether such putative defects were intrinsic to T cells or, rather, secondary to quantitative differences in T cell subset distribution, or to other described disorders, we have used Herpesvirus saimiri (HVS) for the targeted transformation of CVID CD4+ and CD8+ T cells and subsequent functional evaluation by flow cytometry of their capacity to generate cell surface (CD154, CD69) or soluble (IL-2, TNF-alpha, IFN-gamma) help after CD3 engagement. Unexpectedly, the results showed that 40 different CVID blood samples exposed to HVS gave rise with a significantly increased frequency to transformed CD4+ T cell lines, compared to 40 age-matched controls (27% versus 3%, P < or = 0.00002) suggesting the existence of a CVID-specific signalling difference which affects CD4+ cell transformation efficiency. The functional analysis of 10 CD4+ and 15 CD8+ pure transformed T cell lines from CVID patients did not reveal any statistically significant difference as compared to controls. However, half of the CD4+ transformed cell lines showed CD154 (but not CD69) induction (mean value of 46.8%) under the lower limit of the normal controls (mean value of 82.4%, P < or = 0.0001). Exactly the same five cell lines showed, in addition, a significantly low induction of IL-2 (P < or = 0.04), but not of TNF-alpha or IFN-gamma. None of these differences were observed in the remaining CD4+ cell lines or in any of the transformed CD8+ cell lines. We conclude that certain CVID patients show selective and intrinsic impairments for the generation of cell surface and soluble help by CD4+ T cells, which may be relevant for B lymphocyte function. The transformed T cell lines will be useful to establish the biochemical mechanisms responsible for the described impairments.

Topics: Adolescent; Adult; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Brefeldin A; CD3 Complex; CD4-Positive T-Lymphocytes; CD40 Ligand; CD8-Positive T-Lymphocytes; Cell Line, Transformed; Cell Transformation, Viral; Child; Common Variable Immunodeficiency; Female; Flow Cytometry; Gene Expression Regulation, Viral; Herpesvirus 2, Saimiriine; Humans; Immunophenotyping; Interferon-gamma; Interleukin-2; Ionomycin; Lectins, C-Type; Lymphocyte Activation; Male; Middle Aged; T-Lymphocyte Subsets; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

The mammalian Ras GTPase-activating protein (p120Ras-GAP) interacts with activated members of the Ras superfamily of GTP-binding proteins to accelerate their deactivation by sharply increasing their rates of GTP hydrolysis. Among the Ras-family proteins interacting with p120Ras-GAP is Rap1A/Krev1, whose activity is not affected by p120Ras-GAP but which competes with Ras for p120Ras-GAP. A second protein that interacts with p120Ras-GAP is P190Rac-GAP, which activates the GTPase of guanine nucleotide-binding proteins of the Rho family (including Rac1 and Rac2). Both these p120Ras-GAP-binding proteins are of interest in connection with the regulation of the respiratory burst oxidase, Rap1A/Krev1 because it copurifies with cytochrome b558 and p190Ras-GAP because it inhibits the Rac2-dependent activation of the respiratory burst oxidase in a cell-free system. Using an 18-mer antisense oligonucleotide, we were able to decrease the expression of p120Ras-GAP in Epstein-Barr virus-transformed B lymphocytes. Under conditions where p120Ras-GAP expression was significantly depressed by antisense oligonucleotides, we observed a 40% increase in protein kinase C-dependent but not receptor-dependent O2 production. In contrast, sense and scrambled oligonucleotides had no effect on either p120Ras-GAP expression or O2 production. Our results suggest a role for p120Ras-GAP as a negative regulator in the protein kinase C-mediated activation of the respiratory burst oxidase.

Topics: B-Lymphocytes; Base Sequence; Cell Line; Cell Transformation, Viral; Enzyme Inhibitors; GTP-Binding Proteins; GTPase-Activating Proteins; Herpesvirus 4, Human; Humans; Ionomycin; Kinetics; Molecular Sequence Data; NADH, NADPH Oxidoreductases; NADPH Oxidases; Oligonucleotides, Antisense; Protein Kinase C; Proteins; rac GTP-Binding Proteins; ras GTPase-Activating Proteins; Superoxides; Tetradecanoylphorbol Acetate; Time Factors

Posttransplant patients undergoing prolonged cyclosporine A (CsA) immunosuppressive therapy have been reported to have increased incidence of Epstein-Barr virus (EBV)-associated lymphoproliferative disorders. We undertook experiments to analyze the possible actions of CsA during EBV-infection of human peripheral blood mononuclear cells (PBMC). EBV-infected B cells cultured with CsA demonstrated increased EBV B-cell outgrowth as compared with those cultured without CsA. PBMC, after infection with EBV and CsA treatment, demonstrated increased interleukin-6 (IL-6) activity in the culture supernatant. The induction of IL-6 appears to differ within the various lymphocyte populations. In monocytes, IL-6 expression appears preferentially induced by EBV and is initiated by the binding of the two major virion glycoproteins, gp350 and gp220. Expression of IL-6 in T cells appears to be due mainly to CsA. B cells also express IL-6 after EBV exposure, but not after CsA treatment. EBV-immortalized B-cell lines cultured with CsA exhibited both an increased number of cells expressing viral lytic-cycle antigens and increased amounts of lytic-cycle proteins. IL-6, which is induced by CsA in PBMC, was also capable of inducing the lytic viral cycle in several EBV-immortalized cells. CsA, in promoting both increased numbers of lytic EBV B cells and an EBV paracrine factor, IL-6, within the microenvironment of EBV B cell:T cell and EBV B cell:monocyte interactions, may result in increased EBV B-cell immortalization and ultimately lead to the promotion of B-cell lymphomas in immunosuppressed patients.

Topics: Antigens, Viral; B-Lymphocytes; Cell Transformation, Viral; Cells, Cultured; Cyclosporine; Gene Expression Regulation, Viral; Herpesvirus 4, Human; Humans; Immunocompromised Host; Interleukin-6; Ionomycin; Lymphoma, B-Cell; Lymphoproliferative Disorders; Monocytes; Polymerase Chain Reaction; Receptors, Complement 3d; Stimulation, Chemical; Tetradecanoylphorbol Acetate; Tumor Virus Infections; Viral Matrix Proteins; Virus Activation

The development of transformed human airway epithelial cell lines has been important in advancing the understanding of the biochemical and genetic mechanisms underlying the cystic fibrosis (CF) defect. Since the most common mutation associated with CF is a phenylalanine deletion at position 508 (delta F508) in the CF transmembrane conductance regulator (CFTR) gene, a transformed airway epithelial cell line homozygous for this mutation will be important for determining the biologic significance of this mutation in the airways. We report the genotypic and phenotypic characterization of a delta F508 homozygote cell line derived from luminal epithelium in the trachea. The cells were transformed with a plasmid containing an origin of replication defective SV40 genome and have progressed through crisis. Immunocytochemical characterization of the cells shows that they express keratin, indicating epithelial cell origin, and that a calcium-dependent cell adhesion molecule, cellCAM 120/80, is present at plasma membrane junctions between cells. Electrophysiologically, the cells show no cAMP-dependent Cl transport. However, after treatment with the calcium ionophore, ionomycin, cells secrete Cl, albeit at a lower level than that observed in normal cells. Genetically, the cells express CFTR mRNA as determined by polymerase chain reaction amplification and CFTR protein as determined by Western hybridization analysis. Karyotypic analysis shows that 70% of the cells contain two copies of chromosome 7.

Topics: Adult; Base Sequence; Biological Transport; Blotting, Southern; Cell Line, Transformed; Cell Transformation, Viral; Chlorides; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA; Epithelium; Homozygote; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Ionomycin; Kinetics; Male; Membrane Proteins; Molecular Sequence Data; Mutation; Trachea

Circulating renin levels are regulated by release from juxtaglomerular (JG) cells. Here, for the first time, we describe the primary culture of rat juxtaglomerular cells on a reconstituted basement membrane. In addition, primary cultures were transformed with a temperature-sensitive SV40 large T antigen gene to promote the development of a continuous JG cell line. Both primary cultures and transformed JG cells maintain a highly differentiated state and secrete active renin. These preparations now provide a system in which characterization of the cellular mechanisms of regulation of renin synthesis and release is possible.

Topics: Animals; Antigens, Polyomavirus Transforming; Basement Membrane; Cell Division; Cell Transformation, Viral; Cells, Cultured; Cyclic AMP; Cytosol; Ionomycin; Juxtaglomerular Apparatus; Rats; Renin; RNA, Messenger; Second Messenger Systems; Simian virus 40; Thionucleotides

Intestinal epithelial cells were isolated from a fetus with cystic fibrosis (CF) and transfected with a plasmid vector recombined with the ori- mutant of SV40. A population of proliferative cells was then subcloned and designated as CFI-3. These cells had a doubling time of 24 h and were maintained in culture for up to 25 passages. At passage 8, CFI-3 cells did not produce any tumors in nude mice. Northern blot and immunofluorescence studies indicated that the extended lifespan of CFI-3 cells results in genomic insertion of SV40 LT. Intestinal CFI-3 cells are epithelial, according to the expression of the human cytokeratin 18 gene and poorly differentiated by phase-contrast and electron microscopy. Functional membrane receptors activated by vasoactive intestinal peptide (VIP), its natural analogue pituitary adenylate cyclase activating peptide (PACAP-38), and isoproterenol were observed in CFI-3 cells. Restriction fragment length polymorphism analysis of the PstI KM19 site revealed that the cftr locus was identical in the chorionic villi and in CFI-3 cells. The manifestation of CF in this family was not related to the common mutation delta F508, since this fetus was heterozygous for the substitutions S549N and N1303K. Chloride transport, assessed by the 125I efflux, was induced in CFI-3 cells by the calcium inophore ionomycin, but not by the adenylate cyclase activator forskolin, and was inhibited by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid. These results were confirmed in patch clamp studies in which the cpt cAMP analogue failed to stimulate membrane currents, while the calcium ionophore ionomycin stimulated inward currents. We conclude that intestinal CFI-3 cells retain the CF phenotype relating to defective regulation of Cl- channels, and therefore constitute a suitable model, 1) for elucidating the function of CFTR protein, 2) developing new therapeutic agents, and 3) correcting the CF defect by gene replacement therapy in vitro.

Topics: Antigens, Polyomavirus Transforming; Base Sequence; Cell Line; Cell Transformation, Viral; Chloride Channels; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Gene Expression; Humans; In Vitro Techniques; Intestinal Mucosa; Ionomycin; Isoproterenol; Membrane Proteins; Molecular Sequence Data; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Potassium Channels; Transfection; Vasoactive Intestinal Peptide

In many viral infections the host cell carries the viral genome without producing viral particles, a phenomenon known as viral latency. The cellular mechanisms by which viral latency is maintained or viral replication is induced are not known. The modulation of intracellular calcium concentrations by calcium ionophores induced Epstein-Barr viral antigens in lymphoblastoid cell lines that carry the virus. When calcium ionophores were used in conjunction with direct activators of protein kinase C (12-O-tetradecanoyl phorbol-13-acetate and a synthetic diacylglycerol), a greater induction of viral antigens was observed than with either agent alone. Activation of protein kinase C may be required for the expression of the viral genome.

Topics: Aminoquinolines; Burkitt Lymphoma; Calcimycin; Calcium; Cell Line; Cell Transformation, Viral; Culture Media; Ethers; Fluorescent Dyes; Genes, Viral; Herpesvirus 4, Human; Humans; Ionomycin; Kinetics; Tetradecanoylphorbol Acetate

The effects of the calcium channel blocking drug Verapamil and of palmitoyl-carnitine (PTC), an inhibitor of protein-kinase C activity, on human B cell activation were measured. Both Verapamil and PTC inhibited the B cell proliferation induced by costimulation with anti-mu antibody and with 3 different growth factors: interleukin 2, 20-kDa B cell growth factor and 50-kDa B cell growth factor. Both uridine and thymidine incorporation induced by costimulation with ionomycin and phorbol 12-myristate 13-acetate (PMA) were inhibited by Verapamil and PTC. In contrast, B cell proliferation was resistant to Verapamil (while being still inhibited by PTC) in two situations: when B cells were costimulated with PMA and growth factors and when B cells previously activated in vitro (by anti-mu antibody or PMA) were stimulated with growth factors. These results confirm that the late stage (G1----S transition) of B cell activation is independent of Ca2+ entry. More importantly, they show that the initial events induced by anti-mu antibody and by PMA are based on different biochemical pathways: PMA would act on a subpopulation of B cells which has already received an early signal of activation in vivo. This emphasizes the functional and biochemical heterogeneity of the G0 stage among circulating B cells.

Topics: B-Lymphocytes; Calcium; Cell Cycle; Cell Transformation, Viral; DNA; Ethers; Growth Substances; Herpesvirus 4, Human; Humans; Interleukin-4; Ionomycin; Lymphocyte Activation; Lymphokines; Palmitoylcarnitine; Protein Kinase C; Tetradecanoylphorbol Acetate; Verapamil