calcimycin and Cell-Transformation--Viral

Topics: Amiloride; Animals; Calcimycin; Calcium; Carrier Proteins; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Dimethyl Sulfoxide; Electrolytes; Erythropoiesis; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Lipopolysaccharides; Lymphocytes; Lymphoma; Mice; Monensin; Ouabain; Protons; Sodium; Sodium-Hydrogen Exchangers; Sodium-Potassium-Exchanging ATPase

Mutations of the tumor suppressor gene PTEN have been reported in patients with Cowden disease (CD) and in several malignant tumors. We analyzed a germline mutation of the PTEN gene in a patient with CD and identified a 4-bp deletion in exon 8 of the PTEN gene. The same germline mutation was detected in 3 members of her family. The mutated gene was predicted to encode a C-terminal truncated PTEN protein. Immunoblotting analysis revealed that the expression level of the wild-type PTEN protein in the patient's lymphocytes was reduced to almost half the level of the control lymphocytes, and the predicted truncated mutant PTEN could not be detected. Since PTEN is known to function as a negative regulator of the phosphatidylinositol-3-kinase signal pathway that promotes cell survival, the patient's lymphocytes were tested for the resistance against the apoptotic stimulus. It was shown that the patient's lymphocytes were more resistant to apoptosis induced by calcium ionophore than the healthy control lymphocytes. These results indicate that the germline mutation of the PTEN gene and the consequent loss of heterozygous expression may lead to an increase in the survival potential of cells, thereby elucidating a role of PTEN in the pathogenesis of tumor generation and hyperplasia of lymphoid tissue in CD.

Topics: Apoptosis; B-Lymphocytes; Base Sequence; Calcimycin; Cell Survival; Cell Transformation, Viral; Cells, Cultured; DNA Mutational Analysis; DNA, Neoplasm; Dose-Response Relationship, Drug; Female; Germ-Line Mutation; Hamartoma Syndrome, Multiple; Humans; Ionophores; Male; Middle Aged; Molecular Sequence Data; Pedigree; Phosphoric Monoester Hydrolases; PTEN Phosphohydrolase; Tumor Suppressor Proteins

The transverse redistribution of plasma membrane phosphatidylserine is one of the hallmarks of cells undergoing apoptosis and also occurs in cells fulfilling a more specialized function, such as platelets after appropriate activation. Although an increase in intracellular Ca2+ is required to trigger the remodeling of the plasma membrane, little information regarding intracellular signals leading to phosphatidylserine externalization has been provided. Scott syndrome is an extremely rare inherited disorder of the migration of phosphatidylserine toward the exoplasmic leaflet of the plasma membrane of stimulated blood cells. We have studied here the intracellular Ca2+ mobilization and Ca2+ entry involved in tyrosine phosphorylation in Epstein Barr virus (EBV)-infected B cells derived from a patient with Scott syndrome, her daughter, and control subjects. An alteration of Ca2+ entry through the plasma membrane and subsequent tyrosine phosphorylation induced by Ca2+ were observed in Scott EBV-B cells, but the release of Ca2+ from intracellular stores was normal. Furthermore, phosphatidylserine externalization at the surface of stimulated cells does not depend on tyrosine kinases. These results suggest that the defect of phosphatidylserine exposure in Scott syndrome cells is related to the alteration of a particular way of Ca2+ entry, referred to as capacitative Ca2+ entry, although some differences may be related to the cell type. Hence, this genetic mutant testifies to the prime significance of Ca2+ signaling in the regulation of phosphatidylserine expression at the surface of stimulated cells.

Topics: Aged; B-Lymphocytes; Blood Coagulation Disorders; Calcimycin; Calcium Channels; Cell Membrane; Cell Transformation, Viral; Cells, Cultured; Enzyme Inhibitors; Female; Genistein; Humans; Lymphocyte Activation; Phosphatidylserines; Phospholipids; Phosphorylation; Protein-Tyrosine Kinases; Syndrome; Thapsigargin; Tyrosine

Scott syndrome is a bleeding disorder associated with an isolated defect in expression of membrane coagulant activity by stimulated platelets. This defect represents a decrease in platelet membrane binding sites for coagulation factors Va and VIIIa, reflecting diminished surface exposure of phosphatidylserine (PS). To gain insight into the cellular and genetic basis for this disorder, B-lymphocytes from a patient with Scott syndrome and from normal donors were immortalized by EBV-transformation, and tested for their capacity to expose plasma membrane PS in response to the Ca2+ ionophore, A23187. Upon incubation with A23187, EBV-lymphoblasts derived from normal donors consistently induced surface expression of PS in > 70% of all cells, as detected by membrane association of the PS-binding proteins, factor Va or annexin V. PS exposure in these cells was maximal after 5 min, and saturated at < 100 microM external free [Ca2+]. By contrast, < 30% of Scott syndrome lymphoblasts exposed PS, and saturation was not observed at > 1 mM external free [Ca2+]. Single-cell clones derived from the Scott lymphoblasts all exhibited a diminished response to A23187 comparable with that of the parental cells, suggesting that all lymphocytes from this patient share this membrane abnormality. Hybridomas prepared by fusion of Scott lymphoblasts with the myeloma cell line UC-LUC showed responses to Ca2+ ionophore comparable to those observed for normal lymphoblasts and for hybridomas prepared by fusion of normal lymphoblasts with UC-LUC. This correction of the Scott abnormality suggests possible complementation of an aberrant gene(s) responsible for this disorder.

Topics: Annexin A5; B-Lymphocytes; Blood Coagulation Disorders; Calcimycin; Calcium; Cell Death; Cell Membrane; Cell Transformation, Viral; Clone Cells; Dimethyl Sulfoxide; Factor Va; Herpesvirus 4, Human; Humans; Hybridomas; Phosphatidylserines; Protein Binding

The role of latent membrane protein 2 (LMP2) in Epstein-Barr virus (EBV) infection was evaluated by using latently infected primary B lymphocytes that had been growth transformed by wild-type or specifically mutated EBV recombinants. LMP2 null mutant recombinant EBV-infected cells were similar to normal B lymphocytes in their rapid increase in intracellular free calcium after surface immunoglobulin crosslinking. These cells also became more permissive for lytic EBV replication. In sharp contrast, wild-type control infected cells had little or no increase in intracellular free calcium or in permissivity for EBV replication. The block to surface immunoglobulin crosslinking-induced permissivity in cells expressing wild-type LMP2 could be bypassed by raising intracellular free calcium levels with an ionophore and by activating protein kinase C with phorbol 12-myristate 13-acetate. LMP2A, not LMP2B, mediates this effect on calcium mobilization. Genetic and biochemical data are consistent with these effects being due to the interaction of the LMP2A N-terminal cytoplasmic domain with B lymphocyte src family tyrosine kinases.

Topics: Amino Acid Sequence; Antigens, Viral; Calcimycin; Calcium; Capsid; Cell Line, Transformed; Cell Transformation, Viral; Cross-Linking Reagents; Herpesvirus 4, Human; Humans; Molecular Sequence Data; Protein Kinase C; Receptors, Antigen, B-Cell; Tetradecanoylphorbol Acetate; Viral Matrix Proteins; Virus Activation; Virus Replication

Topics: B-Lymphocytes; Calcimycin; Cell Line, Transformed; Cell Transformation, Viral; Herpesvirus 4, Human; Humans; Immunoglobulin M; Kinetics; Phospholipase D; Receptors, Antigen, B-Cell; Tetradecanoylphorbol Acetate

The activated ras oncogene is a key mediator of cellular transformation and is present in a wide variety of primary human neoplasms. The biochemical role of the ras oncogene in cellular transformation is at present unclear, and hence approaches to control its activities in transformed cells have met with limited success. Previous studies have demonstrated the ability of melittin, a 26 amino acid amphipathic peptide from bee venom, to specifically counterselect for cells in culture that express high levels of the ras oncogene product. The biochemical basis for this counterselection is currently unknown. This study demonstrates the ability of melittin to hyperactivate phospholipase A2 (PLA2) in ras-transformed cells by the mediation of enhanced influx of calcium ions (Ca2+). This hyperactivation of PLA2 and Ca2+ mobilization in ras-transformed cells by melittin is mimicked by the calcium ionophore, A23187. Both melittin- and A23187-mediated PLA2 hyperactivation require Ca2+. However, the action of melittin is strongly dependent on extracellular Ca2+, whereas that of A23187 is not. Melittin-induced Ca2+ influx and PLA2 hyperactivation is inhibited by manganese ions (Mn2+). These studies reveal a close correlation between the extent of PLA2 hyperactivation and Ca2+ mobilization, suggesting a causal relationship.

Topics: 3T3 Cells; Animals; Calcimycin; Calcium; Cell Line, Transformed; Cell Transformation, Viral; Cytosol; Enzyme Activation; Genes, ras; In Vitro Techniques; Manganese; Melitten; Mice; Phospholipases A; Phospholipases A2

Topics: Antigens, CD; Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; Calcimycin; Cell Communication; Cell Transformation, Viral; Epoxide Hydrolases; Herpesvirus 4, Human; Humans; Leukotriene A4; Leukotriene B4; Leukotrienes; Monocytes; Receptors, Fc; Receptors, IgE; Thymidine; Tritium; Tumor Cells, Cultured

Ligand binding to B lymphocytes via membrane Ig initiates a cascade of events beginning with the hydrolysis of phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-trisphosphate. Subsequent to the activation of protein kinase C and the induction of a rise in intracellular calcium by diacylglycerol and inositol 1,4,5-trisphosphate, there is gene transcription and eventually cellular activation. By mimicking the initial event of B cell activation with phorbol ester and calcium ionophore one can begin to identify the many mediators used in signaling between the membrane and the nucleus. We have examined the effect of calcium on arachidonic acid (AA) metabolism in several EBV-transformed human B cell lines. The cells were labelled with [3H]AA and stimulated with the calcium ionophore A23187. Analysis of the supernatant by reversed-phase HPLC demonstrated a dose-dependent release of an AA metabolite that coeluted with authentic 5-hydroxyeicosatetraenoic acid (5-HETE). In addition, the AA metabolite coeluted with standard 5-HETE under straight-phase chromatography. Further analysis by RIA confirmed the identification of 5-HETE and revealed an additional metabolite, 5-HETE lactone (5-HL). 5-HL is the intramolecular ester of 5-HETE generated in the presence of acid. We were unable to convert [3H] 5-HETE into 5-HL during sample preparation unless cells were present, suggesting that the 5-HL, is of cellular origin. These results suggest that the AA metabolites 5-HETE and its intramolecular ester 5-HL may play a role in B cell activation because they are produced subsequent to a rise in intracellular Ca2+, an event that occurs during cross-linking of membrane Ig.

Topics: Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; B-Lymphocytes; Calcimycin; Cell Line; Cell Transformation, Viral; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Signal Transduction; T-Lymphocytes

Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with [35S]methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of [32P]orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.

Topics: 2,2'-Dipyridyl; Adenosine Triphosphate; Animals; Arsenic; Arsenites; Calcimycin; Carrier Proteins; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Chick Embryo; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum Chaperone BiP; Fibroblasts; Heat-Shock Proteins; Methionine; Mice; Mice, Inbred BALB C; Precipitin Tests; Sepharose; Sulfur Radioisotopes; Tetradecanoylphorbol Acetate; Tunicamycin

The infection of HeLa cells by poliovirus leads to profound alterations in the activities of both phospholipase C and the A23187-stimulated phospholipase A2. As early as the third hour after poliovirus infection, the activity of phospholipase C is enhanced, as measured by the increase in inositol triphosphate (IP3) in the cells. By the fifth hour post-infection there is a 5-fold increase in IP3 in the infected cells. Therefore, the synthesis of the bulk of poliovirus proteins and poliovirus genomes takes place in cells containing a high and sustained increase in IP3. This augmentation in IP3 is dependent on the multiplicity of infection used. Poliovirus gene expression is required to induce the increase in phospholipase C activity, since the presence of cycloheximide or guanidine blocked it. In contrast to the activation of phospholipase C induced by poliovirus, there is a drastic blockade of the A23187-induced phospholipase A2 activity, measured as the release of [3H]arachidonic acid to the medium. This action on phospholipase A2 is dependent on poliovirus gene expression because it was prevented by cycloheximide or 3-methylquercetin. To our knowledge this is the first report analyzing these two activities in animal virus-infected cells. The findings described may help to explain the profound modifications of both membrane permeability and lipid metabolism undergone by poliovirus-infected cells.

Topics: Antiviral Agents; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Transformation, Viral; Cycloheximide; Flavonols; HeLa Cells; Humans; Hygromycin B; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Phospholipases; Phospholipases A; Phospholipases A2; Poliovirus; Quercetin; Type C Phospholipases

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

Stimulation of protein kinase C in polyoma virus-transformed cells increased the phosphorylation of tyrosine residues of the viral middle T (mT) antigen in mT:pp60c-src complexes precipitated by anti-mT antibodies. This increase might have been due to a stimulation of the complex's pp60c-src tyrosine kinase activity or to an increased ability of the mT protein to be phosphorylated by pp60c-src. These observations suggest that cellular protein kinase C might control the ability of polyoma virus to transform its host cell.

Topics: Animals; Antigens, Polyomavirus Transforming; Antigens, Viral, Tumor; Calcimycin; Cell Transformation, Viral; Densitometry; Mice; Molecular Weight; Oncogene Protein pp60(v-src); Oncogene Proteins, Viral; Phosphorylation; Polyomavirus; Protein Kinase C; Protein-Tyrosine Kinases; Rats; Retroviridae Proteins; Tetradecanoylphorbol Acetate; Threonine; Tyrosine; Vanadates; Vanadium

Previous studies in human foreskin fibroblasts suggested that the mechanism by which serum stimulates Na+ influx is via a Ca2+-calmodulin-mediated event. In the present experiments in normal WI-38 cells (human lung fibroblasts), both the intracellular Ca2+ antagonist 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) and the potent calmodulin antagonist trifluoperazine (TFP) blocked serum-stimulated Na+ influx [TMB-8 concentration causing half-maximal inhibition (Ki) = 15 microM and TFP Ki = 10 microM]. Similar results were obtained in Swiss 3T3 cells. In contrast, in transformed WI-38 or Swiss 3T3 cells neither TMB-8 nor TFP had any effect on serum-stimulated Na+ influx (TMB-8 Ki greater than 100 microM and TFP Ki greater than 100 microM). In addition, when 45Ca2+ efflux measurements were made on normal and transformed cells, serum stimulated significant 45Ca2+ efflux (P less than 0.05) from WI-38 and Swiss 3T3 cells, while having no effect on 45Ca2+ efflux from simian virus 40 (SV40)-WI-38 or SV40-Swiss 3T3 cells. However, an elevation of intracellular Ca2+ can stimulate Na+ influx, since it was found that A23187 mimicked the effects of serum in both normal and transformed cells. These results suggest that the Ca2+-calmodulin-mediated event, which is thought to be involved in serum-stimulated Na+ influx in normal cells, may be bypassed or overridden in transformed cells.

Topics: Amiloride; Animals; Blood; Calcimycin; Calcium; Calcium Channel Blockers; Calcium Radioisotopes; Calmodulin; Cell Line; Cell Transformation, Viral; Culture Media; Fibroblasts; Gallic Acid; Humans; Ion Channels; Lung; Sodium; Trifluoperazine

Binding of phorbol-12,13-dibutyrate (PDBu), a tumor promoter, to quiescent Swiss 3T3 cells increased the number of hexose carriers, resulting in stimulation of membrane transport of 3-O-methylglucose (3MeGlu) in a Ca2+-dependent fashion. Extracellular Ca2+ was necessary to initiate the binding of PDBu to its receptor, and intracellular Ca2+ was required to maintain it. The loss of PDBu-binding, caused by elimination of Ca2+, was accompanied by a loss of stimulation of hexose transport. These results indicated that Ca2+-dependent, continuous binding of PDBu to its receptor was essential to induce the stimulation of hexose transport.

Topics: 3-O-Methylglucose; Animals; Biological Transport, Active; Calcimycin; Calcium; Cell Transformation, Viral; Egtazic Acid; Fibroblasts; Methylglucosides; Methylglycosides; Mice; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Simian virus 40; Time Factors

Topics: Calcimycin; Cell Line; Cell Transformation, Viral; Cells, Cultured; Cytoplasm; Cytoskeleton; Intercellular Junctions; Microtubules; Mitochondria; Picolinic Acids