monensin 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

During papillomavirus infection, the E5 protein localizes in the cell Golgi apparatus and other endomembrane compartments. Cells transformed by E5 do not express major histocompatibility class I complex (MHC I) on the cell surface, while cells transformed by the other transforming proteins E6 and E7 do. In addition, the total amount of both MHC I protein and mRNA is reduced in E5-transformed cells. Here we show that expression of bovine papillomavirus E5 causes the retention of MHC I in the Golgi apparatus, thus preventing its transport to the cell surface. We ascribe this effect to a failure of acidification of the Golgi apparatus, as similar effects are observed in control cells treated with the ionophore monensin. Treatment of E5-transformed cells with either beta- or gamma-interferon increases the synthesis of MHC I, showing that inhibition of MHC I expression by E5 is not irreversible. However, even after interferon treatment, MHC I, although increased in quantity, is not transported to the cell surface. E5 therefore affects MHC I at several levels, but prevention of MHC I transport to the cell surface appears to be the dominant effect. Lack of surface MHC I would have profound consequences for presentation of viral peptides to the immune system.

Topics: Animals; Antiviral Agents; Bovine papillomavirus 1; Cattle; Cell Transformation, Viral; Gene Expression Regulation; Genes, MHC Class I; Genes, Reporter; Golgi Apparatus; Green Fluorescent Proteins; Histocompatibility Antigens Class I; Hydrogen-Ion Concentration; Interferon-beta; Interferon-gamma; Ionophores; Luminescent Proteins; Monensin; Oncogene Proteins, Viral; Protein Transport; Recombinant Fusion Proteins; RNA, Messenger; Viral Proteins

Epidermal growth factor (EGF)-induced down-regulation of its receptor is an obligatory pathway for cellular regulation of EGF-specific receptor (EGF-R) in normal and malignant cells. BNER4 cells are mouse Balb/3T3 cells transfected with the human EGF-R complementary DNA (cDNA). Polyoma middle T antigen-transfectants of BNER4, B4/MT-2, B4/MT-13, B4/MT-23, and B4/MT-24, showed diminished down-regulation of cell surface human EGF-R in response to EGF relative to the parental BNER4 cells. Also, the v-src-transfectants B4/SRC-13 and B4/SRC-24 showed much less down-regulation than BNER4 cells, whereas H-ras-transfectants of BNER4, B4/RAS-24 and B4/RAS-25, showed EGF-induced down-regulation of the cell surface EGF-R similar to that of BNER4. EGF induced DNA synthesis more than 20-fold in BNER4, but induced only about a 1.5- to 6-fold increase in the middle T antigen- and v-src-transfectants. EGF-Rs of the middle T antigen-transfectants were metabolically stable in the presence of EGF in comparison with their parental BNER4 cells. EGF-Rs of BNER4 cells degraded with half-lives of about 2 h in the presence of EGF, but those of the middle T antigen transformants were found to be highly stabilized in the presence of EGF. On the other hand, transfection with polyoma middle T antigen (MTAg) cDNA causes malignant transformation of Balb/3T3 cells, but not its monensin (an ionophoric antibiotic)-resistant mutant MO-5 cells, which have no significant EGF binding activity. Transfection of human EGF-R cDNA into MO-5 leads to the expression of high levels of human EGF-R in MNER31. Unlike the polyoma MTAg transfectants of BNER4, EGF-R in polyoma MTAg cDNA-transfectants into MNER31, M31/MT-13 and M31/MT-14, were down-regulated to levels similar to those of their parental MNER31. Exposure to EGF induced a more than 10-fold increase in DNA synthesis of quiescent BNER4, MNER31, M31/MT-13, and M31/MT-14 cells. Polyoma middle T antigen or v-src appears to modulate EGF-induced down-regulation of EGF-R, possibly through interaction of the receptor with the viral oncogenes, and this interaction may be altered in the mutant.

Topics: 3T3 Cells; Animals; Antigens, Polyomavirus Transforming; Base Sequence; Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA; Down-Regulation; Drug Resistance; Epidermal Growth Factor; ErbB Receptors; Genes, src; Mice; Molecular Sequence Data; Monensin; Mutation; Phosphorylation; Transfection

Both intercellular adhesion and spreading on fibronectin of BHK21 hamster cells are inhibited by vanadate at concentrations that cause specific regulatory effects rather than general metabolic inhibition. Inhibition of aggregation of these cells in suspension (half-maximal in 10(-5) M vanadate) is rapid and reversible. The extent of inhibition, and its decline with culture age parallel inhibition by agents that depolymerize microtubules. Vanadate also reversibly inhibits spreading of both BHK cells and transformed derivatives on fibronectin. If 10(-4) M vanadate is added to BHK cells that have spread in its absence, they remain spread, but transformed derivatives are sensitive to rounding by vanadate at 10(-6) M. The mechanisms by which vanadate inhibits both intercellular adhesion and spreading are unknown, and may be different for the two phenomena. Possible sensitive targets include cytoplasmic dynein for the former, and protein tyrosyl phosphatase for the latter.

Topics: Adenine; Animals; Cell Aggregation; Cell Communication; Cell Line, Transformed; Cell Movement; Cell Transformation, Viral; Clone Cells; Cricetinae; Dyneins; Fibronectins; Microtubules; Monensin; Ouabain; Phosphoprotein Phosphatases; Protein Tyrosine Phosphatases; Vanadates

The BM1A EB-virus transformed human lymphocyte cell line contains approximately 950,000 Na+/K(+)-ATPase sites per cell. The turnover number of each site is approx. 2240 molecules of rubidium per min. When cells are exposed to a low extracellular concentration of potassium the intracellular concentration of sodium rises, and the cells respond in the short term by increasing the Vmax of 86Rb+ uptake. In the longer term the cells respond by increasing both the Vmax of 86Rb+ uptake and the Bmax of [3H]ouabain binding. The suggestion that increases in the intracellular concentration of sodium is responsible for these changes is supported by the finding that monensin, which increases intracellular sodium without affecting intracellular potassium, is capable of inducing both the short- and long-term changes associated with a low external concentration of potassium.

Topics: 5'-Nucleotidase; Biological Transport, Active; Cell Transformation, Viral; gamma-Glutamyltransferase; Herpesvirus 4, Human; Humans; Lymphocytes; Monensin; Ouabain; Potassium; Potassium Channels; Rubidium; Sodium Channels; Sodium-Potassium-Exchanging ATPase; Thymidine

In simian sarcoma virus (SSV)-transformed cells (SSV-NRK, SSV-NIH 3T3, and SSV-NP1 cells), the v-sis gene product was synthesized as a 36-kDa glycopolypeptide with one endoglycosidase (Endo) H-sensitive oligosaccharide chain and formed a dimer (p72) with a half-time of less than 5 min. p72 was proteolytically processed to generate sequentially p68 and p58 in the endoplasmic reticulum/Golgi complex, p44 in the post-Golgi complex compartments, and p27 in an endosomal/lysosomal compartment. A portion (20-30%) of p72 and p68 later became Endo H-resistant but Endo F-sensitive. During processing, the v-sis gene products exhibited rapid turnover, possibly in the endoplasmic reticulum and/or Golgi complex. The rate of turnover correlated with the tumorigenicity previously reported in these SSV-transformed cells. All three SSV-transformed cells secreted v-sis gene product (p44). p44 was secreted but remained tightly associated with the cell surface. This novel secretion provided an efficient system for the interaction of p44 with the cell surface platelet-derived growth factor receptor which resulted in the intracellular formation of p27. A fraction of secreted p44 was converted extracellularly to a 27-kDa product (extracellular p27) after a longer time in culture. The identical N-terminal amino acid sequence of p44 and extracellular p27 (H2N-SLGSLSVAEPAMIA) indicated a preferential site (Lys110-Arg111) for the proteolytic processing. The intracellular turnover of the v-sis gene product and its correlation with tumorigenicity as well as the demonstration of mitogenically active intracellular forms of v-sis gene product support the hypothesis of intracellular loop autocrine transformation.

Topics: Ammonium Chloride; Animals; Blotting, Western; Cell Line; Cell Transformation, Viral; Cytoplasm; Endocytosis; Glycoproteins; Glycoside Hydrolases; Hexosaminidases; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Mice; Molecular Weight; Monensin; Oncogene Proteins v-sis; Protein Processing, Post-Translational; Rats; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; Retroviridae Proteins, Oncogenic; Retroviruses, Simian; Sarcoma Virus, Woolly Monkey; Suramin; Tunicamycin

Lysis of HeLa cells infected with poliovirus revealed intact virus; 135S particles, devoid of VP4 but containing the viral RNA; and 80S empty capsids. During infection the kinetics of poliovirus uncoating showed a continuous decrease of intact virus, while the number of 135S particles and empty shells increased. After 1.5 h of infection conformational transition to altered particles resulted in complete disappearance of intact virions. To investigate the mechanism of poliovirus uncoating, which has been suggested to depend on low pH in endosomal compartments of cells, we used lysosomotropic amines to raise the pH in these vesicles. In the presence of ammonium chloride, however, the kinetics of uncoating were similar to those for untreated cells, whereas in cells treated with methylamine, monensin, or chloroquine, uncoating was merely delayed by about 30 min. This effect could be attributed to a delay of virus entry into cells after treatment with methylamine and monensin, whereas chloroquine stabilized the viral capsid itself. Thus, elevation of endosomal pH did not affect virus uncoating. We therefore propose a mechanism of poliovirus uncoating which is independent of low pH.

Topics: Ammonium Chloride; Cell Transformation, Viral; Chloroquine; Endocytosis; Glutathione; HeLa Cells; Hot Temperature; Humans; Hydrogen-Ion Concentration; Kinetics; Methylamines; Monensin; Poliovirus; Tritium; Virus Replication

The mouse L-cell mutant gro29 was selected for its ability to survive infection by herpes simplex virus type 1 (HSV-1) and is defective in the propagation of HSV-1 and vesicular stomatitis virus (F. Tufaro, M. D. Snider, and S. L. McKnight, J. Cell Biol. 105:647-657, 1987). In this report, we show that gro29 cells harbor a lesion that inhibits the egress of HSV-1 virions during infection. We also found that HSV-1 glycoprotein D was slow to traverse the secretory pathway en route to the plasma membrane of infected gro29 cells. The movement of glycoproteins was not blocked entirely, however, and immunofluorescence experiments revealed that infected gro29 cells contained roughly 10% of the expected amount of glycoprotein D on their cell surface at 12 h postinfection. Furthermore, nucleocapsids and virions assembled inside the cells during infection, suggesting that the lesion in gro29 cells impinged on a late step in virion maturation. Electron micrographs of infected cells revealed that many of the intracellular virions were contained in irregular cytoplasmic vacuoles, similar to those that accumulate in HSV-1-infected cells treated with the ionophore monensin. We conclude from these results that gro29 harbors a defect that blocks the egress of HSV-1 virions from the infected cell without seriously impeding the flux of individual glycoproteins to the cell surface. We infer that HSV-1 maturation and egress require a host cell component that is either reduced or absent in gro29 cells and that this lesion, although not lethal to the host cell, cannot be tolerated by HSV-1 during its life cycle.

Topics: Animals; Blotting, Western; Cell Transformation, Viral; Fluorescent Antibody Technique; Kinetics; L Cells; Mice; Microsomes; Monensin; Mutation; Simplexvirus; Viral Envelope Proteins; Virion; Virus Replication

The Hodgkin-associated Ki-1 antigen was analyzed in different cell lines. In Hodgkin analogous L428 cells, biosynthetically labeled with radioactive amino acids, the Ki-1 antibody precipitated three glycoproteins with 90, 105, and 120 kDa, respectively. Surface-labeling revealed that the two larger components were membrane-associated forms of the Ki-1 antigen, although the 90-kDa molecule was shown in pulse-chase experiments to be the precursor of the 105- and 120-kDa forms. All three forms of the Ki-1 antigen possess a tunicamycin-sensitive 6-kDa N-linked carbohydrate moiety. O-Linked oligosaccharides could not be detected. Thus, the differences in m.w. are probably not due to glycosylation. The ionophore monensin prevented the appearance of the membrane-associated molecules, which demonstrated that they are assembled between the transcompartment of the Golgi complex and their insertion into the cell membrane. The 90-kDa precursor molecule cannot be generated by disulfide reduction from the two larger forms. After internal labeling with P-32, only the 105- and 120-kDa bands became visible, indicating that the Ki-1 molecule is phosphorylated after its processing into the two larger membrane-associated forms. Analysis of the Ki-1 antigens from other cell lines demonstrated that after external labeling of two other Hodgkin-derived cell lines, six Epstein-Barr virus lymphoblastoid cell lines and one human T leukemia virus I-positive T cell line, both the 105- and the 120-kDa membrane molecules could be detected, regardless of the presence or type of virus integrated.

Topics: Antigens, Neoplasm; Antigens, Surface; B-Lymphocytes; Cell Line; Cell Transformation, Viral; Deltaretrovirus; Glycoproteins; Herpesvirus 4, Human; Hodgkin Disease; Humans; Molecular Weight; Monensin; Peptide Hydrolases; Phosphoproteins; T-Lymphocytes; Tunicamycin

Cultured rat Schwann cells transformed by Simian Virus 40 (SV40) have previously been shown to retain their ability to synthesize myelin-associated galactosylceramide and sulfatide. Little is known about the mechanism regulating galactosphingolipid synthesis in Schwann cells. We have found that growing the transformed Schwann cells in the presence of dimethyl sulfoxide (DMSO) markedly inhibits the incorporation of [35S]sulfate into sulfatide, in a time- and dose-dependent manner. The concentration of DMSO which resulted in a half-maximal inhibition after 6 days of incubation was 0.5%, and the incubation time required for a half-maximal effect at 1.0% DMSO was approximately 4 days. In contrast, DMSC did not affect the incorporation of [35S]sulfate into glycosaminoglycans. In addition, DMSO treatment has little effect on the synthesis of cellular DNA, proteins and lipids. When transformed Schwann cells were treated with DMSO, a substantial decrease in the incorporation of [3H]galactose into galactosylceramide was observed. The concentration of DMSO which resulted in a half-maximal inhibition of galactosylceramide synthesis was approximately 0.5%, similar to the concentration required for a similar effect on sulfatide synthesis. However, the incubation time required for a half-maximal inhibitory effect on galactosylceramide synthesis at 1.0% DMSO was less than 1 day, which was substantially shorter than the time required for the inhibition of sulfatide synthesis at this concentration. This finding is consistent with the interpretation that treatment with DMSO inhibits the synthesis of galactosylceramide, a precursor of sulfatide, which results in a decrease in the synthesis of sulfatide during a prolonged incubation of DMSO.

Topics: Animals; Cell Division; Cell Survival; Cell Transformation, Viral; Dimethyl Sulfoxide; Galactose; Galactosylceramides; Glucosamine; Glycoproteins; Glycosaminoglycans; Lipids; Mannose; Monensin; Proteins; Rats; Schwann Cells; Sulfates; Sulfoglycosphingolipids

We have studied the effect of two carboxylic ionophores, monensin and laidlomycin, on the replication of measles virus in KB cells. The yield of infectious virus was markedly depressed at the concentrations of the ionophores which had no effect on overall viral protein synthesis. The ionophores selectively blocked the migration of hemagglutinin (H) glycoprotein from Golgi apparatus to the cell surface. As a result, H glycoprotein is prevented from being converted from incompletely glycosylated form to the mature form. The inhibitory effect on the transport and glycosylation of H was reversed, although gradually, upon the removal of the ionophores.

Topics: Anti-Bacterial Agents; Cell Transformation, Viral; DNA Replication; Fluorescent Antibody Technique; Furans; Hemagglutinins, Viral; Humans; KB Cells; Kinetics; Measles virus; Monensin; Virus Replication

A myoepithelial-like cell line (Rama 401), isolated from rat mammary gland, has been transformed with a temperature-sensitive mutant of Rous sarcoma virus (tsRSV). Rama 401-tsRSV cells adopt a spindle morphology and fail to deposit basement membrane proteins when grown at the permissive temperature (35 degrees C). When switched to the non-permissive temperature (41 degrees C), the cells flatten (with a 5-fold increase in area), and deposit an extracellular matrix containing basement membrane proteins. When the cells are switched from 35 degrees C to 41 degrees C in the presence of monensin (an ionophore that inhibits protein secretion), basement membrane proteins are no longer deposited extracellularly although the cells flatten, their area increasing by ninefold. Cells switched from 35 degrees C to 41 degrees C in the presence of cycloheximide still flatten and deposit basement membrane proteins, whereas the morphological change on switching from 41 degrees C to 35 degrees C is inhibited by cycloheximide. These experiments indicate that the ability of Rama 401-tsRSV cells to spread on a plastic substratum is not dependent on the de novo synthesis and deposition of basement membrane proteins.

Topics: Animals; Basement Membrane; Cell Adhesion; Cell Line, Transformed; Cell Transformation, Viral; Cycloheximide; Epithelium; Extracellular Matrix; Hot Temperature; Mammary Glands, Animal; Membrane Proteins; Microscopy, Electron; Monensin; Rats

Somatostatin is a14-amino acid peptide hormone that inhibits the secretion of a variety of other polypeptide hormones, including growth hormone. Here we describe an experimental system used to determine whether somatostatin can discriminate in its inhibition between secretory and plasma membrane proteins. Growth hormone-secreting cells (GH3) were infected with vesicular stomatitis virus and pulse-chased with [35S]methionine to follow the simultaneous intracellular transit of growth hormone and the viral membrane glycoprotein, G protein. Secretion of growth hormone was monitored by immunoprecipitation of chase media, while appearance of G protein on the plasma membrane was detected by cell surface labeling and virus purification. In the presence of somatostatin (10 micrograms/ml), the secretion of growth hormone was inhibited by 80%. In contrast, G protein appeared on the plasma membrane with slightly enhanced kinetics. When cells were treated with the ionophore monensin (0.2 microM), there was a dramatic inhibition of both the secretion of growth hormone and the incorporation of G protein into plasma membranes. Our results on the differential effect of somatostatin provide evidence for sorting of secretory and membrane proteins into distinct compartments in the secretory pathway. The data further suggest that this sorting event occurs late in the Golgi complex or after proteins exit from that organelle.

Topics: Animals; Biological Transport; Cell Line; Cell Transformation, Viral; Fluorescent Antibody Technique; Growth Hormone; Kinetics; Membrane Glycoproteins; Monensin; Pituitary Gland; Rats; Somatostatin; Viral Envelope Proteins; Viral Proteins

Topics: Cell Transformation, Viral; Coronaviridae; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Humans; Kinetics; Molecular Weight; Monensin; Protein Processing, Post-Translational; Viral Proteins

The carboxylic ionophores monensin and nigericin, at concentrations higher than 10 and 6 muM, respectively, prevent the penetration of the Semliki Forest virus (SFV) genome into the cytosol of baby hamster kidney (BHK-21) cells and thereby inhibit viral replication. In the absence of inhibitors, the entry of SFV is known to proceed by adsorptive endocytosis in coated vesicles, followed by acid-triggered membrane fusion in intracellular vacuoles or lysosomes. The results show that binding of the virus to the cell surface, adsorptive endocytosis, and intracellular transport of viruses to the lysosomes are only marginally affected by the ionophores. No direct virucidal effect is observed, nor is the membrane fusion activity of the virus at low pH directly affected. Sequential addition of monensin and ammonium chloride (a non-related lysosomotropic inhibitor of SFV entry) indicates that both inhibitors affect the same step in the entry pathway. On the basis of these data and the known effects of carboxylic ionophores and lysosomotropic weak bases on cellular pH gradients, we conclude that monensin inhibits penetration by increasing the pH in endocytic vacuoles and lysosomes above pH 6, which is the pH threshold for the viral membrane fusion activity.

Topics: Animals; Cell Line; Cell Transformation, Viral; Cricetinae; Cytosol; Electron Transport; Furans; Kidney; Kinetics; Monensin; Nigericin; Semliki forest virus

Topics: Animals; Cell Transformation, Viral; Cells, Cultured; Endocytosis; Furans; Kinetics; Mice; Monensin; RNA, Viral; Transcription, Genetic; Vesicular stomatitis Indiana virus; Virus Replication

Topics: Animals; Cell Line; Cell Membrane; Cell Transformation, Viral; Cell-Free System; Furans; Hydrogen-Ion Concentration; Ionophores; Kinetics; Mice; Monensin; Simian virus 40; Sodium; Structure-Activity Relationship

The relationship between Na entry and the activity of the Na-K pump has been investigated in a variety of cell types by testing the effect of the Na ionophore monensin, mitogenic stimulation with serum and oncogenic transformation by SV40 and polyoma virus. We found that addition of monensin increases intracellular Na in quiescent cultures of murine, hamster, and human cells. In each case, the rise in intracellular Na by monensin is associated with an increase in the activity of the Na-K pump, which was measured as ouabain-inhibitable 86Rb uptake. The addition of serum to quiescent cultures stimulates 86Rb uptake in all cell types studied. Serum alone causes an increase in intracellular potassium with no consistent change in intracellular Na. In the presence of the Na-K pump inhibitor ouabain, serum causes a marked increase in intracellular Na, with little change in intracellular K. This pattern is interpreted as indicating that the primary effect of serum is to increase Na entry into the cells. A low concentration of monensin (0.2 micrograms/ml) mimics the effect of serum on ion fluxes and content, which supports the conclusion that serum and monensin stimulate 86Rb uptake in the same manner, namely by increasing Na entry into the cells. In addition, a partially purified platelet extract stimulates Na entry and 86Rb uptake in quiescent 3T3 cells. Finally 3T3 cells transformed by SV40 or polyoma virus exhibit a higher rate of Na entry and of Na-K pump activity than their untransformed 3T3 counterparts. All these results indicate that the rate of Na entry plays an important role in the regulation of the activity of the Na-K pump and that an increase in Na and K movements is a rapid response elicited by serum in a variety of cell types.

Topics: Animals; Blood Platelets; Cell Membrane Permeability; Cell Transformation, Viral; Cells, Cultured; Cricetinae; Fibroblasts; Humans; Immune Sera; Mice; Monensin; Polyomavirus; Potassium; Rubidium; Simian virus 40; Skin; Sodium; Tissue Extracts

Topics: Amino Acids; Biological Transport, Active; Cell Line; Cell Membrane; Cell Transformation, Viral; Fibroblasts; Kinetics; Membrane Potentials; Methylglucosides; Monensin; Nigericin; Onium Compounds; Potassium; Simian virus 40; Sodium; Trityl Compounds; Valinomycin