monensin and Cell-Transformation--Neoplastic

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

Fas/CD95 is a type-I membrane glycoprotein, which inducesapoptotic cell death when ligated by its physiological ligand. We generated previously hyperproliferative sublines derived from the human T-cell leukemia Jurkat, Jurkat-ws and Jurkat-hp, which lost Fas/CD95 surface expression. We have now observed that the total amount of Fas protein is similar in the sublines and in the parental cells, indicating that in the sublines Fas remains in an intracellular compartment. We have found that the protein is directed toward lysosomes in the sublines, where it is degraded. This defect in the secretory pathway correlates with loss of polyunsaturated fatty acids from cellular lipids, and with the lack of expression of endophilin-I and CtBP/BARS, enzymes that regulate vesicle fission by catalyzing the acylation of arachidonate into lysophosphatidic acid. In addition, great multillamer bodies, which contained acid phosphatase activity, absent in the parental Jurkat cells, were observed by transmission electron microscopy in the sublines.

Topics: Alcohol Oxidoreductases; Antigens, CD; Arachidonic Acid; Carrier Proteins; CD3 Complex; Cell Division; Cell Membrane; Cell Transformation, Neoplastic; Cytoplasm; DNA-Binding Proteins; fas Receptor; Gene Expression Regulation, Leukemic; Humans; Hydrolases; Jurkat Cells; Leukemia; Lysosomal Membrane Proteins; Lysosomes; Membrane Lipids; Microscopy, Electron; Monensin; Phosphoproteins; Serpins; Transcription Factors

Differences between transformed cells and their normal counterparts were defined by analyzing the cells' three-dimensional distribution of mass. Variables called factors, which explained the covariance of the real variables, were extracted from the data. We found that factors #4 (sharp, tapering projections) and #12 (rounding up), corresponded to G-protein functions. Then, the signature-type mass distribution of transformed cells was defined by factor values. Agents that caused signature-type mimicry could quantitatively shift factor values, for example, those affecting endocytic processing disproportionately reduced values of #4. Signature-type reversal was also observed and may be valuable in predicting the efficacy of chemotherapeutic agents.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Colchicine; Insulin; Microtubules; Monensin; Paclitaxel; Phenotype; Rats; Tetradecanoylphorbol Acetate; Valinomycin

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

Rodent fibroblasts transformed with the Kirsten and Moloney murine sarcoma viruses exhibit increased resistance to the growth inhibitory and cytotoxic action of the carboxylic Na+/H+ ionophore, monensin. The inhibitory effect of monensin on cell proliferation requires exposure for periods longer than 24 hours. The virus-transformed cells also exhibit increased resistance to the K+/H+ ionophore, nigericin. Since monensin is known to have significant effects upon the function and activity of the Golgi apparatus and the intracellular trafficking and processing of endocytosed as well as cell-derived materials, the results suggest that alterations in the activities of the organelles and pathways involved with intracellular protein trafficking and processing likely make an important contribution to the biological and cellular properties of transformed cells.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Drug Resistance; Kinetics; Kirsten murine sarcoma virus; Mice; Moloney murine sarcoma virus; Monensin; Nigericin; Rats

Binding and uptake of transcobalamin II-bound cobalamin by HL-60 promyelocytic leukemia cells proceed through receptor-mediated endocytosis. The affinity constant of the receptor for transcobalamin II-cobalamin was found to be 6.1 liter/nmol and the maximal rate of uptake 12 pmol/10(9) cells/h. This uptake is mediated by about 3000 receptor sites per cell. Evidence is presented that the receptor recirculates from the cell surface to the lysosomes and vice versa. Upon differentiation induction of the cells by either DMSO in granulocytic direction or by 1,25-dihydroxy-vitamin D3 in monocytic direction a rapid decline in cellular uptake and cell surface binding of the protein-bound vitamin ensues. In particular the internalization of the complex decreases faster than all other observed signs of the ongoing differentiation process, such as reduction in the OKT9-reactive transferrin receptor, increase in lineage-specific surface markers, and decrease in [3H]thymidine incorporation and actual cell proliferation. The transcobalamin II receptor on the cell surface appears to be a proliferation-associated membrane component in human leukemic cells.

Topics: Calcitriol; Cell Line; Cell Transformation, Neoplastic; Cycloheximide; Dimethyl Sulfoxide; Endocytosis; Humans; Leukemia, Promyelocytic, Acute; Monensin; Receptors, Cell Surface; Transcobalamins; Tumor Cells, Cultured; Vitamin B 12

A quantitative analysis based on centrifugal force requirements for enucleation was developed to examine the response of a number of untransformed and transformed cell lines to cytochalasin mediated enucleation. Examination of the extent of cell enucleation as a function of centrifugal force resulted in a series of response curves demonstrating that enucleation g force requirements varied between Balb/c 3T3, Swiss 3T3, and Kirsten sarcoma virus transformed Balb/c 3T3 (3T3-K). A four times greater centrifugal force was required to reach 50% enucleation for transformed Balb/c 3T3-K when compared to Swiss 3T3. A qualitative correlation could be observed between ease of enucleation and the existence of a well-formed stress fiber network. A comparison of cytochalasin B and D suggested that cytochalasin D was far more effective in the enucleation of transformed cells. Experiments with 2-deoxyglucose and monensin provided evidence that decreasing cellular ATP levels, either directly or potentially by uncoupling ion transport from ATP generation, can decrease the efficiency of enucleation. It is suggested that the organization of the cytoskeleton is affected by the altered cellular ATP levels which can affect the centrifugal requirements of enucleation.

Topics: Actins; Animals; Cell Line; Cell Nucleus; Cell Transformation, Neoplastic; Cells, Cultured; Centrifugation; Cytochalasin B; Cytochalasin D; Cytochalasins; Fibronectins; Fluorescent Antibody Technique; Mice; Monensin

Several properties of the lysosomal membrane glycoproteins LAMP-1 and LAMP-2 have been analysed. Each molecule was strongly associated with lysosome membranes and was extracted only in the presence of detergent. Studies of the biosynthesis and processing of the glycoproteins showed that each contained a polypeptide core of approx. 43,000 Da as identified by use of tunicamycin and endoglycosidase H. Nascent glycoproteins pulse-labelled for 5 min with [35S]methionine were approx. 92,000 Da. These precursor molecules were processed in 30 min to highly heterogeneous mature glycoproteins of approx. 110,000 Da(LAMP-1) and 105,000 Da(LAMP-2). Concomitant with the increase in apparent Mr the molecules became endoglycosidase H resistant and acquired sialic acid residues, indicating that they were converted to complex-type oligosaccharides. The final maturation of the glycoproteins was blocked by monensin. Immunohistochemical analysis of tissues from Balb/c and Beige/J mice showed that the molecules were present on many types of cells, consistent with their presence in lysosomes. The patterns of tissue expression of LAMP-1 and LAMP-2 in the two mouse strains were the same except that the intensity of staining of LAMP-2 was less than that of LAMP-1. LAMP-2, but not LAMP-1, gave a decreased immunofluorescent staining intensity in transformed HaNIH as compared with NIH/3T3 cells. The marked similarities between the LAMP proteins raise the consideration of common functions, possibly associated with the high oligosaccharide content of the molecules.

Topics: Acetylglucosaminidase; Animals; Antigens, CD; Cell Transformation, Neoplastic; Cells, Cultured; Chemical Phenomena; Chemistry; Glycoproteins; Lysosomal Membrane Proteins; Lysosomes; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Membrane Proteins; Mice; Mice, Inbred Strains; Monensin; Tunicamycin

Electrogenic ionophores have been found to induce membrane permeabilization in Swiss mouse 3T3 cells that had undergone spontaneous transformation (3T6 cells). Cells attached to plastic dishes were loaded with [3H] uridine, and then the medium was replaced by buffered salt solution at pH 7.8. The enhancement of membrane permeability was assayed by following the efflux of uridine nucleotides, normally impermeant substances. Titration with electrogenic ionophores, such as carbonylcyanide m-chlorophenylhydrazone (CCCP), SF-6847 and gramicidin D, markedly increased the membrane permeability within a very narrow range of ionophore concentration. Non-electrogenic ionophores, such as monensin and nigericin, did not affect membrane permeability. Measurements of the distribution of the lipophilic cation tetraphenylphosphonium (TPP+) between the cells and their environment implied that the remarkable increase in permeability took place within a narrow range of membrane potential (delta psi). The data could be explained by a delta psi threshold value, under which aqueous channels are opened in the plasma membrane. The effects exerted by electrogenic ionophores on the plasma membrane were found to be similar to those induced by exogenous ATP. In both cases rapid efflux of K+, influx of Na+ and reduction of delta psi preceded membrane permeabilization to low molecular weight, charged molecules, such as nucleotides. It is suggested that dissipation of delta psi induces conformational alterations in membranal components, and/or topological changes, such as aggregation of protein molecules, to form membranal aqueous channels. Electrogenic ionophores permeabilize both normal (3T3) and transformed (3T6) mouse fibroblasts, whereas ATP effects are specific for transformed cells. Thus, it is postulated that ATP acts via specific sites on the surface of transformed cells.

Topics: Adenosine Triphosphate; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane Permeability; Cell Transformation, Neoplastic; Cells, Cultured; Ionophores; Kinetics; Mice; Monensin; Nigericin; Onium Compounds; Organophosphorus Compounds; Potassium; Sodium; Uridine

Topics: Amphotericin B; Biological Transport, Active; Blood; Blood Platelets; Cell Transformation, Neoplastic; Cells, Cultured; DNA; Fibroblasts; Growth Substances; Melitten; Monensin; Peptides; Platelet-Derived Growth Factor; Potassium; Rubidium; Sodium; Vasopressins

Topics: 4-Chloromercuribenzenesulfonate; Amino Acids; Animals; Biological Transport, Active; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Fibroblasts; Kinetics; Membrane Potentials; Mice; Models, Biological; Monensin; Nigericin; Potassium; Simian virus 40; Sodium; Species Specificity; Valinomycin