Mitochondrial function in oncogene-transfected rat fibroblasts isolated from multicellular spheroids

Twomitochondrion-specific fluorochromes,10-N-nonyl acridineorange (NAO) and rhodamine 123 (Rh123), were used todetermine the mechanism responsible for alterations in energymetabolism of transformed rat embryo fibroblast cells isolated fromdifferent locations within multicellular spheroids. Accumulation ofRh123 depends on intact mitochondrial membrane potential, whereas NAO is taken up by mitochondria independently of their function and thusrepresents mitochondrial distribution only. A reproducible selectivedissociation procedure was used to isolate cells from differentlocations within the spheroids. After isolation, cells weresimultaneously stained with one mitochondrial stain and the DNA dyeHoechst 33342, and several parameters, including cell volume, weremonitored via multilaser-multiparameter flow cytometry. Our dataclearly show a decrease in the uptake of Rh123 in cells from theperiphery to the inner regions of the tumor spheroids, reflecting apersistent alteration in mitochondrial function. However, NAO stainingexperiments showed no reduction in the total mitochondrial mass perunit cell volume. Because cells were exposed to stain under uniformconditions after isolation from the spheroid, these data indicate thatdownregulation of mitochondrial function is associated with cellquiescence rather than a transient effect of reduced nutrientavailability. This result, which is in accordance with data from twoother cell lines (EMT6 and 9L), might reflect a general phenomenon inmulticellular spheroids, supporting the hypothesis that quiescent cellsin the innermost viable spheroid layer stably reduce theirmitochondrial function, presumably to compensate for lower nutrientsupply and/or decreased energy demand.

     

Electrotransformation of Lactobacillus plantarum using linearized plasmid DNA

J.K. THOMPSON, K.J. MCCONVILLE, C. MCREYNOLDS AND M.A. COLLINS. 1997. Evidence is presented that linearized plasmid DNA is capable of electrotransforming Lactobacillus plantarum at a frequency 500-fold lower than with the covalently closed circular molecule. When the linearized plasmid was religated prior to transformation the transformation efficiency was < 10-fold higher, suggesting that open circular plasmid was only slightly more efficient in the transformation of Lact. plantarum than linear DNA. This observation has implications for direct cloning into this species since the high background transformation frequency produced by the linear DNA could potentially obscure the recovery of clones. Nevertheless, using positive selection for enhanced chloramphenicol resistance, cloned fragments of Lact. helveticus DNA were obtained using the shuttle vector pGKV110.     

Direct evidence for intra- and intermolecular disulfide bond formation in the human glucocorticoid receptor. Inhibition of DNA binding and identification of a new receptor-associated protein

We have investigated the potential for the steroid affinity-labeled human glucocorticoid receptor to form both intramolecular and intermolecular disulfide bonds. Glucocorticoid receptors labeled in intact HeLa S3 cells with the covalent affinity label [3H]dexamethasone mesylate ([3H]DM) were analyzed on denaturing 5-12% polyacrylamide gels under both nonreducing and reducing conditions. Under nonreducing conditions the affinity-labeled receptor migrated as a heterogeneous species having an average molecular mass of approximately 96 kDa whereas, under reducing conditions, the receptor migrated as a more discrete form. These data suggest that a reducing environment can influence the structure of the glucocorticoid receptor monomer and further imply that sulfhydryl groups within the affinity-labeled receptor are available for modification. To pursue this observation in greater detail, we tested the effect of oxidizing conditions on the structure of the glucocorticoid receptor. The presence of low concentrations (0.125-0.5 mM) of three oxidizing reagents (sodium tetrathionate, disulfiram, and iodosobenzoate) altered the migration of the affinity-labeled receptor resulting in forms of apparent lower molecular mass (as low as 78 kDa). This altered migration, not seen with most other cytosolic proteins, is consistent with the formation of intramolecular disulfide bonds within the receptor which presumably cause it to assume a folded conformation and migrate faster through the gel. At higher concentrations of these reagents (up to 5.0 mM), we also detect a saturably labeled [3H]DM band which has a higher molecular mass (approximately 140 kDa), indicating the formation of intermolecular disulfide bonds between the [3H]DM-labeled receptor and another closely associated protein(s) having a molecular mass of approximately 40 kDa. The effects which these oxidizing reagents have on glucocorticoid receptor structure are completely reversed upon the addition of dithiothreitol, indicating that the observed changes in migration do not reflect receptor proteolysis but rather a folding and unfolding within the receptor monomeric protein. We have also analyzed the effect of this oxidation/reduction on the function of the glucocorticoid receptor. Oxidation of the [3H]DM-labeled receptor complex with 0.5 mM sodium tetrathionate inhibited activation of receptor to a form capable of binding to DNA-cellulose. This inhibition can be reversed with dithiothreitol at 25 degrees C but not at 0 degrees C, suggesting that these oxidizing reagents are inhibitory at the transformation and/or activation steps.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of membrane potential on divalent cation transport catalyzed by the "electroneutral" ionophores A23187 and ionomycin

Depolarization of plasma membrane potential has a potent inhibitory effect on divalent cation influx catalyzed by the carboxylic ionophores ionomycin and A23187. This effect is observed in different cell models and does not depend on either inhibition of Ca2+-activated cation channels or activation of Ca2+ extrusion mechanisms as suggested previously. A dependence of divalent cation influx on the magnitude of membrane potential is observed also in artificial liposomes. The inhibition of ionophore-dependent divalent cation transport by membrane potential depolarization can be modified varying the ionophore concentration and the external pH. These findings suggest that both neutral and positively charged ionophore-cation complexes can cross the plasma membrane and that their contribution to the overall transport process can be varied according to the experimental conditions.     

Metalloendoprotease inhibitors which block the differentiation of L6 myoblasts inhibit insulin degradation by the endogenous insulin-degrading enzyme

The cultured myoblasts of the rat skeletal muscle cell line L6 proliferate till confluency and then fuse to form myotubes and express a number of muscle-specific proteins. We had shown that this differentiation process is blocked by specific metalloendoprotease inhibitors. We now demonstrate that metabolizing L6 myoblasts and their cell extracts degrade insulin to acid-soluble fragments by a non-lysosomal pathway. About 90% of the insulin-degrading activity residues in the cytoplasm and is due to a 110-kDa enzyme known as the insulin-degrading enzyme. The same metalloendoprotease inhibitors that block the differentiation of L6 myoblasts also inhibit insulin degradation by the metabolizing L6 cells, their cell extracts, and the insulin-degrading enzyme immunoprecipitated from the cytosolic extracts by a monoclonal antibody. These results suggest that the insulin-degrading enzyme is the metalloendoprotease whose activity is required for the initiation of the morphological and biochemical differentiation of L6 myoblasts.     

Glutamate overcomes the salt inhibition of DNA polymerase III holoenzyme

Even though Escherichia coli can grow in media containing up to 1 M NaCl, one-fifth that amount of NaCl will completely inhibit the in vitro activity of DNA polymerase III holoenzyme. It has been established that the major intracellular ionic osmolytes are potassium and glutamate (Richey, B., Cayley, D. S., Mossing, M. C., Kolka, C., Anderson, C. F., Farrar, T. C., and Record, M. T., Jr. (1987) J. Biol. Chem. 262, 7157-7164). We have found that holoenzyme catalyzes replication efficiently in vitro in up to 1 M potassium glutamate. Two salt effects on the replication of single-stranded DNA were observed. At low salt replicative activity was enhanced and at high salt there was anion-specific inhibition. We have found that DNA polymerase III holoenzyme tolerated 10-fold higher concentrations of glutamate than chloride. The ability of various anions to extend the useful range of salt concentrations followed the order: phosphate less than chloride less than N-Ac-glutamate less than acetate less than glycine less than aspartate less than glutamate. With the exception of phosphate, this order followed the Hofmeister series indicating that the anion-specific effects were due to anions interacting at the protein-water interface at weak anion binding sites. Glutamate did not reverse the inhibition by chloride. The low salt enhancement and high salt inhibition effects were additive for the two anions indicating that they competed for common anion binding sites. The major salt-sensitive step was holoenzyme binding to template rather than the subsequent elongation reaction.     

Specific overproduction of very late antigen 1 integrin in two human neuroblastoma cell lines selected for resistance to detachment by an Arg-Gly-Asp-containing synthetic peptide

Very late antigen (VLA) 1 is a member of the family of integral plasma-membrane glycoproteins known as integrins. It is a heterodimer composed of an alpha subunit of Mr 200,000, noncovalently associated with a beta subunit of Mr 110,000 which is shared by other VLA molecules (VLA-2-5). Unlike most of the other VLA proteins which have been shown to be receptors for various extracellular matrix proteins, the ligand for VLA-1 is unknown. Utilizing polyclonal antisera against the human fibronectin receptor as well as alpha subunit-specific monoclonal antibodies and cDNA probes, we have been able to demonstrate that in two human neuroblastoma cell lines, IMR-32 and SK-N-SH, the common beta subunit is associated with alpha 1, alpha 2, alpha 3, and alpha 5 subunits. By culturing these two cell lines in the presence of a synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, which contains the Arg-Gly-Asp cell attachment promotion tripeptide, we have isolated variant cell lines resistant to the detachment effects of this peptide. Peptide-resistant SK-N-SH and IMR-32 neuroblastoma cells exhibit weaker attachment to type I collagen and laminin, but a similar level of attachment to fibronectin as compared to the parental cells. Although the peptide-resistant variant cell lines proliferate at a rate similar to that of the parental cell lines, they stably overproduce (up to 20-fold) the alpha 1 subunit (VLA-1) specifically; and in the IMR-32 variant cells, the common beta 1 subunit is also overproduced. The level of expression of alpha 2 and alpha 3 subunits, however, is considerably reduced and that of the alpha 5 subunit is unchanged relative to the parental cells. These data suggest that the expression of integrin alpha subunits can be regulated differentially and independently of the beta subunit and that the VLA-1 heterodimer has an important function in mediating Arg-Gly-Asp-dependent cell adhesion or other phenotypic properties in human neuroblastoma cells.     

Proteoglycan biosynthesis in murine monocytic leukemic (M1) cells before and after differentiation

Murine monocytic leukemic (M1) cells were cultured in the presence of [3H]glucosamine and [35S]sulfate. Labeled proteoglycans were purified by anion exchange chromatography and characterized by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with chemical and enzymatic degradation. M1 cells synthesize a single predominant species of proteoglycan which distributes almost equally between the cell and medium after 17 h labeling. The cell-associated proteoglycan has an overall size of about 135 kDa and contains three to five chondroitin sulfate chains (28-31 kDa each) attached to a chondroitinase-generated core protein of 28 kDa. The synthesis and subsequent secretion of this proteoglycan was enhanced 4-5-fold in cells induced to differentiate into macrophages. This was not a phenomenon of arrest in the G0/G1 stage of the cell cycle, since density inhibited undifferentiated cells arrested at this stage did not increase proteoglycan synthesis. The chondroitin sulfate chains contained exclusively chondroitin 4- and 6-sulfate; however, the ratio of these two disaccharides differed between the medium- and cell-associated proteoglycans, and changed during progression of the cells into a fully differentiated phenotype. Pulse-chase kinetics indicate the presence of two distinct pools of proteoglycan; one that is secreted very rapidly from the cell after a approximately 1-h lag, and a second pool that is turned over in the cell with a half-time of approximately 3.5 h. Subtle differences in the glycosylation patterns of the medium- and cell-associated species are consistent with synthesis of two pools. Papain digestion suggests that the chondroitin sulfate chains are clustered on a small protease resistant peptide. The data suggest that this proteoglycan is similar to the serglycin proteoglycan family.