Antisense inhibition of glial S100 beta production results in alterations in cell morphology, cytoskeletal organization, and cell proliferation.

The phenotypic effects of selectively decreasing the levels of S100 beta in cultured glial cells were analyzed. Two separate antisense approaches were utilized for inhibition of S100 beta production: analysis of clonal isolates of rat C6 glioma cells containing an S100 beta antisense gene under the control of a dexamethasone-inducible promoter, and analysis of C6 cells treated with S100 beta antisense oligodeoxynucleotides. Both antisense methods resulted in a decrease in S100 beta levels in the cell, as measured by RIA. The inhibition of S100 beta production correlated with three alterations in cellular phenotype: (a) a flattened cell morphology; (b) a more organized microfilament network; and (c) a decrease in cell growth rate. The studies describe here provide direct evidence for an involvement of S100 beta in glial cell structure and function, and suggest potential in vivo roles for S100 beta in regulation of glial cell morphology, cytoskeletal organization, and cell proliferation.

Effects of 12-O-tetradecanoylphorbol-13-acetate on epidermal growth factor receptors in BALB/c-3T3 cells: relationship between receptor modulation and mitogenesis.

The addition of medium containing 0.1% serum and 12-O-tetradecanoylphorbol-13-acetate (TPA) to quiescent cultures of BALB/c-3T3 cells caused a rapid decrease in the subsequent binding and accumulation of epidermal growth factor (EGF) during a 30-min exposure at 37 degrees C. With further incubation in medium containing TPA, the rate of EGF accumulation steadily increased over time, until by 24 h it had recovered to control values. The addition of TPA with low serum did not stimulate cell cycle traverse. In contrast, when TPA was added directly to spent medium or with fresh medium containing insulin, there was a marked increase in DNA synthesis. Under these conditions the initial TPA-induced decrease in EGF accumulation persisted over a 24-h period. A Scatchard analysis indicated that TPA caused an initial decrease in receptor affinity that persisted if the cells were also stimulated to enter the cell cycle. Thus TPA appeared to have multiple effects on the modulation of the EGF receptor. The pattern of binding following addition of TPA depended on both direct actions of the promoter and an indirect action which depended on whether the promoter was added under conditions in which cell cycle traverse was stimulated.

Isolation of cell membrane for epidermal growth factor receptor studies.

The cell membrane isolation procedure we developed here can be scaled up from four to several hundred plates of cultured cells. Transmission electron microscopy, membrane marker enzyme analysis, binding study, EGF-dependent receptor autophosphorylation, and Western blots all demonstrate the biological activity of the purified cell membranes. The membrane purification procedure has been adapted by others in assessing EGF kinase activity and has been used for the purification of cell membranes from other types of cultured cells.

Interaction of beta-endorphin, naloxone and dopamine: effects on melanocyte-stimulating hormone secretion of amphibian pituitaries in vitro.

Neurointermediate lobes from amphibians (Rana pipiens) were incubated in Medium 199 containing dopamine, beta-endorphin or dopamine plus beta-endorphin. Dopamine inhibited melanocyte-stimulating hormone (MSH) secretion as measured by bioassay in hypophysectomized frogs, an effect which was transiently reversed by beta-endorphin. The effects of endorphin were in turn partially suppressed by the opiate antagonist, naloxone hydrochloride. Cells treated with all three agents exhibited expanded rough endoplasmic reticulum and decreased secretory granule content, indicative of peptide release and new synthesis. Beta-Endorphin alone did not stimulate MSH secretion above control levels, and at one time period was seen to reduce MSH secretion. The findings indicate a complex interaction between beta-endorphin and dopamine directly upon MSH secretion at the level of the neurointermediate lobe.

Insulin-like growth factor-I induces a rapid increase in calcium currents and spontaneous membrane activity in clonal pituitary cells.

The role of growth factors in the adult brain is largely unknown, although receptors for factors such as insulin-like growth factor-I (IGF-I) have been localized on nondividing mature neurons. Because neurons use the frequency and pattern of action potentials to encode information, we assessed the ability of IGF-I to modulate rapidly the electrical properties of GH4C1 cells, a spontaneously active pituitary line with neuronal L- and T-type calcium currents. Electrical quiescence (the absence of spontaneous activity) was induced by culture in serum-depleted conditions. IGF-I, which is synthesized locally in mammalian brain, induced a rapid increase in electrical activity that was accompanied by increased activation of calcium channel currents. These effects were dose and time dependent. The spontaneous activity of cells exposed to 20 ng/ml IGF-I increased in approximately 10 sec and, after a brief exposure, continued increasing for at least 8 hr. Currents carried by calcium channels doubled within 10 sec. Both the increase in spontaneous activity and the increased activation of calcium channel currents were blocked by tyrosine kinase inhibitors. These results suggest that IGF-I can act as a rapid neuromodulator of calcium currents.

Rapid and efficient purification of plasma membrane from cultured cells: characterization of epidermal growth factor binding.

We have devised a rapid and simple protocol for the purification of the plasma membrane from several lines of transformed cultured cells. A431 or KB plasmalemma was purified in 90 min with a two-step centrifugation cycle after selectively inducing microsomal aggregation by the addition of calcium to homogenized cells. Relative specific activity analysis using membrane marker enzymes on the various fractions indicated that the isolated plasmalemma was purified 8-12-fold over the starting homogenate and contained a high density of epidermal growth factor (EGF) receptors. Transmission electron microscopy showed the final membrane suspension consisted of unilamellar vesicles with an average diameter of approximately 100 A. The purified membrane vesicles avidly bound to 125I-EGF and reached equilibrium within 30 min. Microfiltration assays indicated more than 90% of the total binding can be displaced by excess unlabeled ligand. Equilibrium binding analysis showed a single class of high-affinity 125I-EGF binding site, with Kd = 0.14 nM and Bmax = 0.1 pmol/mg of protein for purified KB membrane and Kd = 1.2 nM and Bmax = 5.26 pmol/mg of protein for purified A431 membrane. Gel electrophoresis of 125I-EGF cross-linked to membrane EGF receptors showed a distinct autoradiographic band at 170 kilodaltons, which could be displaced with excessive amounts of unlabeled EGF. Finally, EGF-dependent autophosphorylation of the EGF receptor was clearly demonstrated with the purified membrane preparation. Membrane vesicles purified in this manner can be stored in liquid nitrogen for several months without losing their biological activity.

Interaction between monensin and lysosomotropic amines in the regulation of the processing of epidermal growth factor by BALB/c 3T3 cells.

Monensin, like the lysosomotropic amines chloroquine and methylamine, caused a large accumulation of 125I-EGF in BALB/c-3T3 cells that was due to specific increases in the amount of intracellular intact hormone. However using a pulse-chase paradigm of 125I-EGF accumulation, marked differences were observed between monensin and the amines. When EGF was accumulated in the presence of monensin, there was a gradual loss of cell-bound radioactivity during a chase in the absence of the drug, and the labeled material recovered in the medium primarily consisted of degraded hormone. The continued presence of monensin in the chase medium substantively prevented the loss of cell bound material, and what little was recovered in the medium consisted of intact 125I-EGF. In contrast, when 125I-EGF was accumulated in the presence of methylamine, predominantly intact peptide was lost from the cells at a relatively high rate during the chase whether or not methylamine remained in the medium. When monensin was present in the chase medium following accumulation in the presence of either chloroquine or methylamine, the loss of intracellular 125I-EGF was essentially blocked.

Western blot detection of epidermal growth factor receptor from plasmalemma of culture cells using 125I-labeled epidermal growth factor.

We have developed a novel Western blot procedure for the detection of epidermal growth factor (EGF) receptors within a complex mixture of membrane proteins. Purified cell membranes from either human placenta or cultured A431 cells were solubilized, resolved by electrophoresis, and electroblotted onto nitrocellulose paper. With 5-15% gradient gels, electroblotting was completed in 2 h and both the high- and low-molecular-weight proteins were transferred evenly onto the nitrocellulose, as indicated by the radiolabeled protein markers. Upon hybridization with 125I-EGF, the membrane receptor was identified as two adjoining bands on the nitrocellulose of 150 and 170 kDa. Binding of 125I-EGF to the immobilized membrane receptor was specific and was displaced by excess unlabeled EGF. The receptor signal on the autoradiogram was optimized when 1% hemoglobin and 0.05% Tween 20 were present during the hybridization. The ligand-binding activity of the immobilized receptor was not affected by sodium dodecyl sulfate detergent or ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, but was drastically reduced by either heat denaturation or the addition of dithiothreitol to the membrane samples. Using this method, we were able to demonstrate that no noticeable difference was observed between the pre- and postphosphorylated EGF receptors in their ability to bind to 125I-EGF. Because it allows both identification and purification of a receptor from a mixture of proteins, this protocol should have general application in characterizing various receptor-ligand systems.

Neurotrophic protein S100 beta stimulates glial cell proliferation.

Nervous system development involves a coordinated series of events, including regulation of cell proliferation and differentiation by specific extracellular factors. S100 beta is a neurotrophic protein that has been implicated in regulation of cellular proliferation, but direct evidence was lacking. In this report, nanomolar concentrations of S100 beta are shown to stimulate proliferation of rat C6 glioma cells and primary astrocytes. An S100 mutant with a single amino acid change was inactive. S100 beta also stimulated increases in the steady-state levels of c-myc and c-fos protooncogene mRNAs and complemented the effects of platelet-derived growth factor. Two neuroblastoma cell lines did not proliferate in response to S100 beta, suggesting that the mitogenic activity of S100 beta is selective for astroglial cells. These results suggest that S100 beta may be involved in the coordinate development and maintenance of the central nervous system by synchronously stimulating the differentiation of neurons and the proliferation of astroglia.

Effects of phenothiazines on binding and processing of epidermal growth factor in 3T3 cells.

Chlorpromazine (CPZ) or the functionally related N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide caused a rapid decrease in binding of 125I-epidermal growth factor (EGF) that was due to a specific decrease in receptor affinity. The decrease in ligand binding was observed when cells were exposed to CPZ at either 4 degrees C or 37 degrees C but a rapid reversal of CPZs effects was observed only during a 37 degrees C incubation. In contrast to the decrease in 125I-EGF binding seen after short (30 min) accumulations at 37 degrees C, the presence of CPZ caused a large increase in the amount of cell-associated radioactivity after longer periods (over 1 h) of accumulation. Although the CPZ-induced effect was similar in extent to that observed after the addition of methylamine, the increased accumulation after CPZ was probably not due to a nonspecific ionic neutralization of the lysosomes. CPZ did not lower EGF binding in cultures chronically treated with a phorbol ester to reduce protein kinase C levels, although the CPZ-induced increases in accumulation were still observed in cells with reduced protein kinase C activity.

Rapid isolation of plasmalemma from cultured A431 cells: characterization of epidermal growth factor receptors.

A rapid method for the purification of plasma membrane from a relatively small number of A431 cells is described. The method is a simple, two-step differential centrifugation in the presence of Ca2+ that requires a total centrifugation time of 7 min. The membrane preparations contained a high level of epidermal growth factor (EGF) receptor activity demonstrated by both the quantity of specific ligand binding and the amount of EGF-dependent phosphorylation of the receptor and an exogenous substrate. EGF-dependent autophosphorylation identified the EGF receptor in the purified membranes as an undegraded 170-kDa protein.