Search for radiative transitions in the hypernucleus Lambda 10B.
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Journal Article↗
Biomedical subjects
W Wharton
Publications and source records attributed to W Wharton.
Rapid minipreparation of lambda DNA.
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Animals↗
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.
Angiotensin II↗
Characterization of the rebinding of 125I-epidermal growth factor released from BALB/c-3T3 cells following accumulation in the presence of chloroquine.
Lysosomotropic amines, such as chloroquine and methylamine, increase the intracellular accumulation of 125I-EGF by inhibiting lysosomal degradation. It has been shown previously that BALB/c-3T3 cells, prelabeled at 4 degrees C with 125I-EGF for 3 h and subsequently chased at 37 degrees C in the presence of chloroquine, internalized the surface bound 125I-EGF which was subsequently released into the extracellular medium in a high molecular weight form which co-migrated with native 125I-EGF. The secreted 125I-EGF rebound to the cells from which it was released more efficiently than does peptide in the extracellular media. We now show that when the BALB/c-3T3 cells were prelabeled at 37 degrees C for 2 h in the presence of chloroquine, the internalized 125I-EGF released into the medium was in a high molecular weight form which co-migrated with native 125I-EGF and did not rebind anymore efficiently than did peptide in the extracellular media. This lack of rebinding was not due to an alteration in the 125I-EGF molecule since it was still capable of rebinding to naive A431 cells, nor was it due to the exhaustion of EGF receptors on the BALB/c-3T3 cells. The inhibition of rebinding was observed only when the cells were treated with EGF in the presence of chloroquine, and was not due to a general down-regulation of membrane receptors. The differences between the rebinding of 125I-EGF at 4 degrees C and 37 degrees C suggest that EGF may be processed via different pathways in the cell.
Animals↗
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.
Cell Membrane↗
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.
Cell Fractionation↗
Platelet-derived growth factor stimulation of [3H]-glucosamine incorporation in density-arrested BALB/c-3T3 cells.
G0/G1 traverse in density-arrested BALB/c-3T3 cells is controlled by multiple serum-derived growth factors. Platelet-derived growth factor (PDGF) initiates a proliferative response, whereas factors present in plasma facilitate progression through G0/G1. In the absence of competence formation, progression factors are unable to stimulate cell cycle traverse. We have identified the stimulation of a biochemical process specific to competence formation in BALB/c-3T3 cells. PDGF treated BALB/c-3T3 cells incorporated 5-10-fold more [3H]-glucosamine (GlcN) into acid-insoluble material as compared to platelet-poor plasma (PPP) treated cultures. Increased GlcN incorporation occurred in density-arrested BALB/c-3T3 cells in response to treatment with other competence factors, fibroblast growth factor, and Ca3 (PO4)2 and was not due to cell-cycle traverse. Stimulation of [3H]-GlcN incorporation by PDGF was time dependent, and increased incorporation of [3H]-GlcN into protein required de novo protein synthesis. Several mechanisms through which PDGF could increase GlcN incorporation into cellular material were examined. Results of these studies suggest an increase in the cellular capacity to glycosylate proteins is a response to or a part of competence formation.
Animals↗
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.
Animals↗
Dissociation of cellular transformation from platelet-derived growth factor independence.
ST2-3T3, a spontaneously transformed BALB/c-3T3 cell line which does not require platelet-derived growth factor (PDGF) for growth, was fused to THO2, a PDGF-responsive non-transformed BALB/c-3T3 cell line, in order to learn whether transformation is expressed coordinately with PDGF independence. Hybrid cells were selected and grown in medium containing both HAT (hypoxanthine-aminopterin-thymidine) and ouabain; unfused cells of each parental type were killed in HAT-ouabain medium. Five independently isolated ST2-3T3xTHO2 hybrid cell lines were established and characterized for both transformation and PDGF responsiveness. All five were transformed, having a disorganized growth pattern and achieving a final cell density similar to that of ST2-3T3 cells. Two of these lines did not respond to a brief treatment with PDGF: the mitogen neither induced the synthesis of a PDGF-modulated lysosomal protein (termed MEP), nor stimulated the cells to enter the S phase; one line responded to PDGF by synthesizing both MEP and DNA, whereas two others synthesized MEP but not DNA. In contrast, four independently isolated cell lines obtained by fusing PDGF-responsive non-transformed BALB/c-3TC cells to the THO2 line were all PDGF-responsive for both MEP and DNA synthesis and were not transformed. It appears that PDGF independence is not required for the transformation of BALB/c-3T3 cells.
Animals↗
The relationship between intracellular cyclic AMP concentrations and the in vitro growth of macrophages.
The addition of cholera toxin, prostaglandins, or one of a series of xanthine phosphodiesterase inhibitors to bone marrow-derived macrophages maintained in liquid culture caused a dose-dependent decrease in colony formation measured 7-10 days following seeding. The growth inhibitory effects of xanthines were in the same order of potency (caffeine less than theophylline less than isobutylmethylxanthine) as their reported ability to inhibit cyclic AMP phosphodiesterase. The relationship between the magnitude of the increases in intracellular concentrations of cyclic AMP observed following the addition of the drugs and the degree of growth inhibition was complex. Combinations of cholera toxin and phosphodiesterase inhibitors caused synergistic elevations in cyclic AMP levels after a lag of approximately 3 days. However, the growth rate was decreased immediately following the addition of the combination of drugs, and thus seemed to be independent of the nucleotide levels. A cyclic AMP-resistant variant of a cloned nontransformed macrophage cell line was found to be also resistant to the growth inhibitory actions of both cholera toxin and prostaglandins. However, resistance to the inhibitory effects of cyclic AMP did not render the cells resistant to a xanthine-induced growth inhibition.
1-Methyl-3-isobutylxanthine↗
Effects of cholera toxin and isobutylmethylxanthine on growth of human fibroblasts.
Cholera toxin produced a dose-dependent decrease in the restimulation of G0/G1 traverse in density-arrested human fibroblasts but did not inhibit the stimulation of cells arrested in G0 after serum starvation at low density. In addition, cholera toxin did not inhibit the proliferation of sparse logarithmically growing human fibroblasts, even when low concentrations of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) were also present. However, the final density to which sparse cells grew was limited by cholera toxin, when added either alone or together with low concentrations of IBMX. In contrast, high concentrations of the phosphodiesterase inhibitor alone produced a profound inhibition in the growth of sparse human fibroblasts. IBMX produced an inhibition both in the G1 and in the G2 phases of the cell cycle by a mechanism(s) that was not related to the magnitude of the increases in adenosine 3',5'-cyclic monophosphate concentrations.
1-Methyl-3-isobutylxanthine↗
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.
Animals↗
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.
Animals↗
Weak decay of 1 Lambda 2C and 1 Lambda 1B hypernuclei.
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Journal Article↗
Late G1 amino acid restriction point in human dermal fibroblasts.
Human dermal fibroblasts arrested in G0 by maintenance in medium supplemented with 0.1% serum were not restimulated to divide when fresh medium containing 10% dialyzed serum but lacking group B amino acids (cystine, isoleucine, lysine, phenylalanine and tyrosine) was added. Unlike rodent cells, the addition of fresh serum-supplemented medium lacking only isoleucine did not cause a growth arrest. The amino acid sensitive growth arrest in human fibroblasts was dependent both on presynchronization in G0 as well as a prestarvation for amino acids prior to stimulation with high serum. When cells were restimulated in the absence of amino acids, they arrested predominantly in G1, although a small percentage of cells entered early S phase. When medium containing a complete complement of amino acids was then added, cells initiated DNA synthesis following a minimum lag of 2-3 hr. Growth arrested cells initiated DNA synthesis even when complete unsupplemented medium was added, although the addition of high concentrations of insulin or 10% serum increased the rate of entry.
Amino Acids↗
Chloroquine allows the secretion of internalized 125I-epidermal growth factor from fibroblasts.
Incubation of cells with labelled hormone in the presence of the lysosomotropic agent chloroquine produces an enhanced intracellular accumulation of hormone and receptor. Using a pulse-chase paradigm in which cell surface receptors were labelled with 125I-EGF at 4 degrees C, it was found that when 100 microM chloroquine was present in the 37 degrees C chase medium intact hormone was accumulated in the medium. Without chloroquine, low molecular weight (mw) degradation products were found in the medium. The processes of receptor-mediated endocytosis and subcellular distribution of 125I-EGF-receptor complexes were unchanged by chloroquine. The source of the intact hormone accumulating in the medium was therefore an intracellular compartment(s). The 125I-EGF released from the cells could rebind to surface receptors and be re-internalized; rebinding was inhibited by unlabelled EGF or Concanavalin A in the incubation medium. The concentration of unlabelled EGF required to inhibit rebinding was more than three orders of magnitude greater than the amount of 125I-EGF whose rebinding was inhibited. Thus, the 125I-EGF released from intracellular sites was rebound preferentially over exogenous EGF. The possible pathways for secretion of intact 125I-EGF and mechanisms of its preferential rebinding are discussed.
Animals↗
Stabilized complexes of epidermal growth factor and its receptor on the cell surface stimulate RNA synthesis but not mitogenesis.
Treatment of mouse fibroblasts, prelabeled at 4 degrees C with 125I-labeled epidermal growth factor (EGF), with the lectin concanavalin A (Con A) stabilized the 125I-labeled EGF-receptor complex to dissociation and prevented receptor-mediated endocytosis; after 5 hr at 37 degrees C, approximately 50% of the 125I-labeled EGF initially bound at 4 degrees C remained cell associated, compared to less than 15% in control cells. The radioactivity lost from the Con A-treated cells was found as intact hormone in the medium, with almost no hormone degradation evident, whereas in control cells most of the medium radioactivity was in the form of low molecular weight degradation products. The trimolecular complex Con A-EGF-receptor was capable of stimulating RNA synthesis to levels greater than control (untreated) or EGF alone and maintained this stimulation for prolonged periods of time. However, there was no effect of Con A treatment on the stimulation of DNA synthesis induced by EGF prebound at 4 degrees C. Thus, maintaining the EGF-receptor complex at the cell surface allows enhanced stimulation of an acute biological response to EGF (RNA synthesis) but not stimulation of DNA synthesis. These data support the idea that processing subsequent to receptor binding is necessary to produce the mitogenic signal.
Animals↗
Multistep change in epidermal growth factor receptors during spontaneous neoplastic progression in Chinese hamster embryo fibroblasts.
Whole Chinese hamster embryo lineages have been shown to undergo multistep spontaneous neoplastic progression during serial passage in culture. We have studied the binding, internalization, and degradation of 125I-labeled epidermal growth factor at four different stages of transformation. The whole Chinese hamster embryo cells lost cell surface epidermal growth factor receptors gradually during the course of neoplastic progression until only 10% of the receptor number present in the early-passage cells (precrisis) were retained in the late-passage cells (tumorigenic). No differences in internalization rates, chloroquine sensitivity, or ability to degrade hormone between the various passage levels were seen. No evidence for the presence in conditioned medium of transforming growth factors which might mask or down-regulate epidermal growth factor receptor was obtained. These results suggest that a reduction in cell surface epidermal growth factor receptor might be an early event during spontaneous transformation in whole Chinese hamster embryo cells.
Animals↗