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Biomedical subjects

J A Hanover

Publications and source records attributed to J A Hanover.

At least 73 records · Page 4Linked to original sources
PubMed1985

Transit of receptors for epidermal growth factor and transferrin through clathrin-coated pits. Analysis of the kinetics of receptor entry.

Selective enrichment of clathrin-coated membranes by anticlathrin immunoadsorption was used to examine the internalization of receptor-ligand complexes through coated pits. Using Staphylococcus aureus-anticlathrin antibody and [35S]methionine-labeled KB cells, the kinetics of association of the epidermal growth factor (EGF-R) and transferrin receptors (TF-R) with coated membranes were directly examined. The accumulation of EGF-R in coated pits at the cell surface was dependent upon EGF binding. EGF-R then passed sequentially through a compartment which did not react with anticlathrin antibody and a second clathrin-coated compartment. The EGF-R was degraded in lysosomes with a half-life of approximately 41-55 min. The tumor promoter, 4 beta-phorbol 12-myristate 13-acetate, appears to mimic the action of EGF in inducing EGF-R accumulation in coated pits at the cell surface and receptor internalization. In contrast to the results with EGF-R, the TF-R was found in clathrin-coated membranes in the presence or absence of TF, and the concentration of TF-R in clathrin-coated membranes did not significantly change with time. The method presented should be of great utility for examining the biochemical changes that occur during the receptor-mediated endocytosis and sorting of ligands and receptors.

Animals↗
PubMed1985

Isolation and genetic characterization of human KB cell lines resistant to multiple drugs.

Human KB cell lines resistant to high levels of colchicine were isolated by several successive single-step selections. Most of these selection steps resulted in cross-resistance to vincristine, vinblastine, adriamycin, actinomycin D, and puromycin; however, at the highest levels of colchicine resistance, increased cross-resistance to other drugs was not observed. There was no major change in protein synthesis or alteration in protein phosphorylation or [14C]glucosamine labeling patterns accompanying the development of multiple drug resistance as measured by analysis of metabolically labeled proteins on SDS gels. Cell-cell hybridization experiments showed that the colchicine-resistant and multiple drug-resistant phenotypes were incompletely dominant. In addition, colchicine resistance was found to segregate independently from resistance to other drugs in one somatic cell hybrid, suggesting that complex genetic loci are involved in the development of the multiple drug-resistant phenotype. These mutants should be useful for the study of the clinically important problem of multiple drug resistance in human cancer.

Antineoplastic Agents↗
PubMed1985

Inhibition of phosphatidylcholine synthesis does not alter uptake of transferrin by LM fibroblasts.

The receptor-mediated endocytosis of diferric transferrin by LM fibroblasts has been examined to determine if de novo synthesis of phosphatidylcholine is required for this process. To test this possibility, LM cells were allowed to internalize [125I]transferrin in the presence or absence of exogenous choline. Under conditions in which de novo synthesis of phosphatidylcholine was reduced from 51% of the total to less than 10%, the initial rate of transferrin uptake was unaffected. These data suggest that the process of receptor-mediated endocytosis of transferrin can proceed normally in the absence of de novo phosphatidylcholine synthesis.

Animals↗
PubMed1985

Phorbol esters induce transient internalization without degradation of unoccupied epidermal growth factor receptors.

4 beta-Phorbol 12-myristate 13-acetate (PMA) treatment of KB cells at 37 degrees C rapidly induces a 50% reduction in epidermal growth factor (EGF) binding that is maximal by 30 min. EGF binding activity returns to the original value by 1 hr and remains constant for 2 hr thereafter. Using a polyclonal antibody directed against the cytoplasmic domain of the EGF receptor (EGF-R), we examined the fate of the receptor after PMA treatment. Immunofluorescent and electron microscopic localization of the EGF-R after PMA treatment demonstrated that about 50% of the receptor became internalized into endocytic vesicles (receptosomes) and Golgi-associated structures. Unlike EGF-induced internalization, PMA-induced internalization did not cause delivery of EGF-R to lysosomes or receptor degradation. Rather, receptor reappeared on the cell surface. No stimulation of EGF-R synthesis was observed after 1 hr of PMA treatment. Loss of cell surface binding correlated with the internalization of the EGF-R observed morphologically. A possible explanation for these observations is that PMA, an activator of protein kinase C, confers a signal sufficient for EGF-R clustering and internalization but not for transport to lysosomes.

Carcinoma, Squamous Cell↗
PubMed1984

Verapamil enhances the toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin and antitransferrin receptor with Pseudomonas exotoxin.

Verapamil, a clinically important calcium channel blocker, has been found to cause a 40-fold enhancement of killing of the human KB cell line by a cytotoxic conjugate of epidermal growth factor with Pseudomonas exotoxin (EGF-PE). Synergistic effects of verapamil and EGF-PE are also seen on HeLa D98 cells and a human epidermal carcinoma cell line, A431. Verapamil also potentiates the effect of a toxic conjugate formed between Pseudomonas exotoxin and a monoclonal antibody to the human transferrin receptor (anti-TFR-PE) and enhances the effect of Pseudomonas exotoxin (PE) alone. Two other calcium antagonists were tested. Diltiazem enhances the cytotoxic effect of EGF-PE, but nifedipine does not. Verapamil does not affect the binding and uptake of 125I-EGF by KB cells, but it significantly delays the disappearance of internalized 125I-EGF from the cells. Density gradient fractionation studies using cell homogenates suggest that 125I-EGF accumulates in an undegraded form in lysosomes when cells are treated with verapamil. By immunofluorescence microscopy using an antibody to PE, EGF-PE was found to accumulate in lysosomes; by electron microscopy the lysosomes had an abnormal appearance. The effects of verapamil on toxicity of EGF-PE and lysosomal function appear to be related. However, it is not known whether the enhanced toxicity of EGF-PE in the presence of verapamil is due to its delayed degradation in lysosomes or some more general effect of verapamil on membrane permeability.

ADP Ribose Transferases↗
PubMed1984

Kinetics of transit of transferrin and epidermal growth factor through clathrin-coated membranes.

The kinetics of association of epidermal growth factor (EGF) and diferric-transferrin (TF) with clathrin-coated membranes of KB cells were examined using anti-clathrin antibody bound to Staphylococcus aureus. The ligands were bound to cells at 4 degrees C and after warming to 30 degrees C for 5 min, both ligands were found concentrated in a membrane fraction immunoadsorbed with anti-clathrin antibody. Fifteen minutes after entry both ligands moved to a non-clathrin-associated compartment. Twenty to 25 min after entry, EGF, but not TF, appeared in a second clathrin-associated compartment. In parallel morphologic experiments, EGF-horseradish peroxidase (EGF-HRP) and TF-HRP were both found at 6 min in coated pits associated with the plasma membrane. At 22 min EGF-HRP, but not TF-HRP, was localized in Golgi-associated coated pits. These studies provide biochemical and morphological evidence suggesting that coated membranes in the Golgi region are involved at some stage in the transfer of EGF to lysosomes. The method should be of general utility in the study of receptor-mediated endocytosis.

Biological Transport↗
PubMed1984

Morphologic characterization of the pathway of transferrin endocytosis and recycling in human KB cells.

The pathway of transferrin uptake and recycling was investigated in KB cells to attempt to identify the organelles involved in the return of transferrin to the cell surface. Comparison was made with the pathway of internalization of epidermal growth factor (EGF), which has been shown to terminate in lysosomes. A horseradish peroxidase conjugate of transferrin (TF-HRP) was incubated with KB and at 4 degrees C and, at various times after warming to 37 degrees C, the location of TF-HRP was examined at the electron microscopic level. Transferrin, like EGF, was found to enter cells via coated pits and to move to the Golgi region in receptosomes (endosomes). Transferrin was located in the tubular elements of the transreticular portion of the Golgi but not in Golgi stacks. Interestingly, transferrin was not concentrated in the coated pits of the Golgi. In contrast, EGF was highly concentrated there. Transferrin was next detected in tubular elements (approximately equal to 600 A in width and up to 5,000 A in length) that were closely associated with microtubules and in dumbbell-shaped structures. In contrast, EGF was not detected in these structures. These results suggest that those organelles containing transferrin, but not EGF, participate in the return of receptor-bound transferrin to the cell surface.

Biological Transport↗
PubMed1983

Prelysosomal divergence of transferrin and epidermal growth factor during receptor-mediated endocytosis.

The routes followed by epidermal growth factor and transferrin during their endocytosis by human epithelial cells were compared in double-label studies by using density gradient centrifugation of cell homogenates and fluorescence microscopy with intact cells. Gradient centrifugation studies of cells incubated with radioactively labeled epidermal growth factor and transferrin indicated that both ligands initially were associated with a class of vesicles having a density of 1.037 g/mL and then were rapidly transferred to a membrane compartment having a slightly higher density (1.039 g/mL). Subsequently, the two ligands diverged. Epidermal growth factor ultimately was transferred to a membranous compartment containing lysosomal enzymes (density (1.08 g/mL) where it was degraded. Transferrin was released intact from the cells; very little was transferred to lysosomes. Using fluorescently labeled ligands, it was observed that after cells were warmed to 37 degrees C for 5 min, transferrin and epidermal growth factor gave coincident, punctate fluorescent patterns, strongly suggesting they were localized within the same endocytic vesicles. Subsequently, the epidermal growth factor signal was observed in lysosomes whereas the transferrin signal became weaker and diffuse and did not coincide with the punctate epidermal growth factor fluorescence. The time course of the divergence of the radioactive and fluorescent ligands coupled with the previous morphologic studies on the pathway of epidermal growth factor internalization [Willingham, M. C., & Pastan, I. (1982) J. Cell Biol. 94, 207-212] suggests that the sorting process is prelysosomal and possibly Golgi associated.

Centrifugation, Density Gradient↗
PubMed1983

alpha 2-Macroglobulin binding to cultured fibroblasts. Solubilization and partial purification of binding sites.

Binding sites having the characteristics of receptors for "activated" alpha 2-macroglobulin (alpha 2M) have been solubilized with octyl-beta-D-glucoside from fibroblast membranes. When the detergent was removed by dialysis, the resulting insoluble extract was shown to bind 125I-alpha 2M specifically. Analysis of the binding data using a nonlinear curve-fitting program suggests that the solubilized preparation contains two classes of binding sites (KD = 0.34 nM and KD = 104 nM). Membranes or solubilized extracts from KB cells which lack alpha 2M binding sites did not specifically bind 125I-alpha 2M. The solubilized binding sites from fibroblasts were inactivated by boiling and trypsin treatment, and required Ca+2 for maximal binding. In addition, the high affinity binding of 125I-alpha 2M to the solubilized receptor was inhibited by bacitracin and by alpha-bromo-5-iodo-4-hydroxy-3-nitroacetophenone, two agents which interfere with the uptake of alpha 2M in cultured fibroblasts. Using a combination of ion exchange and gel permeation chromatography, we have purified the high affinity alpha 2M binding site approximately 100-fold from membrane derived from NIH-3T3 (spontaneously transformed) fibroblasts grown as tumors in mice. The receptor is apparently an acidic protein and the receptor octyl-beta-D-glucoside complex has a Stokes radius of 45-50 A as measured by gel filtration.

Animals↗
PubMed1983

Alpha 2-macroglobulin binding to cultured fibroblasts: identification by affinity chromatography of high-affinity binding sites.

Binding sites having the properties of high-affinity receptors for activated alpha 2-macroglobulin (alpha 2M) have been purified over 100-fold from membranes of spontaneously transformed NIH-3T3 cells (J. A. Hanover, S.-y. Cheng, M. C. Willingham, and I. H. Pastan [1983] J. Biol. Chem. 258, 370-377). To identify the molecular species involved in high-affinity binding, the solubilized receptor has been purified 500-fold by conventional procedures and further purified by affinity chromatography. After radioiodination of the 500-fold-purified preparation, the detergent-solubilized extract was applied to alpha 2M-Sepharose and an 85,000 +/- 5000 Mr species was selectively retained by the column. Binding of the 85,000 +/- 5000 Mr species to the affinity resin was inhibited by EDTA and by excess alpha 2M. Elution from the affinity column could be accomplished with bacitracin, a competitive inhibitor of alpha 2M binding, or with EDTA. Consistent with the previously reported characteristics of the high-affinity alpha 2M receptor, the 85,000 Mr species bound much more efficiently to methylamine-activated alpha 2M-Affigel than to alpha 2M-Affigel which had not been amine-activated. The present data suggest that a protein with a subunit Mr of 85,000 +/- 5000 may represent a component of the high-affinity alpha 2M receptor present on cultured fibroblasts.

Animals↗
PubMed1983

Isolation and characterization of a highly enriched preparation of receptosomes (endosomes) from a human cell line.

Receptor-mediated endocytosis proceeds by transfer of receptor-ligand complexes from clathrin-coated pits at the cell surface to uncoated endocytic vesicles termed receptosomes (or endosomes). These vesicles have now been purified more than 37-fold based on their content of newly internalized epidermal growth factor. 125I-labeled EGF was bound to human KB carcinoma cells at 4 degrees C, and then the cells were warmed to 37 degrees C for 8 min and disrupted. The purification scheme involved density gradient centrifugation on colloidal silica and sucrose and gel filtration on Sephacryl S-1000. Relative to homogenate, receptosomes are enriched 4.3-fold in their cholesterol content and depleted in enzyme markers for plasma membranes (2- to 3-fold) and lysosomes (9-fold). Receptosomes have a polypeptide composition that is different from plasma membrane, lysosome, and other homogenate fractions. They are enriched in transferrin receptors (30-fold) and in unidentified Mr 70,000-75,000 glycoprotein(s); they contain phosphomannosyl receptors. They do not contain detectable amounts of clathrin.

Carcinoma↗
PubMed1983

Receptor-mediated endocytosis of alpha 2-macroglobulin: solubilization and partial purification of the fibroblast alpha 2-macroglobulin receptor.

Recent studies in our laboratory have been aimed at biochemically characterizing the alpha 2M receptor present on fibroblast membranes. The approach we have taken is to develop a means of assessing binding to solubilized alpha 2M binding sites. The binding activity was not removed by treatment with high salt concentration or treatment with chaotropic agents. Removal of the binding activity from membranes did occur using a variety of detergents which suggests that the receptor molecules may be "intrinsic" membrane proteins. The most useful detergent for solubilizing the alpha 2M receptor was octyl-beta-D-glucoside. The alpha 2M binding activity could be removed from NRK fibroblasts and Vero cells using this detergent and was found to remain in solution at 100,000 X g. Removal of the octyl-beta-D-glucoside by extensive dialysis resulted in formation of protein-lipid aggregates that bind to 125I-alpha 2M specifically and with high affinity. Such binding sites were not generated when KB cells (which lack receptors) were extracted with the detergent. Significantly, the observed affinities detected for both high- and low-affinity binding sites were similar to those reported with intact cells or membranes. In addition, binding to the solubilized sites could be inhibited using compounds known to block the receptor-mediated endocytosis of alpha 2M (bacitracin, IBHNA). Other compounds (monensin, dansylcadaverine), which did not directly inhibit the high-affinity binding sites, may exert their effects at different stages in receptor-mediated endocytosis (i.e., receptor recycling). alpha 2M binding sites from NIH-3T3 (spontaneous) tumors have been purified approximately 95-fold by a combination of ion exchange and gel permeation chromatography. The receptor appears to be an acidic protein that approximately coelutes with aldolase (45 A, 158,000 daltons) on gel filtration. Ion exchange chromatography appears to remove an endogenous inhibitor of alpha 2M binding and may also remove binding sites having lower affinity for 125I-alpha 2M. Recent studies using immobilized alpha 2M as an affinity resin suggest that the high-affinity alpha 2M receptor may have a subunit molecular weight of approximately 85,000. Studies are now in progress aimed at further characterizing these high-affinity binding sites. Once bound to the alpha 2M receptor, alpha 2M enters cells via coated pits and is rapidly transferred to receptosomes. These organelles carry the ligand into the Golgi region, from which it is transferred to lysosomes where it is slowly degraded.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1982

Temporal aspects of the N- and O-glycosylation of human chorionic gonadotropin.

The glycoprotein hormone, human chorionic gonadotropin (hCG), contains both N- and O-linked oligosaccharide chains linked to its beta-subunit. Using the human choriocarcinoma cell line, BeWo, we have examined the temporal relationship between N- and O-glycosylation of hCG and the subsequent processing of both types of oligosaccharide chains. The results indicate that, as observed in related cell lines, mature, completely glycosylated forms of the subunits of hCG cannot be detected intracellularly in BeWo cells during pulse-chase experiments with [35S]methionine. To more directly study the temporal relationship between N- and O-glycosylation of hCG in BeWo cells, 14C-amino acids and [3H]glucosamine (which also serves as a precursor to N-acetylgalactosamine) were used to label hCG. The results of these studies are consistent with a model for the N- and O-glycosylation of hCG in which 1) N-glycosylation of hCG occurs co-translationally or very shortly after translation, and 2) the addition of O-linked GalNAc residues to the polypeptide and the addition of peripheral GlcNAc residues to the N-linked oligosaccharide chains occur just prior to secretion, presumably in the Golgi complex.

Acetylglucosamine↗
PubMed1982

Transmembrane assembly of N-linked glycoproteins. Studies on the topology of saccharide synthesis.

The mechanism of synthesis of dolichol-linked saccharides has been examined using sealed hen oviduct microsomes. N,N'-Diacetylchitobiosylpyrophosphoryldolichol (chitobiosyl-lipid) was shown to be inaccessible to a soluble galactosyltransferase during its synthesis from UDP-GlcNac. In contrast, exogenous chitobiosyl-lipid added to microsomes is accessible to galactosyltransferase and does not appear to be unidirectionally translocated to the lumen. These results imply that assembly of chitobiosyl-lipid does not occur on the cytoplasmic face of the rough endoplasmic reticulum followed by a rapid unidirectional translocation to the lumen. Two lines of evidence rule out sugar nucleotide uptake and utilization within the lumen as a mechanism for chitobiosyl-lipid synthesis. GDP-mannose, which is involved in the elongation of chitobiosyl-lipid to form oligosaccharide-lipid, also does not permeate microsomal vesicles. This observation regarding GDP-mannose raises two important questions. 1) What is the topology of the oligosaccharide in sealed microsomes? and 2) how is the chitobiosyl-lipid elongated by addition of mannosyl units to form an oligosaccharide-lipid? During the synthesis of oligosaccharide-lipid in freeze-thawed microsomes, approximately 40% of the oligosaccharide is released from the lipid by hydrolysis and remains entrapped within the sealed microsomes. These data suggest that oligosaccharide-lipid is transiently present at the luminal face of the rough endoplasmic reticulum during its synthesis. Most of the enzymes involved in the synthesis of oligosaccharide-lipid can be inactivated by external proteolysis of microsomal vesicles. These data are discussed in terms of a model in which synthesis of chitobiosyl-lipid and its elongation to oligosaccharide-lipid are coupled processes that occur in a transmembrane multienzyme complex.

Animals↗
PubMed1980

Synthesis of N- and O-linked glycopeptides in oviduct membrane preparations.

A hen oviduct membrane preparation that catalyzes both the N- and O-glycosylation of exogenous acceptor peptides was used to examine the possible involvement of lipid intermediates in enzymatic O-glycosylation. The results indicate that, under a variety of experimental conditions in which the dolichol-linked saccharides involved in N-glycosylation are readily observed, no lipid-linked intermediates for O-glycosylation could be detected. Whereas N-glycosylation is abolished by tunicamycin treatment and stimulated by dolichol phosphate addition, O-glycosylation is unaffected by such treatments. Further, the results of subcellular fractionation of oviduct membranes suggest that N-acetylgalactosaminyl:polypeptide transferase is localized primarily in membranes derived from the smooth endoplasmic reticulum and Golgi apparatus. This is in contrast to the subcellular site of N-glycosylation, which has previously been shown to be primarily the rough endoplasmic reticulum. These findings are discussed in relation to the function of dolichol phosphate in protein glycosylation.

Animals↗