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M C Willingham

Publications and source records attributed to M C Willingham.

At least 163 records · Page 9Linked to original sources
PubMed1986

The organelles of the trans domain of the cell. Ultrastructural localization of sialoglycoconjugates using Limax flavus agglutinin.

The subcellular distribution of sialic acid was determined at the ultrastructural level using Limax flavus agglutinin (LFA). This lectin, which is specific for N-acetylneuraminic acid and N-glycolylneuraminic acid, was covalently conjugated to horseradish peroxidase (HRP). The conjugates (LFA-HRP) were applied to aldehyde-fixed, saponin-permeabilized 3T3 cells in pre-embedding labeling electron microscopy. Peroxidase label was detected in a patchy distribution at the cell surface, and in plasma-membrane-coated pits, endocytic vesicles (receptosomes), multivesicular bodies, and lysosomes. Smooth-surfaced tubular and vesicular structures, similar to those that participate in membrane recycling, were labeled. In the Golgi complex, more than half of the cisternae contained label--typically only one cisterna on the cis side was unlabeled. Heavily labeled structures of the trans Golgi included a reticular membranous system with coated regions--50-80 nm diameter vesicular or pit-like profiles and larger coated vacuoles. Smooth 200-300 nm vacuoles were labeled on the trans side of the Golgi stack. Similar structures have been previously shown to participate in the exocytosis of plasma membrane and secretory glycoproteins from the Golgi stacks. These findings identify those intracellular organelles that are functionally at the level of, or distal to, the sialyltransferase-containing membranes of the Golgi, and distinguish them from the pre-Golgi membranous structures. The LFA-HRP conjugate is an indicator for this functional trans domain of the cell, and should be applicable for ultrastructural double-label experiments as a cis versus trans marker of the exocytic pathway.

Cell Membrane↗
PubMed1986

Single cell analysis of daunomycin uptake and efflux in multidrug-resistant and -sensitive KB cells: effects of verapamil and other drugs.

The accumulation of daunomycin in drug-sensitive and multidrug-resistant human KB cells was examined using light microscopy to detect the inherent fluorescence of daunomycin. Intracellular accumulation of fluorescent drug occurred rapidly in parental KB cells and was markedly reduced in several multidrug-resistant mutants. The addition of verapamil, which reverses multidrug resistance, resulted in increased accumulation of daunomycin in resistant cells. In living cells, most of the daunomycin was found in the nucleus, but significant amounts were detected associated with the plasma membrane, in the cytoplasm, in organelles of the Golgi region, and in lysosomes. The nuclear fluorescence was measured using a photometer system, and the loss of daunomycin from the cells was determined under various conditions. When sensitive cells were allowed to accumulate daunomycin for 5 min at 37 degrees C and then placed in medium without the drug, daunomycin remained inside the nuclei for longer than 1 day. When resistant cells were loaded in the presence of verapamil and the verapamil was removed, the resistant cells lost daunomycin with a half-time of about 1 min. The continuous presence of verapamil markedly inhibited the loss of daunomycin from the cells. Similar results were obtained in separate experiments using [3H]daunomycin. Vinblastine, vincristine, and quinidine were also effective in stimulating daunomycin accumulation in multidrug-resistant cells and in preventing the loss of daunomycin from these resistant cells. This effect required half-maximal concentrations of 1-2 microM for verapamil, vinblastine, and quinidine. Ouabain, lanthanum, colchicine, amiloride, probenecid, and 1-beta-D-arabinofuranosylcytosine had no effect on this process. Quinine was effective at 10 microM and nifedipine was effective at 50 microM. Depletion of cellular adenosine triphosphate levels by preincubation of cells with azide and 2-deoxyglucose partially inhibited daunomycin loss from resistant cells. These single-cell measurements indicate that diminished daunomycin accumulation in multidrug-resistant cells results from accelerated energy-dependent efflux across the plasma membrane, and this efflux is inhibited by verapamil, quinidine, vincristine, and vinblastine.

Biological Transport↗
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

Binding of adenovirus and its external proteins to Triton X-114. Dependence on pH.

35S-Labeled adenovirus type 2 (Ad2) (10 ng/ml) was incubated with 1% Triton X-114 at various pH values varying from 3.0 to 8.0. The detergent phase was separated from the aqueous phase by centrifugation, and the amounts of Ad2 were determined in the two phases. At pH 7.0-8.0, less than 5% of Ad2 was associated with the detergent phase; at pH 5.0 or below, about 60% of Ad2 was associated with the detergent phase. When a mixture of 35S-labeled capsid proteins was used at pH 7.0, 60-70% of the total proteins were associated with the detergent at pH 5.0, but less than 5% of the proteins interacted with detergent at pH 7.0. Among the three major external proteins (hexon, penton base, and fiber), penton base had the highest association with Triton X-114 at pH 5.0. Both intact virus and the capsid proteins that were associated with Triton X-114 at pH 5.0 were released into the aqueous phase on subsequent incubation at pH 7.0. On the basis of these results, it is suggested that mildly acidic pH induces amphiphilic properties in adenovirus capsid proteins and may help Ad2 escape from acidic endocytic vesicles.

Adenoviruses, Human↗
PubMed1985

Adenovirus-dependent increase in cell membrane permeability.

When KB cells were labeled with either 51Cr (1 microCi/ml) or [35S]methionine (5 microCi/ml) and treated with 10 micrograms/ml of adenovirus type 2 (Ad2) at pH 6.0 for 60 min at 37 degrees C, about 25% of the cell-associated 51Cr and 5% of the [35S]methionine were released into the medium. The 51Cr was mainly associated with molecules of 1500 Da or less. When KB cells were labeled with either [3H] choline, alpha-[3H]aminobutyric acid, or [3H]deoxy-2-fluoro-D-glucose and exposed to Ad2, these molecules were released in amounts much higher than 51Cr. The Ad2-dependent release of choline was found to be dependent on Ad2 concentration, with maximum release (nearly 60%) at 10 micrograms/ml of Ad2, on the length of the incubation with Ad2, with maximum release at about 90 min, and on the medium pH with maximum activity at pH 6.0 to 6.5. Greater than 95% of the choline released was water-soluble and identified as choline phosphate. Less than 5% of the choline released was associated with lipids, and none was released as a phospholipid vesicle or micelle. The ability of Ad2 to release choline was abolished by incubating Ad2 for 10 min at 45 degrees C, whereas the binding of Ad2 to the cells was not affected. Fetal calf serum also blocked Ad2-dependent choline release.

Adenoviridae↗
PubMed1985

EGF induces receptor down-regulation with no receptor recycling in KB cells.

Several ligands, including epidermal growth factor (EGF), have been found to negatively modulate or down-regulate their specific plasma membrane receptors. Using both 125I-EGF and a monoclonal antibody against the EGF-receptor (EGF-R1), we studied the down-regulation of the EGF-receptor in the human adenocarcinoma cell line KB. The results presented here demonstrate that incubating KB cells at 37 degrees C with EGF rapidly decreases the number of plasma membrane EGF-receptors. In addition, there is a concomitant rise of equal magnitude in the number of EGF molecules taken up. The latter result argues strongly that there is negligible recycling of the EGF-receptor in KB cells and that the major portion of internalized EGF-receptor complexes are transported to lysosomes and subsequently degraded. The fate of the EGF-receptor is markedly different from that of receptors not subject to down-regulation. The biochemical signals that operate to regulate such diverse receptor traffic in cells remains to be elucidated.

Carcinoma↗
PubMed1985

Serial section analysis of clathrin-coated pits in rat liver sinusoidal endothelial cells.

Electron microscopy and serial sections were used to examine the shape of clathrin-coated pits in sinusoidal endothelial cells of rat livers. Livers were perfused at 4 degrees C with either concanavalin A-horseradish peroxidase (conA-HRP), or HRP alone, followed by warm-up to 37 degrees C and fixation with glutaraldehyde. Alternatively, the livers were perfused with HRP at 37 degrees C, followed by fixation. All tissue was preserved using a membrane contrast enhancement technique (R-OTO) consisting of sequential osmium-ferrocyanide, thiocarbohydrazide, and osmium-ferrocyanide treatment. Peroxidase reaction product was used to identify structures participating in endocytosis. One hundred and ninety-three clathrin-coated structures were examined. Sixty-six were from livers perfused with conA-HRP at 4 degrees C, 63 were from livers perfused with only HRP at 4 degrees C, and 64 were from livers perfused with HRP at 37 degrees C. These coated structures were morphologically classified into three categories: (a) flat pits; (b) cup-shaped pits; (c) pits with a narrow neck. No isolated coated vesicles were found. In cells perfused at 4 degrees C followed by warming to 37 degrees C, the percentage of coated pits found connected to the cell surface by narrow necks was 31%, using conA-HRP, and 27% using HRP alone. In cells perfused continuously at 37 degrees C, the percentage of coated pits with narrow neck connections was 21% using HRP alone. These results suggest that the formation of coated pits connected to the surface by narrow necks is not an artifact of cell type, of experimental protocol or of incubation with a lectin.

Animals↗
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↗
PubMed1985

Processing and lysosomal localization of a glycoprotein whose secretion is transformation stimulated.

The major excreted protein (MEP) of transformed mouse fibroblasts is a mannose 6-phosphate-containing glycoprotein whose synthesis and secretion are increased in malignantly transformed 3T3 cells and whose synthesis is increased by treatment of 3T3 cells with tumor promoters or growth factors. When pulse-labeled extracts from Kirsten virus-transformed NIH 3T3 (KNIH) cells were immunoprecipitated using an antibody against secreted MEP, one cellular protein was immunoprecipitated that had the same molecular weight and tryptic peptide map as the secreted protein. Pulse-chase labeling experiments showed that 50-60% of this 39,000-mol-wt form was secreted in transformed cells. Of the 40-50% remaining, approximately 5% was processed into two lower molecular weight forms (29,000 and 20,000) which are sequestered within the cell. Similar processing of these proteins was observed in the nontransformed parent NIH 3T3 (NIH) cells. However, in NIH cells, much less of the synthesized MEP was secreted. Measurements of steady-state levels of these three forms of cellular MEP by Western blot immunolocalization revealed approximately fourfold more MEP in KNIH cells than in NIH cells as well as differences in the relative distribution of MEP forms in transformed and nontransformed cells. Subcellular fractionation of KNIH cells on a Percoll gradient demonstrated a distribution of total MEP similar to that of several lysosomal enzymes. The light lysosomal/Golgi peak from these gradients contained both the precursor 39,000-mol-wt form of MEP and the 20,000-mol-wt form, whereas the heavy lysosomal peak was enriched in the 20,000-mol-wt form. The distribution of MEP forms was found to be similar in NIH cells except that the 29,000-mol-wt form was also seen to be enriched in the heavy lysosomal peak. This biochemical localization of MEP was confirmed by immunolocalization with light and electron microscopy. These data support the hypothesis that MEP is a lysosomal protein that is secreted by transformed cells.

Animals↗
PubMed1985

Identification of two lysosomal membrane glycoproteins.

Two murine lysosome-associated membrane proteins, LAMP-1 of 105,000-115,000 D and LAMP-2 of 100,000-110,000 D, have been identified by monoclonal antibodies that bind specifically to lysosomal membranes. Both glycoproteins were distinguished as integral membrane components solubilized by detergent solutions but not by various chaotropic agents. The lysosome localization was demonstrated by indirect immunofluorescent staining, co-localization of the antigen to sites of acridine orange uptake, and immunoelectron microscopy. Antibody binding was predominantly located at the limiting lysosomal membrane, distinctly separated from colloidal gold-labeled alpha-2-macroglobulin accumulated in the lumen during prolonged incubation. LAMP-1 and LAMP-2 also appeared to be present in low concentrations on Golgi trans-elements but were not detected in receptosomes marked by the presence of newly endocytosed alpha-2-macroglobulin, or in other cellular structures. LAMP-1 and LAMP-2 were distinguished as different molecules by two-dimensional gel analysis, 125I-tryptic peptide mapping, and sequential immunoprecipitations of 125I-labeled cell extracts. Both glycoproteins were synthesized as a precursor protein of approximately 90,000 D, and showed a marked heterogeneity of apparent molecular weight expression in different cell lines. LAMP-2 was closely related or identical to the macrophage antigen, MAC-3, as indicated by antibody adsorption and tryptic peptide mapping. It is postulated that these glycoproteins, as major protein constituents of the lysosomal membrane, have important roles in lysosomal structure and function.

Animals↗
PubMed1985

Characterization of immunotoxins active against ovarian cancer cell lines.

The purpose of the present study was to develop immunotoxins directed against human ovarian carcinoma cells. Four monoclonal antibodies (260F9, 454C11, 280D11, and 245E7) were chosen because they were found to bind to various ovarian carcinoma cell lines. These antibodies were covalently linked to either Pseudomonas exotoxin (PE) or ricin A chain (RTA), and the conjugates were tested against five ovarian cancer cell lines (OVCAR-2, -3, -4, -5; A1847). The ability of the immunotoxins to inhibit both protein synthesis and colony formation was evaluated. Qualitatively similar results were obtained for both types of assays. Usually, PE conjugates were more toxic than their corresponding RTA conjugates. 454C11-PE was very toxic for all ovarian carcinoma lines, whereas 454C11-RTA had low activity. Both 260F9-PE and 260F9-RTA were active in all OVCAR cell lines but not in A1847 cells. 280D11-PE was toxic for OVCAR-4; otherwise, 280D11-PE and RTA conjugates of both 280D11 and 245E7 had little activity. Specificity of immunotoxin action was shown by competition by excess antibody, nontoxicity in nontarget cells, and inactivity of an irrelevant immunotoxin. To investigate the basis of antibody-dependent differences in activity of the various immunotoxins, antibody uptake was studied in OVCAR-2 cells, and the results indicate that antibody internalization is one important factor in the activity of immunotoxins.

ADP Ribose Transferases↗
PubMed1985

Ultrastructural immunocytochemical localization of the transferrin receptor using a monoclonal antibody in human KB cells.

Using a monoclonal antibody (HB21) against the human transferrin receptor, we have localized this receptor in cultured KB human carcinoma cells by fluorescence and ultrastructural immunocytochemistry. The receptor was found diffusely distributed on the cell surface, concentrated in clathrin-coated pits of the cell surface, in intracellular endocytic vesicles (receptosomes) derived from coated pits, in tubular elements of the trans-reticular Golgi system, and in microtubule-associated membranous elements thought to be part of the constitutive exocytic system. This distribution is the same as that previously shown for labeled transferrin in these same cells (Willingham MC, Hanover JA, Dickson BB, Pastan J: Proc Natl Acad Sci USA 81:175, 1984). No significant amounts of receptor were found in lysosomes. An aggregation of membranous elements containing this receptor was found in the pericentriolar region of cells during mitosis. Together with the previous data on the immunocytochemical localization of transferrin, these results suggest that the transferrin receptor may constitutively enter and exit KB cells by endocytosis and exocytosis, carrying bound transferrin into and out of the cell for the purpose of supplying iron from the extracellular environment for cell growth.

Antibodies, Monoclonal↗
PubMed1985

Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins.

Immunofluorescence microscopy of cultured animal cells is often performed after detergent permeabilization of formaldehyde-fixed cellular membranes so that antibodies may have access to intracellular antigens. A comparison was made of the ability of several detergents, after formaldehyde fixation, to affect localization of intracellular proteins or to permeabilize different organelles to antibodies. Saponin, a detergent-like molecule that can permeabilize cholesterol-containing membranes, was also used. Four monoclonal antibodies were found to have a bright, discrete fluorescence localization with saponin alone, but were almost undetectable when the cells were treated with nonionic detergents such as Triton X-100 or NP-40. These immunoglobulin G antibodies included two against lysosomal membrane glycoproteins, one against an integral membrane protein found in the plasma membrane and endocytic vesicles, and one against a membrane protein in the endoplasmic reticulum and the nuclear envelope. However, antigens localized in mitochondria and the nucleus required the use of a detergent such as Triton X-100 for their detection. The detection of a number of other membrane or cytoplasmic proteins was unaffected by Triton X-100 treatment. It was concluded that nonionic detergents such as Triton X-100 cause artifactual loss of detection of some membrane proteins, and saponin is a favorable alternative reagent for immunofluorescence detection of intracellular membrane antigens in many organelles.

Animals↗
PubMed1985

Anti-transferrin receptor antibody linked to Pseudomonas exotoxin as a model immunotoxin in human ovarian carcinoma cell lines.

The present in vitro study was performed to evaluate the potential usefulness of immunotoxins in treating human ovarian carcinomas. A monoclonal antibody against the human transferrin receptor was covalently linked to Pseudomonas exotoxin. The activity of this immunotoxin (anti-TFR-PE) was studied in five ovarian carcinoma cell lines, a breast carcinoma cell line (MCF-7), and in KB cells. The ovarian carcinoma cell lines included one previously established cell line (A1847) and four recent isolates obtained from the malignant ascites of patients with metastatic ovarian carcinoma (OVCAR cell lines). While all cell lines showed inhibition of protein synthesis by anti-TFR-PE, there were quantitative differences when the level of protein synthesis was assayed after a 12-hr incubation with the immunotoxin. These differences resulted from different kinetics of anti-TFR-PE activity in the various cell lines. Higher levels of cellular binding and internalization of anti-TFR were shown to contribute to increased toxicity of anti-TFR-PE. Verapamil increased the rate of protein synthesis inhibition and thus enhanced the toxicity of anti-TFR-PE in the OVCAR cell lines.

ADP Ribose Transferases↗
PubMed1984

Adenovirus-dependent release of 51Cr from KB cells at an acidic pH.

When KB cells labeled with 51Cr (1 muCi/ml) were treated with adenovirus type 2 (Ad2) at pH 6, 51Cr was released in a concentration-dependent manner with half-maximal release at 1 microgram/ml of virus. The 51Cr release was maximum at 10 micrograms/ml and represented nearly 20% of cell-associated 51Cr. 51Cr release depended on the length of incubation with Ad2 and the pH of the medium; maximum release was at pH 6 with very little release at pH 7.5. An antiserum against the penton base of Ad2 specifically neutralized the ability of Ad2 to release 51Cr. Chloroquine up to 200 microM did not block Ad2-dependent 51Cr release. DEAE-dextran stimulated Ad2-dependent 51Cr release; 4-5-fold stimulation was observed at 50 micrograms/ml of DEAE-dextran. We have compared the ability of Ad2 to release 51Cr with its ability to disrupt endocytic vesicles. Vesicle disruption was independent of pH of the medium in the range of pH 6 to 7.5 and was blocked by chloroquine, whereas, 51Cr release was much greater at pH 6 than at pH 7.5 and was not affected by chloroquine. These results suggest that Ad2 possess a lytic activity which ordinarily lyses acidic endocytic vesicles but, if the cells are maintained at acidic pH, can also directly disrupt the plasma membrane.

ADP Ribose Transferases↗