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We have used electron microscopic immunocytochemistry to compare the distribution of LAMP-1, a marker for lysosomal membranes, with the intracellular localization of alpha 2-macroglobulin (alpha 2-M) and transferrin at various time points after their endocytosis into cultured NIH 3T3 cells. The purposes of this study were (a) to determine how soon endocytic ligands reach lysosomal organelles, (b) to examine whether the intermediate endocytic vesicles gained lysosomal markers gradually or in a precipitous, discrete event, and (c) to examine the relationship, if any, between the pathway of recycling ligands and lysosomes. At early time points (0-5 min) after initiation of endocytosis, most structures containing alpha 2-M labeled with colloidal gold (receptosomes) were not labeled by anti-LAMP-1 detected using ferritin bridge or peroxidase immunocytochemistry. At late time points (greater than or equal to 15 min), the structures containing alpha 2-M (lysosomes) were strongly labeled by anti-LAMP-1. In contrast, transferrin that was directly labeled with ferritin was mostly located in LAMP-1-negative structures at all time points studied. The proportion of alpha 2-M-gold containing vesicles strongly labeled for LAMP-1 roughly paralleled the proportion of alpha 2-M-gold-containing structures positive for cytochemically detectable acid phosphatase. Our data indicate that ligands such as transferrin that are internalized through coated pits and receptosomes, but not delivered to lysosomes, do not traverse a lysosomal organelle compartment as marked by LAMP-1 content. Ligands such as alpha 2-M that are destined for lysosomal delivery reach a LAMP-1-positive organelle compartment only after they traverse LAMP-1-negative, non-lysosomal vesicles previously described as receptosomes.

Monoclonal antibody 101 produced by a hybridoma obtained by fusion with NS-1 myeloma cells of spleen cells from a mouse immunized with the human epidermoid carcinoma cell line, A431, specifically precipitates epidermal growth factor receptor, a glycoprotein of 170,000 Mr solubilized from A431 cell membranes (Richert, N. D., Willingham, M. C., and Pastan, I. H. (1983) J. Biol. Chem. 258, 8902-8907). The antibody also binds to neutral glycolipids extracted from A431 cells as evidenced by solid phase radioimmunoassay and by autoradiography. Binding of antibody to its target is inhibited by lacto-N-fucopentaose I but not by 2'-fucosyllactose or related oligosaccharides. Thus, antibody 101 is probably directed against the human blood group H type 1 sugar sequence Fuc alpha 1-2Gal beta 1-3GlcNAc . . .. This sequence presumably occurs on the epidermal growth factor receptor. Monoclonal antibody 102 produced by another hybridoma from the same fusion has the same cell specificity as antibody 101 and also binds to neutral glycolipids. However, binding of antibody 102 to its target is inhibited by 2'-fucosyllactose and not by lacto-N-fucopentaose I or related oligosaccharides. Thus, antibody 102 is probably directed against the human blood group H type 2 sugar sequence Fuc alpha 1-2Gal beta 1-4GlcNAc . . .. Antibody 102 does not precipitate solubilized epidermal growth factor receptor. Both antibodies bind to neutral glycolipids extracted from human erythrocytes belonging to blood group O but not to neutral glycolipids extracted from human erythrocytes with the "Bombay" phenotype.

A full-length cDNA for the human multidrug resistance gene 1 (MDR1) has been inserted into a retroviral vector containing a murine Harvey sarcoma virus from which the viral oncogene was deleted. Ecotropic and amphotropic virus was produced after transfection of this vector into psi-2 and PA-12 packaging cell lines. This virus conferred the full phenotype of multidrug resistance on mouse and human cell lines. Viral titers of up to 2 X 10(5) drug-resistant colonies per ml were observed. Infected cells became resistant to colchicine, vinblastine, doxorubicin, VP16 (etoposide), and puromycin, but not cisplatin, indicating that the presence of the human MDR1 gene is sufficient to cause multidrug resistance. When the dog kidney cell line MDCK was infected with the MDR1 virus, P-glycoprotein was expressed in a polarized manner on the upper surface of the cells, showing that the cloned cDNA also encodes information for polarized expression of P-glycoprotein. The MDR1 virus should be useful for introducing this drug resistance gene into a variety of cell types for biological experiments in vitro and in vivo.

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.

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.

The binding and internalization of a model lysosomal enzyme, beta-galactosidase, was visualized by use of rabbit anti-beta-galactosidase and goat anti-rabbit IgG; the second antibody was labeled with rhodamine or fluorescein (for detection by fluorescence) or with horseradish peroxidase (for electron microscopy). Chinese hamster ovary cells were incubated with beta-galactosidase at 4 degrees C, and then were washed and sequentially incubated in the cold with the two antibodies. The beta-galactosidase was found primarily in coated pits. The binding of the enzyme was completely inhibited by 5 mM mannose 6-phosphate. After the reaction with enzyme and antibodies, the cells were warmed to 37 degrees C; within 1 minute, the beta-galactosidase--antibody complex had begun to move to uncoated vesicles (receptosomes). After 8 min, the beta-galactosidase--antibody complex was seen in receptosomes near tubular elements in the Golgi/GERL area, within such tubular elements and at times, in vesicular elements that may correspond to coated structures of the GERL system. After 15 min, the enzyme--antibody complex was found in lysosomes near the Golgi/GERL are and a half-hour later it was in lysosomes distributed throughout the cytoplasm. Double-label experiments using beta-galactosidase and gold/alpha 2-macroglobulin showed the presence of the two ligands in the same coated pits and receptosomes. Thus, the pathway for internalization of beta-galactosidase via the mannose 6-phosphate receptor is similar to the pathway established for other ligands such as low density lipoprotein and alpha 2-macroglobulin.

A small number of Escherichia coli-derived human interferon alpha A molecules (up to approximately 800/cell) bind specifically to high-affinity cell surface receptors on bovine kidney cells at 4 degrees. When the cells are subsequently warmed to 37 degrees, the amount of surface-bound interferon alpha (IFN-alpha) as recognized by a radiolabeled monoclonal antibody rapidly decreases. Using 125I-IFN-alpha and treatment of cells with acetic acid/sodium chloride to remove surface-bound IFN, a similar decrease of surface-bound IFN and a corresponding increase in intracellular radiolabeled material is observed following the temperature shift. An analysis of the trichloroacetic acid precipitability of the radiolabeled material in the medium, removed by acid treatment or internalized, shows that IFN is degraded intracellularly and that its degradation products are then released into the medium. The process of uptake, degradation, and release of degraded material can be inhibited by the lysosomotropic agent chloroquine. A stable conjugate of IFN with 5-nm colloidal gold was prepared without a detectable loss of antiviral activity. As shown by transmission electron microscopy, the conjugate, bound to cells at 4 degrees, was found in clathrin-coated pits and later in receptosomes following a temperature shift to 37 degrees. Morphometric quantitation showed that an excess of native IFN added during binding of the conjugate in the cold reduced the appearance of conjugate in receptosomes by 80%. These studies demonstrate that at least a portion of receptor-bound IFN enters cells by receptor-mediated endocytosis.

We are currently investigating the morphologic aspects of two areas of the basic cell biology of cancer: tumor-specific surface antigens as targets for immunotoxins, and the phenomenon of multidrug resistance in chemotherapy of human tumors. Colloidal gold cytochemistry has provided a useful method for the electron-microscopic cytochemical detection of materials endocytosed by cells in culture. This technique has been used to study the internalization pathway of ligands bound to the surface of cancer cells, particularly antibodies for use as immunologic targeting reagents for the construction of immunotoxins. These colloidal gold conjugates with monoclonal antibodies have demonstrated the internalization of these immunologic reagents through coated pits and receptosomes, which is a necessary step in the delivery of immunotoxins into the cell where they can mediate their cell-killing functions. Morphologic methods have been employed for the screening and selection of monoclonal antibodies reactive with the surface of human ovarian cancer cells for use as immunotoxins and have demonstrated the in vivo activity of immunotoxins made with these antibodies and Pseudomonas exotoxin in a nude mouse model system. In other studies, we have employed such reagents for the immunocytochemical detection of the surface expression of P170, the cell-surface efflux pump protein responsible for the phenotype of multidrug resistance in tumor cells, and to investigate the distribution of this protein by using immunocytochemistry in normal human tissues. These results have suggested a role for P170 in normal cell membrane transport of metabolites in various organ systems.

Antibodies to barin tubulin were created in rabbits and used to localize tubulin in cultured fibroblasts. Cells were fixed and permeabilized using the EGS procedure (Willingham et al: J Cell Biol 79:256a, 1978). Tubulin was localized and quantified using a ferritin-bridge labeling method. Antibody localization was generally confined to morphologically identifiable microtubules, with a smaller concentration in a diffuse cytoplasmic distribution. Following treatment with colchicine, morphologically identifiable microtubules disappeared and the amount of diffuse localization in the cytosol increased. However, a significant concentration of tubulin was found associated with the microfilament mat under the plasma membrane after colchicine treatment, whereas in untreated cells, very little tubulin was found in the microfilament mat. These results demonstrate that tubulin is a component of microtubules and that colchicine treatment results in an association of this tubulin with microfilaments.

Double-label fluorescence microscopy was used to demonstrate the efflux activity of the multidrug transporter in single cultured cells. NIH3T3 cells expressing a transfected MDR1 gene (NIH3T3-MDR) were treated with vinblastine or daunomycin. The accumulation of vinblastine was monitored by examining the morphology of tubulin in cells, using immunofluorescence. Overnight treatment of drug-sensitive cells caused disassembly of microtubules and formation of paracrystals; the absence of vinblastine effects was evident by the presence of intact microtubules. Daunomycin accumulation was detected in nuclei using the inherent fluorescence of the drug with rhodamine epifluorescence microscopy. Drug efflux in multidrug-resistant cells was inhibited with verapamil. When multidrug-resistant cells were treated overnight in vinblastine, an effect of 0.5 microM vinblastine on microtubules was seen only in the presence of verapamil. Similarly, when cells were treated with daunomycin, this drug accumulated in nuclei only when verapamil was present. When cells incubated with vinblastine and verapamil were washed free of drugs, they did not accumulate daunomycin in a subsequent incubation, indicating that the multidrug transporter was still active; this occurred even though the morphologic effects of vinblastine persisted. Cells incubated with vinblastine alone showed an immediate inhibition of efflux activity when verapamil was subsequently added with daunomycin. These results show that the efflux activity of the multidrug transporter can be rapidly manipulated by agents such as verapamil, despite a prior history of drug treatment, and that the effects of inhibition of the transporter are rapidly reversible.

An immunotoxin composed of an antibody to the human transferrin receptor (454A12) and ricin A chain (RTA) was shown to inhibit the growth of NIH:OVCAR-3 tumors in a nude mouse model of human ovarian cancer. Inhibition of tumor growth by 454A12-RTA was related to the dose administered. The antitumor activity of the immunotoxin was blocked by coinjection of excess antibody with immunotoxin. An immunotoxin made using 454A12 and recombinant ricin A chain (rRTA) had an activity similar to that made with native RTA. The administration of 10 micrograms or greater of the immunotoxin 454A12-RTA/rRTA had significant antitumor activity. The injection of 30 micrograms of an irrelevant immunotoxin, MOPC21-RTA, or 30 to 500 micrograms of the 454A12 antibody had no antitumor activity.

Although phorbol esters can enhance formation of an active, catalytic domain of protein kinase C (PKC) in intact cells, little is known about the actual importance of the proteolytic pathway in mediating cellular responses to the phorbol esters or other PKC activators. To explore this issue, we examined the effect of microinjected catalytic fragment of PKC on Swiss 3T3 cell morphology. In contrast to the dramatic, rapid response upon phorbol ester treatment, catalytic fragment microinjected in the presence of bovine serum albumin or normal goat immunoglobulin G as carrier protein had no effect. A morphological response similar but not identical to the effect of phorbol ester treatment was obtained, however, if catalytic fragment was microinjected in the presence of normal rabbit immunoglobulin G rather than the usual carrier proteins. The normal rabbit immunoglobulin by itself was inactive. Although the mechanism remains undefined, normal rabbit immunoglobulin but not other carrier proteins modulated PKC activity in vitro. We conclude that the generation of free catalytic fragment of PKC cannot account for the morphological response of Swiss 3T3 cells to the phorbol esters; secondary factors may, however, potentiate its action.

Concentrative receptor-mediated endocytosis of many specific ligands by cultured fibroblasts occurs through the coated pit-receptosome pathway. The formation of receptosomes was studied using two impermeant electron-dense labels for the cell surface, ruthenium red and concanavalin A-horseradish peroxidase. These studies show that at 4 degrees C, virtually all coated structures near the plasma membrane are in communication with the cell surface, and are not isolated coated vesicles. On warming cells to 37 degrees C for only 1 minute, a major portion of these structures become cryptic, that is, not labeled by these surface markers. However, on cooling cells immediately back to 4 degrees C, virtually all of these structures are again in communication with the surface. Many images showed that membrane of these cryptic pits to be continuous with the cell surface when caught in the appropriate plane of section; often there was a very narrow entrance that excluded extracellular label. At 37 degrees C, receptosomes could be occasionally seen forming as an invagination of membrane adjacent to the coated region. Mechanisms by which receptosomes may form and other evidence demonstrating the failure of coated pits to pinch off to form isolated coated vesicles during endocytosis are discussed.

LysPE40 is a modified form of Pseudomonas exotoxin that lacks the cell-binding domain and has a chemically reactive lysine residue near the amino terminus. LysPE40 is made in Escherichia coli and secreted into the medium from which it is readily purified. Two immunotoxins were constructed by coupling LysPE40 to an antibody to the human transferrin receptor (TFR) or to an antibody to the human interleukin-2 receptor. These immunotoxins were selectively cytotoxic to receptor-bearing cells in tissue culture. Anti-TFR-LysPE40 given intraperitoneally to mice appeared rapidly in the blood and caused regression of A431 tumors growing as subcutaneous xenografts. These results show that it is possible to cause regression of a solid carcinoma by an immunotoxin if proper targeting can be achieved.

A mutant line of 3T3 (mouse fibroblast) cells with temperature-sensitive cyclic AMP (cAMP) metabolism was isolated by selecting for low substratum adhesiveness after a change in temperature. Although the mutant is identical in behavior to the parent cell at constant temperature, a rise or fall in temperature causes a fall in intracellular cAMP levels within seconds, followed by loss of adherence to the culture dish and retraction of cell processes. The mechanism of this fall in cAMP level is at least in part excretion into the medium. The decrease in adhesiveness and retraction of processes can be blocked by analogues of cAMP or agents that elevate intracellular cAMP. The properties of this mutant imply that cAMP is a direct regulator of cell shape and adhesiveness.