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.
The preservation and contrast of membranous structures in cultured cells using various postfixation procedures prior to embedding have been investigated. These include routine 0504, ferrocyanide-reduced 0504, osmiumthiocarbohydrazide-osmium (OTO), and ferrocyanide-reduced osmium-thiocarbohydrazide-ferrocyanide-reduced osmium (R-OTO). With standard ethanol-Epon dehydration/embedding techniques, a dramatic improvement in both membrane contrast and preservation of bilayer membrane structure was achieved using preembedding OTO in cultured cells. R-OTO yielded similar enhanced preservation and contrast of membranes. Both of these methods also resulted in an increase in the contrast of
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.
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