Biomedical subjects

I Pastan

Publications and source records attributed to I Pastan.

At least 523 records · Page 29Linked to original sources

PubMed1987

Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues.

Monoclonal antibody MRK16 was used to determine the location of P-glycoprotein, the product of the multidrug-resistance gene (MDR1), in normal human tissues. The protein was found to be concentrated in a small number of specific sites. Most tissues examined revealed very little P-glycoprotein. However, certain cell types in liver, pancreas, kidney, colon, and jejunum showed specific localization of P-glycoprotein. In liver, P-glycoprotein was found exclusively on the biliary canalicular front of hepatocytes and on the apical surface of epithelial cells in small biliary ductules. In pancreas, P-glycoprotein was found on the apical surface of the epithelial cells of small ductules but not larger pancreatic ducts. In kidney, P-glycoprotein was found concentrated on the apical surface of epithelial cells of the proximal tubules. Colon and jejunum both showed high levels of P-glycoprotein on the apical surfaces of superficial columnar epithelial cells. Adrenal gland showed high levels of P-glycoprotein diffusely distributed on the surface of cells in both the cortex and medulla. These results suggest that the protein has a role in the normal secretion of metabolites and certain anti-cancer drugs into bile, urine, and directly into the lumen of the gastrointestinal tract.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1987

Pseudomonas exotoxin coupled to a monoclonal antibody against ovarian cancer inhibits the growth of human ovarian cancer cells in a mouse model.

In an effort to devise an alternative treatment for human ovarian cancer, we have isolated a monoclonal antibody (OVB-3) that reacts with all ovarian cancers tested (10/10) but few normal tissues. An immunotoxin produced by coupling OVB-3 to Pseudomonas exotoxin kills ovarian cancer cells in tissue culture and prolongs the life of animals bearing human ovarian cancers. These data suggest that this immunotoxin should be evaluated as a treatment for ovarian cancer in women.

ADP Ribose Transferases↗

PubMed1987

Expression of a full-length cDNA for the human "MDR1" gene confers resistance to colchicine, doxorubicin, and vinblastine.

Intrinsic and acquired multidrug resistance (MDR) is an important problem in cancer therapy. MDR in human KB carcinoma cells selected for resistance to colchicine, vinblastine, or doxorubicin (former generic name adriamycin) is associated with overexpression of the "MDR1" gene, which encodes P-glycoprotein. We previously have isolated an overlapping set of cDNA clones for the human MDR1 gene from multidrug-resistant KB cells. Here we report the construction of a full-length cDNA for the human MDR1 gene and show that this reconstructed cDNA, when inserted into a retroviral expression vector containing the long terminal repeats of Moloney leukemia virus or Harvey sarcoma virus, functions in mouse NIH 3T3 and human KB cells to confer the complete multidrug-resistance phenotype. These results suggest that the human MDR1 gene may be used as a positive selectable marker to introduce genes into human cells and to transform human cells to multidrug resistance without introducing nonhuman antigens.

Colchicine↗

PubMed1987

ATP-binding properties of P glycoprotein from multidrug-resistant KB cells.

The photoaffinity reagent 8-azido-alpha-[32P]ATP was used to label a protein of 170 kDa in membrane vesicle preparations from a highly multidrug-resistant cell line, KB-V1, but not from the drug-sensitive parental cell line KB-3-1. The 170-kDa labeled protein was immunoprecipitated with a monoclonal antibody (MRK-16) to P glycoprotein. Both ATP and GTP inhibited labeling by 8-azido-alpha-[32P]ATP. Labeling of P170 was not inhibited by 5 mM ADP, 5 mM ribose-5-phosphate, or 100 microM vinblastine. These data directly demonstrate that P glycoprotein has a nucleotide-binding site that could supply energy for drug transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1987

Adenovirus-dependent changes in cell membrane permeability: role of Na+, K+-ATPase.

Adenovirus-dependent release of choline phosphate from KB cells at pH 6.0 was partially blocked by ouabain. In K+-containing medium, maximum inhibition of release was obtained by 10(-5) M ouabain and half-maximal inhibition was achieved by about 0.5 X 10(-6)M ouabain. Ouabain did not block either the binding or the uptake of adenovirus by KB cells. Without K+, about 25% of cell-associated choline phosphate was released by adenovirus, whereas with 1 mM K+ about 50% was released. This activation by K+ was blocked by 0.1 mM ouabain. HeLa cells behaved like KB cells, but a mutant of HeLa cells resistant to ouabain (D98-OR) released much lower amounts of choline phosphate in response to human adenovirus type 2 (Ad2). Wild-type D98-OR cells bound nearly the same amount of adenovirus as did normal HeLa cells. Ad2 also increased the activity of Na+,K+-ATPase in KB cells, with maximum activation at 50 micrograms of Ad2 per ml. In D98-OR cells, Ad2 failed to activate Na+,K+-ATPase activity. Ad2-dependent lysis of endocytic vesicles (receptosomes) was assayed by measuring Ad2-dependent enhancement of epidermal growth factor-Pseudomonas exotoxin toxicity. This action of adenovirus was increased when K+ was present in the medium. Under the conditions used, K+ had no effect on the amount of Ad2 or epidermal growth factor taken up by the cells. On the basis of these results, it is suggested that Ad2-dependent cellular efflux of choline phosphate and adenovirus-dependent lysis of receptosomes may require Na+,K+-ATPase activity.

Adenoviruses, Human↗

PubMed1987

Comparison of the intracellular pathways of transferrin recycling and vesicular stomatitis virus membrane glycoprotein exocytosis by ultrastructural double-label cytochemistry.

Transferrin is taken up by receptor-mediated endocytosis into intracellular vesicles and tubules, and then recycles rapidly to the plasma membrane (diacytosis). We applied double-label cytochemistry to study whether the recycling structures containing transferrin fuse with the intracellular membranous structures that deliver newly synthesized membrane glycoproteins from the ER to the plasma membrane (exocytosis) or whether they remain independent. KB and Vero cells were infected with the temperature-sensitive transport mutant 0-45 of vesicular stomatitis virus (VSV). Temperature-regulated exocytosis of membrane glycoprotein "G" occurred simultaneously with diacytosis of transferrin. The exocytic "G" protein, as detected by immunoperoxidase electron microscopy, passed through the cisternal Golgi stacks and vacuolar, tubular, vesicular, and pit-like structures of the Golgi system. A transferrin-ferritin conjugate used in ultrastructural double-label experiments was detected in diacytic vesicles and tubules that accumulated in the proximal (trans-reticular) Golgi area of the cell. The ferritin-labeled vesicles/tubules were often close to and intermixed with the VSV-"G" containing membranous structures, but in most cases at early times (15-20 min) the transferrin and VSV-"G" containing vesicular structures remained distinct. At later times (30-45 min), the two labels were occasionally found in the same structures. These results indicate that rapid recycling of endocytosed materials and exocytosis of membrane glycoproteins to the cell surface usually occur in distinct vesicles, possibly along the same general morphologic exit pathway.

Exocytosis↗

PubMed1987

Intrinsic drug resistance in human kidney cancer is associated with expression of a human multidrug-resistance gene.

The cloning of the cDNA for the mdr1 gene, whose expression is associated with the development of multidrug-resistance in cultured cells, has made it possible to explore the mechanism of multidrug resistance in human tumors. We have found that normal human kidney, six of eight adenocarcinomas of the kidney, and four cell lines derived from kidney adenocarcinomas express high levels of mdr1 mRNA. Two criteria suggest that primary multidrug resistance in human adenocarcinomas of the kidney results, at least in part, from expression of the mdr1 gene: (1) mdr1 mRNA levels are elevated in four unselected kidney adenocarcinoma cell lines that show a multidrug-resistant phenotype; and (2) multidrug resistance in these kidney cancer cell lines is reversed by verapamil and quinidine, agents known to reverse mdr1-associated drug resistance in cell lines selected for multidrug resistance in vitro. These results suggest that appropriate pharmacological intervention to reverse multidrug resistance might make adenocarcinomas of the kidney more sensitive to chemotherapy with agents such as Adriamycin (Adria Laboratories, Columbus, OH) and the vinca alkaloids.

Cell Line↗

PubMed1986

The mdr1 gene, responsible for multidrug-resistance, codes for P-glycoprotein.

The development of simultaneous resistance to multiple drugs in cultured cells occurs after selection for resistance to single agents. This multidrug-resistance phenotype is thought to mimic multidrug-resistance in human tumors treated with chemotherapy. Both the expression of a membrane protein, termed P170 or P-glycoprotein, and the expression of a cloned DNA fragment, termed mdr1, have been shown independently to be associated with multidrug-resistance in cultured cells. In this work, we show that human KB carcinoma cells which express the mdr1 gene also express P-glycoprotein, and that cDNAs encoding P-glycoprotein cross-hybridize with mdr1 cDNAs. Thus, the mdr1 gene codes for P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1986

Monoclonal antibodies against a glycoprotein localized in coated pits and endocytic vesicles inhibit alpha 2-macroglobulin binding and uptake.

Eight monoclonal antibodies, all IgG2a, which recognize a 180/90-kDa glycoprotein similar in properties to the receptor for alpha 2-macroglobulin of mouse embryo 3T3 cell plasma membranes, have been tested for their effect on the binding and uptake of alpha 2-macroglobulin by live cells. One antibody directly inhibited binding of 125I-alpha 2-macroglobulin under conditions in which 125I-transferrin binding to the transferrin receptor was unaffected. Another monoclonal antibody decreased alpha 2-macroglobulin binding when preincubated with cells at 37 degrees C. This antibody was also capable of specifically binding to ligand-receptor complexes formed by preincubating 125I-alpha 2-macroglobulin with detergent extracts of Swiss 3T3 cells. Immunoelectron microscopy showed that the 180/90-kDa glycoprotein was localized in coated pits of the cell surface and in intracellular endocytic vesicles (receptosomes/endosomes). The data suggest that the 180/90-kDa glycoprotein is a component of the receptor for alpha 2-macroglobulin.

Animals↗

PubMed1986

Immunotoxins.

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Animals↗

PubMed1986

Characterization of a membrane-associated 3,3',5-triiodo-L-thyronine binding protein by use of monoclonal antibodies.

Four mouse hybridoma cell lines have been isolated which secrete antibodies to the membrane-associated thyroid hormone binding protein (Mr 55,000) from human epidermoid carcinoma A431 cells. J6 is rat specific; J2 is human and monkey specific; J8 and J9 have a wider specificity and react with similar thyroid hormone binding proteins (p55) from human, monkey, rat, and hamster. None of these antibodies reacts with mouse cells. J2, J6, and J9 are of the IgG1k class, and J8 is an IgAk antibody. p55 was characterized by using these monoclonal antibodies. It is not posttranslationally processed by glycosylation, phosphorylation, or sulfation. It has a cellular degradation rate t1/2 approximately equal to 3.2 h. Using immunofluorescence and electron microscopic immunocytochemistry, p55 was found to be associated with the lumenal face of the endoplasmic reticulum and nuclear envelope. When cell homogenates were prepared, significant amounts of p55 were released into the 110000g supernatant, indicating that p55 is loosely associated with the endoplasmic reticulum and nuclear envelope.

Animals↗

PubMed1986

Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells.

Resistance of tumor cells to multiple cytotoxic drugs is a major impediment to cancer chemotherapy. Multidrug resistance in human cells is determined by the mdr1 gene, encoding a high molecular weight membrane glycoprotein (P-glycoprotein). Complete primary structure of human P-glycoprotein has been determined from the cDNA sequence. The protein, 1280 amino acids long, consists of two homologous parts of approximately equal length. Each half of the protein includes a hydrophobic region with six predicted transmembrane segments and a hydrophilic region. The hydrophilic regions share homology with peripheral membrane components of bacterial active transport systems and include potential nucleotide-binding sites. These results are consistent with a function for P-glycoprotein as an energy-dependent efflux pump responsible for decreased drug accumulation in multidrug-resistant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1986

Reduction of EGF receptor synthesis by antisense RNA vectors.

An association has been observed between squamous cell carcinomas and elevated EGF receptor levels. The approach taken here to analyze this problem was to construct vectors which express antisense RNA for the EGF receptor. The plasmid vector used was derived from pSV2-neo, which is readily selectable. Three EGF receptor cDNA fragments, a 5'-, a middle and a 3'-region were tested for differences in efficiency as antisense RNA. The results suggest that the 5'-fragment was the most efficient.

Carcinoma, Squamous Cell↗

PubMed1986

Synthesis of epidermal growth factor (EGF) receptor in vitro using SP6 RNA polymerase-transcribed template mRNA.

The epidermal growth factor (EGF) receptor plays a key role in the control cellular proliferation, and its homology to the avian erythroblastosis virus erb B oncogene implicates its involvement in cellular transformation. The establishment of a correlation between the various structural domains of the EGF receptor and their functional counterparts would greatly advance our understanding of these processes. To this end, we have constructed an expression vector containing the SP6 viral promoter and an adjacent cDNA fragment encoding the full-length EGF receptor. Upon addition of SP6 RNA polymerase, this DNA is capable of generating large amounts of EGF receptor mRNA; this RNA can then be translated in vitro into immunoprecipitable EGF receptor protein. The translational efficiency of this EGF receptor RNA was found to be relatively low: approx. 100-fold lower than globin RNA synthesized using SP6 RNA polymerase. Use of these tools should now permit the synthesis and analysis of mutated EGF receptor protein in an effort to clarify the role of this receptor in growth control.

Cell-Free System↗

PubMed1986

Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins.

We have established four cell lines derived from the human KB carcinoma cell line which express high-level multiple drug resistance. One of these lines was selected for resistance to colchicine, one was selected for resistance to colchicine in the presence of the tumor promoter, mezerein, one for resistance to vinblastine, and one for resistance to adriamycin. All of these cell lines are cross-resistant to the other selective agents. The development of multidrug resistance in these cultured human carcinoma cells is associated with a limited number of specific protein alterations revealed by high resolution two-dimensional gel electrophoresis and Western blot analysis. These protein alterations in multidrug-resistant lines include the decreased prevalence of members of a family of proteins of molecular mass 70,000 to 80,000 daltons, pI 4.8-5.0, the increased synthesis of a protein of molecular mass 21,000 daltons, pI 5.0, in the colchicine-resistant cell lines only, and the increased expression of a 170,000-dalton protein in membrane preparations from all of the resistant cells. The loss of the 70,000- to 80,000-dalton proteins in the multidrug-resistant lines, which can also be demonstrated by immunoprecipitation of these proteins with specific antisera, is associated with a loss of translatable mRNA for these proteins. These studies suggest that only a limited number of protein changes occur in multidrug-resistant cell lines.

Animals↗

PubMed1986

Binding of the Sp1 transcription factor by the human Harvey ras1 proto-oncogene promoter.

Members of the ras gene family encode proteins that when overproduced or mutated can transform immortalized mammalian cells. It is therefore important to understand the mechanisms by which the ras genes are regulated. The promoter region of the human Harvey ras proto-oncogene c-Ha-ras1 initiates RNA transcription at multiple sites and contains repeated copies of the hexanucleotide GGGCGG and its inverted complement CCGCCC, referred to as GC boxes. These GC boxes consist of sequences identical to those found in the SV40 early promoter, where the human cellular transcriptional factor Sp1 binds. Footprinting analysis with deoxyribonuclease I was used to show that Sp1 binds to six GC box sequences within the c-Ha-ras1 promoter. An in vivo transfection assay showed competition between the 21-base pair repeats of the SV40 promoter and the c-Ha-ras1 promoter for common regulatory factors. In this system the presence of Sp1 is apparently required for c-Ha-ras1 transcription. Analysis of deletions of the c-Ha-ras1 promoter region by means of a transient expression assay revealed that the three Sp1 binding sites closest to the RNA start sites were sufficient for full transcriptional activity.

Base Sequence↗

PubMed1986

Human multidrug-resistant cell lines: increased mdr1 expression can precede gene amplification.

The development of simultaneous resistance to multiple structurally unrelated drugs is a major impediment to cancer chemotherapy. Multidrug resistance in human KB carcinoma cells selected in colchicine, vinblastine, or Adriamycin is associated with amplification of specific DNA sequences (the multidrug resistance locus, mdr1). During colchicine selection resistance is initially accompanied by elevated expression of a 4.5-kilobase mdr1 messenger RNA (mRNA) without amplification of the corresponding genomic sequences. During selection for increased levels of resistance, expression of this mRNA is increased simultaneously with amplification of mdr1 DNA. Increased expression and amplification of mdr1 sequences were also found in multidrug-resistant sublines of human leukemia and ovarian carcinoma cells. These results suggest that increased expression of mdr1 mRNA is a common mechanism for multidrug resistance in human cells. Activation of the mdr1 gene by mutations or epigenetic changes may precede its amplification during the development of resistance.

Animals↗

PubMed1986

pH-dependent lysis of liposomes by adenovirus.

Purified adenovirus induced a dose-dependent release of the water-soluble markers calcein and carboxyfluorescein from liposomes. Marker release was strongly dependent on pH, and at temperatures below 5 degrees C, the rate of release showed an optimum at a pH of about 6. This pH dependence parallels disruption of endocytic vesicles by adenovirus and the permeabilization that adenovirus induces on the cell surface. There did not seem to be a striking dependence on the lipid composition of the liposomes. Electron microscopy using a negative stain shows liposomes bound to adenovirus. In some cases, the liposomes were still intact, but many liposomes, which were attached to the vertices of the virus, appeared lysed. These data support the notion that adenovirus, which enters the host cell by receptor-mediated endocytosis, gains access to the cytoplasm by a subsequent pH-dependent disruption of the membrane of the endocytic vesicle.

Adenoviruses, Human↗