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Biomedical subjects

I Pastan

Publications and source records attributed to I Pastan.

At least 541 records · Page 30Linked to original sources
PubMed1986

Adenylate cyclase activity of NIH 3T3 cells morphologically transformed by ras genes.

The observed homology between G-proteins which regulate adenylate cyclase and ras proteins and the suggested role of ras in the regulation of adenylate cyclase in yeast prompted us to examine the regulation of adenylate cyclase in three cell lines: (i) NIH 3T3 cells, (ii) NIH 3T3 cells transformed by high levels of the normal rasH gene product and (iii) NIH 3T3 cells transformed by a mutated rasH gene product. We found that the regulation of adenylate cyclase by G-proteins is identical in the three cell lines, although the response of the transformed NIH 3T3 cells to agonists is strongly attenuated. Our data suggest that mammalian ras products do not interact directly with adenylate cyclase, although their increased expression may indirectly inhibit the interaction of adenylate cyclase stimulatory receptors with G-proteins.

Adenylyl Cyclases↗
PubMed1986

Functional studies on the EGF receptor with an antibody that recognizes the intracellular portion of the receptor.

An antibody against the human epidermal growth factor receptor (EGF), capable of activating its tyrosine kinase has been produced. Antibody 2913 recognizes only the cytoplasmic portion of the EGF receptor in A431 carcinoma cells, in normal human fibroblasts, and in a variety of other human tumor cell lines (Xu, Y.-A., Richert, N., Ito, S., Merlino, G. T., and Pastan, I. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7308-7313). Indirect immunofluorescence and electron microscopy show that the antibody binds to intact cells only after membrane permeabilization. Moreover the antibody immunoprecipitates the v-erb-B gene product in avian myeloblastosis virus-infected cells but does not recognize the secreted form (105 kDa) of the A431 cell EGF receptor which lacks the cytoplasmic domain. Antibody 2913 activates the EGF receptor kinase in solubilized A431 membranes causing autophosphorylation on tyrosine residues only. Tryptic peptide maps suggest that antibody 2913 and EGF stimulate phosphorylation of the same amino acid residues. By electron microscopy, the cytoplasmic portion of the receptor was followed throughout its endocytotic pathway. The results show that the kinase domain is rapidly degraded in lysosomes with no accumulation in the cytoplasm or in the nucleus.

Alpharetrovirus↗
PubMed1986

The role of intracellular pH in ligand internalization.

Internalization of EGF and transferrin measured as the rate of uptake of 125I-labeled ligands was compared in the cell line CCL39 and a mutant derivative, PS-120, lacking the Na+/H+ antiport system. No significant alteration was detected between the two cell lines. In contrast, pretreatment of the mutant cells PS-120 with 20 mM NH4Cl for 30 min to decrease persistently intracellular pH resulted in an increase in 125I-EGF and 125I-transferrin uptake by 60% and 25%, respectively. However, similar NH4Cl pretreatment of the parental cell line, CCL-39, which only affected intracellular pH very transiently did not cause an increase of ligand uptake. The binding of 125I-EGF to CCL-39 and PS-120 cells with or without NH4Cl pretreatment showed that NH4Cl pretreatment did not affect EGF binding in either CCL-39 or PS-120 cells. Since cells regulate intracellular pH by ion transport systems, we also examined the role of Na+, K+-ATPase. Ouabain, an inhibitor of Na+, K+-ATPases, showed no effect on 125I-EGF uptake in either of the cell types with or without NH4Cl pretreatment. Taken together, these results suggest that the plasma membrane-bound Na+/H+ antiport, a major pHi-regulating system in vertebrates, indirectly plays a role in ligand internalization through regulation of intracellular pH.

Ammonium Chloride↗
PubMed1986

Localization of multidrug resistance-associated DNA sequences to human chromosome 7.

Multidrug resistance in several human cell lines correlates with amplification or increased expression of two related DNA sequences, designated mdr1 and mdr2. These DNA sequences were used as probes for hybridization with DNA with a panel of human-mouse somatic cell hybrids and from individual human chromosomes separated by fluorescence-activated chromosome sorting. By these assays, both mdr1 and mdr2 sequences were localized to chromosome 7.

Animals↗
PubMed1986

Membrane vesicles from multidrug-resistant human cancer cells contain a specific 150- to 170-kDa protein detected by photoaffinity labeling.

Multiple drug resistance of tumor cells is a common problem in cancer therapy. We have demonstrated that membrane vesicles from highly multidrug-resistant human KB carcinoma cell lines exhibit increased specific and saturable binding of vinblastine. To identify the molecules that bind vinblastine, membrane vesicles from multidrug-resistant cells were exposed to two analogs of vinblastine, N-(p-azido-[3,5-3H]benzoyl)-N'-(beta-aminoethyl)vindesine and N-(p-azido-[3-125I]salicyl)-N'-(beta-aminoethyl)vindesine, that could be photoactivated. Our studies show the specific labeling of a 150- to 170-kDa protein in membrane vesicles from two independently selected multidrug-resistant KB cell lines, which was not seen in drug-sensitive parental or revertant cell lines. The labeling of the high molecular weight protein was inhibited in a dose-dependent manner by vinblastine with half-maximal inhibition at about 1 microM. Photolabeling was also inhibited by 100 microM vincristine or 100 microM verapamil but not by 100 microM colchicine or 100 microM dexamethasone. The data suggest that the 150- to 170-kDa protein may play an important role in the multidrug-resistance phenotype.

Affinity Labels↗
PubMed1986

Isolation of human mdr DNA sequences amplified in multidrug-resistant KB carcinoma cells.

The ability of tumor cells to develop simultaneous resistance to structurally different cytotoxic drugs constitutes a major problem in cancer chemotherapy. It was previously demonstrated that multidrug-resistant Chinese hamster cell lines contain an amplified, transcriptionally active DNA sequence designated mdr. This report presents evidence that multidrug-resistant sublines of human KB carcinoma cells, selected for resistance to either colchicine, vinblastine, or Adriamycin (doxorubicin), display amplification of two different DNA sequences homologous to the hamster mdr gene. Segments of the human mdr DNA sequences, designated mdr1 and mdr2, have been cloned. mdr1 sequences were amplified in all of the highly drug-resistant sublines and were expressed as a poly(A)+ RNA species of 4.5 kilobases that was detected in the resistant cells but not in the parental cell line. No expression of mdr2 sequences was detected. mdr2 sequences were coamplified with mdr1 in some of the multidrug-resistant sublines and, in two independently derived cell lines, underwent very similar rearrangements. The data suggest that the mdr1 gene is involved in multidrug resistance in human cells.

Carcinoma↗
PubMed1986

Antitumor effects of an immunotoxin made with Pseudomonas exotoxin in a nude mouse model of human ovarian cancer.

An immunotoxin composed of Pseudomonas toxin coupled to an antibody to the human transferrin receptor was evaluated for its effect on ovarian cancer. In the tumor model employed, 60 million human ovarian cancer cells were injected into the peritoneal cavity of an immunodeficient nude mouse. By day 5, cancer cells were implanted and growing in small clusters throughout the peritoneal cavity. On days 5-8, 0.3-2 micrograms of immunotoxin was injected into the peritoneal cavity. Control mice died with malignant ascites at 34-58 days after the implantation of tumor cells, whereas immunotoxin-treated mice lived to 100 days or longer. Irrelevant immunotoxins or antibody alone had no antitumor activity. These findings suggest that intraperitoneal injection of immunotoxins may have a role in the treatment of ovarian cancer.

ADP Ribose Transferases↗
PubMed1986

Purification and characterization of a membrane-associated 3,3',5-triiodo-L-thyronine binding protein from a human carcinoma cell line.

A membrane-associated binding protein for 3,3',5-triiodo-L-thyronine (T3) was purified to apparent homogeneity from A431 human epidermoid carcinoma cells. A431 cells were specifically labeled with the N-bromoacetyl derivative of T3 labeled with 125I at the 3' position (BrAc[125I]T3) and were extracted with 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent. The solubilized BrAc[125I]T3-labeled protein was successively purified by chromatography on Sephadex G-200 and QAE-Sephadex followed by NaDod-SO4/PAGE. Approximately 0.2 mg of purified protein was obtained from 2.5 X 10(9) cells, which represents a 3000-fold purification. The membrane-associated T3 binding protein is an acidic protein with a pI of 5.1 and an apparent molecular mass of 55,000 daltons determined by NaDodSO4/PAGE. Polyclonal antibodies against the 55-kDa protein were prepared and used in indirect immunofluorescence to show that the 55-kDa protein was mainly found in the nuclear envelope and endoplasmic reticulum.

Carcinoma, Squamous Cell↗
PubMed1986

Multidrug resistance of DNA-mediated transformants is linked to transfer of the human mdr1 gene.

Mouse NIH 3T3 cells were transformed to multidrug resistance with high-molecular-weight DNA from multidrug-resistant human KB carcinoma cells. The patterns of cross resistance to colchicine, vinblastine, and doxorubicin hydrochloride (Adriamycin; Adria Laboratories Inc.) of the human donor cell line and mouse recipients were similar. The multidrug-resistant human donor cell line contains amplified sequences of the mdr1 gene which are expressed at high levels. Both primary and secondary NIH 3T3 transformants contained and expressed these amplified human mdr1 sequences. Amplification and expression of the human mdr1 sequences and amplification of cotransferred human Alu sequences in the mouse cells correlated with the degree of multidrug resistance. These data suggest that the mdr1 gene is likely to be responsible for multidrug resistance in cultured cells.

Animals↗
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

High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma cell lines.

Southern blot-hybridization analysis of DNAs from human tumors demonstrated amplification of the epidermal growth factor (EGF) receptor gene in 10 of 12 squamous cell carcinoma cell lines tested and in none of 18 tumor cell lines of nonsquamous cell carcinomas. The degree of amplification in the squamous cells varied from 2- to 50-fold relative to the epidermal keratinocyte. Hybridization analysis of the RNA showed that the amplification of the EGF receptor gene is accompanied with an increase of the 5.6 kilobases of EGF receptor mRNA. Scatchard plot analysis and sodium dodecyl sulfate-polyacrylamide gel analysis of the EGF receptor revealed that the synthesis of the EGF receptor is also greater in the cells with amplified EGF receptor gene. In contrast, Southern blot analysis of DNAs of primary tumors showed that incidence of amplification of the EGF receptor gene in squamous cells (1 of 6) was almost as frequent as in nonsquamous cells (1 of 4). These results show that amplification of the EGF receptor gene is commonly found in various tumors. In addition, our data suggest that primary squamous cell carcinomas with amplified EGF receptor gene may readily adapt to growth in tissue culture.

Carcinoma, Squamous Cell↗
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

Solubilization and characterization of a membrane 3, 3', 5-triiodo-L-thyronine binding protein from rat pituitary tumor GH3 cells.

To understand the mechanism by which T3 enters cells and carries out its biological functions membrane binding sites for 3, 3', 5-triiodo-L-thyronine were solubilized from rat pituitary tumor GH3 cells by detergents. Among three detergents tested, CHAPS is the best in preserving hormonal binding affinity and specificity. Least square analysis of the binding data show one class of binding site with a Kd of (6.35 +/- 1.27) nM and Bmax of (0.84 +/- 0.056) pmoles/50 micrograms protein. Hormone binding activity is lost by heating, pronase digestion and in the absence of NaCl. The pH optimum for binding is 7.0 and the binding activity is enhanced by dithiothreitol. The solubilization of membrane-associated thyroid hormone binding proteins will facilitate further characterization and exploration of their biological functions.

Animals↗
PubMed1985

Promoter region of the human Harvey ras proto-oncogene: similarity to the EGF receptor proto-oncogene promoter.

Regulation of transcription of members of the ras gene family undoubtably plays an important role in controlling cellular growth. Examination of this level of regulation requires identification of the promoter regions of the ras proto-oncogenes. Four major transcriptional start sites were detected in the human Harvey ras 1 proto-oncogene. The promoter region contains neither a TATA box nor a CAAT box in their characteristic upstream positions, has an extremely high G+C content (80 percent), and contains multiple GC boxes including seven CCGCCC repeats and three repeats of the inverted complement, GGGCGG. This region has strong promoter activity when placed upstream from the chloramphenicol acetyl transferase gene and transfected into monkey CV1 cells. In these ways the Harvey ras 1 proto-oncogene promoter resembles the promoter of the gene encoding the epidermal growth factor (EGF) receptor. The similarity between the two proto-oncogene promoters may be relevant to the mechanism by which the expression of such "growth control" genes is regulated.

DNA Restriction Enzymes↗
PubMed1985

Modulation of fibronectin gene activity in chick embryo fibroblasts transformed by a temperature-sensitive strain (ts68) of Rous sarcoma virus.

Transcriptional regulation of the fibronectin gene is a major mechanism for lowering steady-state levels of fibronectin mRNA in chick embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSV) (1). In the present study, we have measured the change of transcriptional activity of the fibronectin gene in CEF transformed by a temperature-sensitive strain of RSV (ts68). Ts68-CEF maintained at either 35 degrees C or 41 degrees C were shifted to 41 degrees C or 35 degrees C, respectively, at 5-hour intervals, and isolated nuclei were used in runoff transcription assays. Nuclear RNA labeled with [alpha-32P]UTP was hybridized to DNA fragments encoding the src gene, the beta-actin gene and the fibronectin gene. In shift-up (35 degrees C----41 degrees C) and shift-down (41 degrees C----35 degrees C) experiments, src gene and beta-actin gene activities in ts68-CEF nuclei remained relatively unchanged. In ts68-CEF shifted to the nonpermissive temperature (41 degrees C), a lag time of at least 5 hours was followed by a 4- to 5-fold increase in fibronectin specific RNA 15 hours after the shift. When cells were shifted to the permissive temperature (35 degrees C), a 4- to 5-fold decrease in fibronectin RNA was apparent within 5 hours of the temperature shift and a 17- to 18-fold decrease was observed 15 hours after the shift. The relatively slow rates of changes of fibronectin gene activity in shift-up experiments suggest that the effect of p60src on fibronectin gene activity is indirect.

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↗