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Biomedical subjects

I Pastan

Publications and source records attributed to I Pastan.

At least 577 records · Page 32Linked to original sources
PubMed1985

Potentiation of cytotoxic activity of immunotoxins on cultured human cells.

The cytotoxic activity against human tumor cells of toxic conjugates of Pseudomonas exotoxin with anti-transferrin receptor antibody or epidermal growth factor was potentiated up to 10 to 20-fold by the calcium antagonists verapamil, D-600, and diltiazem and by the lysosomotropic agent beta-glycylphenyl-naphthylamide. The potentiating activity of these agents could be predicted by measuring the inhibition of protein synthesis by the immunotoxins on various cell lines. The use of potentiating agents such as these in combination with immunotoxins may prove useful in the treatment of some human cancers.

Antibodies, Neoplasm↗
PubMed1985

Hyperproduction and gene amplification of the epidermal growth factor receptor in squamous cell carcinomas.

Human squamous cell carcinoma tissue was screened to determine the epidermal growth factor (EGF) receptor level. In 9 out of 15 cases, increased EGF binding was observed relative to normal adjacent tissue. In two tissue samples (SCE1 and SCL1), amplification of the EGF receptor gene and increased mRNA level were also observed. In two other cases (SCE2 and SCL3), EGF receptor gene amplification did not occur. We propose that there is an alternative mechanism for EGF receptor hyperproduction, independent of gene content, active in these tissues. A possible role of EGF receptor hyperproduction is envisioned in human neoplasia.

Carcinoma, Squamous Cell↗
PubMed1985

Reduced drug accumulation in multiply drug-resistant human KB carcinoma cell lines.

Human KB cells with increasing resistance to colchicine and other chemotherapeutic agents have been isolated in four sequential steps. This report describes the characterization of drug uptake in the parent and four mutant cell lines. Drug uptake in these cell lines occurred via a nonsaturable process. In general, drug accumulation decreased with increasing drug resistance; this relationship was seen best with colchicine, vincristine, vinblastine, and daunomycin and, to a lesser extent, with actinomycin D. The accumulation of dexamethasone, an agent to which all lines were equally sensitive, was similar for the parent and the four mutants. Drug efflux occurred rapidly, and differences among the various cell lines could be detected within the first minute. In the more resistant lines, a greater percentage of the drug was released more rapidly, although the absolute amount of drug released was less. Verapamil partially reversed the multiple drug-resistance phenotype by increasing the initial rate of uptake and accumulation of drugs in the resistant cell lines without an apparent effect on drug efflux. The results suggest that, in this human epithelial cell, the development of resistance to multiple drugs is complex, with changes in drug uptake, accumulation, and efflux.

Antineoplastic Agents↗
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↗
PubMed1984

Antibodies against the plasma membrane 3,3',5-triiodo-L-thyronine binding protein of rat pituitary GH3 cells: partial characterization and cross-species immunoreactivity.

To develop antibodies against the plasma membrane 3,3',5-triiodo-L-thyronine (T3) binding protein (M.W. 55,000), rabbits were immunized with formalin-fixed GH3 cells or highly purified plasma membranes from these cells. Antibodies were screened by immunoprecipitation using detergent solubilized N-bromoacetyl-[125I]T3-labeled 55K protein. Among the nine detergents tested, 0.18% CHAPS was found to be the best in its solubilization efficiency and its ability to maintain the integrity of the antigenicity of the 55K protein. The N-bromoacetyl-[125I]T3-labeled 55K protein was also immunoprecipitated by anti-T3 antibodies. The anti-55K protein antibodies cross-reacted with plasma membrane T3 binding proteins from cultured cells and tissues of human and rodent origin. These results indicate that structural similarities exist in human and rodent plasma membrane T3 binding proteins. These antibodies should provide a powerful tool in the characterization and in probing the function(s) of the plasma membrane T3 binding protein in cells.

Animals↗
PubMed1984

Site-specific antibodies to the erbB oncogene product immunoprecipitate epidermal growth factor receptor.

Antiserum to a defined region (residues 373-383) of the erbB oncogene product immunoprecipitated a 170,000 dalton protein that was phosphorylated in an EGF-sensitive fashion as well as the 125I-EGF-receptor complex from A431 human epidermoid carcinoma cells. Preincubation of the antiserum with an excess of the synthetic peptide corresponding to the defined region blocked the immunoprecipitation of this protein. A partial proteolytic peptide map of this immunoprecipitated 170,000 dalton protein was identical to that of the authentic EGF receptor. These results suggest immunological similarity between the erbB gene product and the EGF receptor.

Alpharetrovirus↗
PubMed1984

Amplification and enhanced expression of the epidermal growth factor receptor gene in A431 human carcinoma cells.

The sequence of the human epidermal growth factor (EGF) receptor shows great homology with the avian erythroblastosis virus v-erb B oncogene, raising the possibility that the receptor gene is identical to the c-erb B protooncogene. Human A431 epidermoid carcinoma cells, which have an unusually high number of EGF receptors, were examined to determine whether elevated EGF receptor levels correlate with gene amplification. Southern blots of genomic DNA's from A431 and other human cell lines were probed with either a v-erb B gene fragment or a human EGF receptor complementary DNA clone (pE7), previously isolated from an A431 complementary DNA library. When either probe was used to analyze Eco RI- or Hind III-generated DNA fragments, EGF receptor DNA sequences were amplified about 30-fold in A431. Differences in the banding pattern of A431 DNA fragments relative to normal fibroblast DNA indicate the occurrence of a rearrangement in the region of the receptor gene. Furthermore, A431 cells contain a characteristic, prominent 2.9-kilobase RNA. These results are consistent with the hypothesis that, in A431 cells, gene amplification, possibly associated with a translocation event, may result in the overproduction of EGF receptor protein or the appearance of the transformed phenotype (or both).

Alpharetrovirus↗
PubMed1984

DNase I sensitivity of the alpha 2(I) collagen gene: correlation with its expression but not with its methylation pattern.

The chromatin structure of the chick alpha 2(I) collagen gene was probed with DNase I. Because our previous work strongly suggested that the 5' end of this gene is not methylated whereas the rest of the gene is methylated whether or not the gene is expressed, we compared the relative DNase I sensitivity of the methylated and unmethylated segments. Both regions demonstrate similar relative DNase I sensitivities within a given tissue. In chromatin of chick embryo fibroblasts, we find a DNase I hypersensitive site which maps between 100 and 300 bp preceding the start of transcription. This site is not found in brain chromatin but is present in chick embryo fibroblasts transformed by Rous Sarcoma virus although the rate of transcription of the alpha 2(I) collagen gene is greatly reduced in these cells. Hence, the mechanism responsible for the large decrease in alpha 2(I) collagen gene expression in RSV transformed cells is different from the mechanism that is responsible for the presence of a DNase I hypersensitive site in the promoter. Furthermore, changes in the DNase I sensitivity of the chromatin of the alpha 2(I) collagen promoter occur without changes in the methylation pattern of the gene.

Animals↗
PubMed1984

Vinculin phosphorylation in response to calcium and phorbol esters in intact cells.

Vinculin phosphorylation in both chick embryo fibroblasts and Swiss 3T3 cells was increased by either calcium or biologically active phorbol esters. Increased phosphorylation of vinculin was noted as early as 10 min following phorbol 12-myristate 13-acetate treatment and was maximal at about 1 h. Maximal increases in phosphorylation were noted at approximately 100 nM phorbol 12-myristate 13-acetate. Phorbol 12,13-dibutyrate (80 nM), a less potent phorbol ester, resulted in smaller increases in vinculin phosphorylation than phorbol 12-myristate 13-acetate at equimolar concentrations. Phorbol, dibutyryl cAMP, and dibutyryl cGMP had no significant effect on phosphorylation. No correlation was found between vinculin phosphorylation and the morphological changes induced by phorbol esters. Tryptic peptide analysis of vinculin revealed multisite phosphorylation. Phosphorylation of only three of the peptides was significantly increased following phorbol 12-myristate 13-acetate treatment. Phosphoamino acid analysis revealed increases at both serine and threonine residues. The low level of phosphotyrosine present in control cells was not significantly increased by phorbol 12-myristate 13-acetate treatment. These findings combined with studies of vinculin phosphorylation by purified protein kinase C (Werth, D. K., Niedel, J. E., and Pastan I. (1983) J. Biol. Chem. 258, 11423-11426) suggest the hypothesis that protein kinase C may be involved in regulation of phosphorylation of vinculin, a cytoskeletal protein.

Animals↗
PubMed1984

Phosphorylation of the solubilized insulin receptor by the gene product of the Rous sarcoma virus, pp60src.

Both the insulin receptor and the gene product of the Rous sarcoma virus, pp60src, are protein kinases which phosphorylate themselves and other proteins on tyrosine residues. Addition of the solubilized insulin receptor to purified pp60src increased the phosphorylation of the beta-subunit of the insulin receptor. Phosphorylation of the insulin receptor by pp60src occurred both in the absence and presence of insulin but did not alter the insulin dose response for autophosphorylation of the receptor. Increasing concentrations of pp60src increased the phosphorylation of the receptor and at high concentrations equaled the maximal effect produced by insulin. Our observations suggest a possible mechanism by which the metabolically regulated insulin receptor tyrosine kinase could be altered by other tyrosine kinases such as that associated with pp60src. Further studies will be required to determine if the insulin receptor is phosphorylated by pp60src in Rous sarcoma virus-infected cells.

Animals↗
PubMed1984

Verapamil enhances the toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin and antitransferrin receptor with Pseudomonas exotoxin.

Verapamil, a clinically important calcium channel blocker, has been found to cause a 40-fold enhancement of killing of the human KB cell line by a cytotoxic conjugate of epidermal growth factor with Pseudomonas exotoxin (EGF-PE). Synergistic effects of verapamil and EGF-PE are also seen on HeLa D98 cells and a human epidermal carcinoma cell line, A431. Verapamil also potentiates the effect of a toxic conjugate formed between Pseudomonas exotoxin and a monoclonal antibody to the human transferrin receptor (anti-TFR-PE) and enhances the effect of Pseudomonas exotoxin (PE) alone. Two other calcium antagonists were tested. Diltiazem enhances the cytotoxic effect of EGF-PE, but nifedipine does not. Verapamil does not affect the binding and uptake of 125I-EGF by KB cells, but it significantly delays the disappearance of internalized 125I-EGF from the cells. Density gradient fractionation studies using cell homogenates suggest that 125I-EGF accumulates in an undegraded form in lysosomes when cells are treated with verapamil. By immunofluorescence microscopy using an antibody to PE, EGF-PE was found to accumulate in lysosomes; by electron microscopy the lysosomes had an abnormal appearance. The effects of verapamil on toxicity of EGF-PE and lysosomal function appear to be related. However, it is not known whether the enhanced toxicity of EGF-PE in the presence of verapamil is due to its delayed degradation in lysosomes or some more general effect of verapamil on membrane permeability.

ADP Ribose Transferases↗
PubMed1984

Morphologic demonstration of clathrin-coated pits in frog and turkey erythrocytes.

We have examined nucleated erythrocytes of frog and turkey for the presence of clathrin-coated structures using electron microscopy and immunocytochemistry. By electron microscopy, coated pits were found on the plasma membrane of peripheral blood erythrocytes of both species. These structures had an appearance similar to coated pits seen in non-erythroid mammalian cells. Using immunofluorescence with anti-(bovine) clathrin antibody, erythrocytes of both species showed punctate membrane fluorescence similar to the pattern of coated pits seen in other cells. By both methods, frog erythrocytes showed considerable heterogeneity, such that only about 50% of the cells showed significant numbers of coated pits, usually fewer than 20-50 per cell. In contrast, the vast majority of turkey erythrocytes showed no detectable coated pits, but occasional cells (less than 10%) showed large numbers of coated structures. These results suggest that a functional endocytic system may be present in a subpopulation of these nucleated erythrocytes. These findings may be of significance in understanding the ligand-induced loss of some receptors from the surface of these cells, and may serve as an indication of morphologic differentiation.

Animals↗
PubMed1984

Initial steps in receptor-mediated endocytosis. The influence of temperature on the shape and distribution of plasma membrane clathrin-coated pits in cultured mammalian cells.

We have examined the shape and distribution of clathrin-coated pits in Swiss 3T3 cells at 4 or 37 degrees C using electron microscopy with serial sections and immunofluorescence light microscopy. Both groups were fixed in glutaraldehyde and preserved further using a membrane contrast enhancement technique consisting of sequential osmium-ferrocyanide, thiocarbohydrazide and osmium-ferrocyanide treatment in situ. Concanavalin A-horseradish peroxidase (conA-HRP) was used to identify these structures participating in endocytosis. Two hundred twenty-two clathrin-coated structures were analysed; 126 from cells fixed at 4 degrees C, and 96 from cells fixed after a 3 min warm-up to 37 degrees C. All coated structures labeled with conA-HRP had demonstrable connections to the plasma membrane. These coated structures were morphologically classified into three categories: (a) flat pits; (b) curved pits; and (c) pits with narrow-neck connections to the plasma membrane. At 37 degrees C, 27% of coated pits had narrow neck connections to the plasma membrane whereas at 4 degrees C only 1% had such connections. Receptosomes (endosomes) labeled with conA-HRP were found only after incubation at 37 degrees C, indicating that active endocytosis was occurring in cells at 37 degrees C, but not at 4 degrees C. Immunofluorescence with anti-clathrin antibody was used to quantitate the number of clathrin-coated pits in Swiss 3T3 cells incubated at 4 and 37 degrees C prior to fixation. No difference was detected. There were 426 +/- 122 pits per cell at 37 degrees C and 441 +/- 106 at 4 degrees C. These results support the hypothesis that formation of a narrow neck connected a coated pit to the cell surface is an early step in the mechanism of receptor-mediated endocytosis.

Animals↗
PubMed1984

Kinetics of transit of transferrin and epidermal growth factor through clathrin-coated membranes.

The kinetics of association of epidermal growth factor (EGF) and diferric-transferrin (TF) with clathrin-coated membranes of KB cells were examined using anti-clathrin antibody bound to Staphylococcus aureus. The ligands were bound to cells at 4 degrees C and after warming to 30 degrees C for 5 min, both ligands were found concentrated in a membrane fraction immunoadsorbed with anti-clathrin antibody. Fifteen minutes after entry both ligands moved to a non-clathrin-associated compartment. Twenty to 25 min after entry, EGF, but not TF, appeared in a second clathrin-associated compartment. In parallel morphologic experiments, EGF-horseradish peroxidase (EGF-HRP) and TF-HRP were both found at 6 min in coated pits associated with the plasma membrane. At 22 min EGF-HRP, but not TF-HRP, was localized in Golgi-associated coated pits. These studies provide biochemical and morphological evidence suggesting that coated membranes in the Golgi region are involved at some stage in the transfer of EGF to lysosomes. The method should be of general utility in the study of receptor-mediated endocytosis.

Biological Transport↗
PubMed1984

Endocytosis and exocytosis: current concepts of vesicle traffic in animal cells.

Animal cells have specific pathways to transport macromolecules from their surrounding environment to their interior, and from internal compartments to the cell surface or other intracellular locations. Many of these movements appear to be receptor-dependent processes in which specific membrane receptors bind macromolecules, segregate them into discrete membrane-limited compartments, and move the molecules to new locations. Such processes include the clustering and internalization of receptor-bound ligands at the cell surface in clathrin-coated pits, the formation of endocytic vesicles (receptosomes) from coated pits, the movement of receptosomes by saltatory motion to the Golgi system, the concentration of materials in the coated pits of the Golgi system that are destined for delivery to lysosomes, and the directed traffic of materials destined for exocytosis out of the Golgi to the cell surface. This review describes some of the experiments which have led to our current understanding of the various organelles involved in this traffic and some of the biochemical mechanisms involved.

Animals↗
PubMed1984

Morphologic characterization of the pathway of transferrin endocytosis and recycling in human KB cells.

The pathway of transferrin uptake and recycling was investigated in KB cells to attempt to identify the organelles involved in the return of transferrin to the cell surface. Comparison was made with the pathway of internalization of epidermal growth factor (EGF), which has been shown to terminate in lysosomes. A horseradish peroxidase conjugate of transferrin (TF-HRP) was incubated with KB and at 4 degrees C and, at various times after warming to 37 degrees C, the location of TF-HRP was examined at the electron microscopic level. Transferrin, like EGF, was found to enter cells via coated pits and to move to the Golgi region in receptosomes (endosomes). Transferrin was located in the tubular elements of the transreticular portion of the Golgi but not in Golgi stacks. Interestingly, transferrin was not concentrated in the coated pits of the Golgi. In contrast, EGF was highly concentrated there. Transferrin was next detected in tubular elements (approximately equal to 600 A in width and up to 5,000 A in length) that were closely associated with microtubules and in dumbbell-shaped structures. In contrast, EGF was not detected in these structures. These results suggest that those organelles containing transferrin, but not EGF, participate in the return of receptor-bound transferrin to the cell surface.

Biological Transport↗
PubMed1984

Characterization of epidermal growth factor receptor gene expression in malignant and normal human cell lines.

To investigate the possibility that the epidermal growth factor (EGF) receptor functions as an oncogene product, we have determined the levels of EGF receptor protein and RNA in a variety of malignant and normal human cells, using a specific polyclonal antibody to the EGF receptor and a cDNA clone (plasmid pE7) that encodes the EGF receptor, respectively. Besides A431 epidermoid carcinoma cells, which are known to make large amounts of EGF receptor, cell lines from two ovarian cancers, two cervical cancers, and one kidney cancer were found to contain substantial amounts of receptor protein (11-22% of A431). Normal human fibroblasts (Detroit 551), a human lymphocyte line (IM-9), and a leukemic lymphocyte line (CEM) contained low or undetectable levels of EGF receptor. RNA blot analysis showed that among the human cell lines examined the levels of a 10- and a 5.6-kilobase species of pE7-specific RNA generally correlated with the amount of the EGF receptor protein. Genomic DNA blot analysis revealed that except for A431 none of these cell lines expressing high levels of EGF receptor protein possessed amplified receptor gene sequences. A431 cells are known to secrete a truncated form of the EGF receptor. An abundant 2.9-kilobase RNA is found only in A431 cells; it could encode the truncated form of the EGF receptor.

Cell Line↗