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Biomedical subjects

I Pastan

Publications and source records attributed to I Pastan.

At least 559 records · Page 31Linked to original sources
PubMed1985

EGF induces receptor down-regulation with no receptor recycling in KB cells.

Several ligands, including epidermal growth factor (EGF), have been found to negatively modulate or down-regulate their specific plasma membrane receptors. Using both 125I-EGF and a monoclonal antibody against the EGF-receptor (EGF-R1), we studied the down-regulation of the EGF-receptor in the human adenocarcinoma cell line KB. The results presented here demonstrate that incubating KB cells at 37 degrees C with EGF rapidly decreases the number of plasma membrane EGF-receptors. In addition, there is a concomitant rise of equal magnitude in the number of EGF molecules taken up. The latter result argues strongly that there is negligible recycling of the EGF-receptor in KB cells and that the major portion of internalized EGF-receptor complexes are transported to lysosomes and subsequently degraded. The fate of the EGF-receptor is markedly different from that of receptors not subject to down-regulation. The biochemical signals that operate to regulate such diverse receptor traffic in cells remains to be elucidated.

Carcinoma↗
PubMed1985

Isolation and genetic characterization of human KB cell lines resistant to multiple drugs.

Human KB cell lines resistant to high levels of colchicine were isolated by several successive single-step selections. Most of these selection steps resulted in cross-resistance to vincristine, vinblastine, adriamycin, actinomycin D, and puromycin; however, at the highest levels of colchicine resistance, increased cross-resistance to other drugs was not observed. There was no major change in protein synthesis or alteration in protein phosphorylation or [14C]glucosamine labeling patterns accompanying the development of multiple drug resistance as measured by analysis of metabolically labeled proteins on SDS gels. Cell-cell hybridization experiments showed that the colchicine-resistant and multiple drug-resistant phenotypes were incompletely dominant. In addition, colchicine resistance was found to segregate independently from resistance to other drugs in one somatic cell hybrid, suggesting that complex genetic loci are involved in the development of the multiple drug-resistant phenotype. These mutants should be useful for the study of the clinically important problem of multiple drug resistance in human cancer.

Antineoplastic Agents↗
PubMed1985

Translocation chromosome 7 of A431 cells contains amplification and rearrangement of EGF receptor gene responsible for production of variant mRNA.

The epidermal growth factor (EGF) receptor, along with several oncogene protein products, possesses tyrosine-specific protein kinase activity. Furthermore, the EGF receptor has structural similarity to the putitive v-erb-B transforming protein. Because of these closely shared characteristics, it is important to elucidate the possible involvement of the EGF receptor in malignant transformation. The epidermal carcinoma cell line A431 exhibits an abnormally high number of EGF receptors, which is associated with the presence of translocation chromosome M4. Recently, A431 cells have been shown to contain amplified sequences for the EGF receptor gene(s) and also to produce a variant mRNA which diverges from the normal EGF receptor mRNA at the 3' end. Here we report, using the human EGF receptor cDNA probe pE7, that the chromosome M4 has a six- to sevenfold amplification of the EGF receptor gene. Furthermore, the presence of M4 in somatic cell hybrids correlates with the production of the variant 2.9-kb mRNA. This aberrant mRNA is apparently generated by an intrachromosomal rearrangement which was detected using as a probe a fragment of the pE15cDNA encoding the variant mRNA.

Animals↗
PubMed1985

Serial section analysis of clathrin-coated pits in rat liver sinusoidal endothelial cells.

Electron microscopy and serial sections were used to examine the shape of clathrin-coated pits in sinusoidal endothelial cells of rat livers. Livers were perfused at 4 degrees C with either concanavalin A-horseradish peroxidase (conA-HRP), or HRP alone, followed by warm-up to 37 degrees C and fixation with glutaraldehyde. Alternatively, the livers were perfused with HRP at 37 degrees C, followed by fixation. All tissue was preserved using a membrane contrast enhancement technique (R-OTO) consisting of sequential osmium-ferrocyanide, thiocarbohydrazide, and osmium-ferrocyanide treatment. Peroxidase reaction product was used to identify structures participating in endocytosis. One hundred and ninety-three clathrin-coated structures were examined. Sixty-six were from livers perfused with conA-HRP at 4 degrees C, 63 were from livers perfused with only HRP at 4 degrees C, and 64 were from livers perfused with HRP at 37 degrees C. These coated structures were morphologically classified into three categories: (a) flat pits; (b) cup-shaped pits; (c) pits with a narrow neck. No isolated coated vesicles were found. In cells perfused at 4 degrees C followed by warming to 37 degrees C, the percentage of coated pits found connected to the cell surface by narrow necks was 31%, using conA-HRP, and 27% using HRP alone. In cells perfused continuously at 37 degrees C, the percentage of coated pits with narrow neck connections was 21% using HRP alone. These results suggest that the formation of coated pits connected to the surface by narrow necks is not an artifact of cell type, of experimental protocol or of incubation with a lectin.

Animals↗
PubMed1985

Inhibition of phosphatidylcholine synthesis does not alter uptake of transferrin by LM fibroblasts.

The receptor-mediated endocytosis of diferric transferrin by LM fibroblasts has been examined to determine if de novo synthesis of phosphatidylcholine is required for this process. To test this possibility, LM cells were allowed to internalize [125I]transferrin in the presence or absence of exogenous choline. Under conditions in which de novo synthesis of phosphatidylcholine was reduced from 51% of the total to less than 10%, the initial rate of transferrin uptake was unaffected. These data suggest that the process of receptor-mediated endocytosis of transferrin can proceed normally in the absence of de novo phosphatidylcholine synthesis.

Animals↗
PubMed1985

Characterization and sequence of the promoter region of the human epidermal growth factor receptor gene.

The promoter region of the epidermal growth factor (EGF) receptor has been identified by in vitro transcription using EGF receptor genomic DNA fragments as template and by primer extension and nuclease S1 mapping using EGF receptor mRNA. Six transcriptional start sites were identified. DNA sequence analysis shows that the promoter region contains neither a "TATA box" nor a "CAAT box," has an extremely high G+C content (88%), and contains five CCGCCC repeats and four (TCC)TCCTCCTCC repeats. This promoter region is situated close to or within a DNase I-hypersensitive site in A431 human epidermoid carcinoma cells, which overproduce the EGF receptor. The EGF receptor gene promoter has some resemblance to the promoter of the hydroxymethylglutaryl-CoA reductase gene and the early promoter of simian virus 40. This similarity may offer a clue to the mechanism by which the receptor gene is regulated.

Base Sequence↗
PubMed1985

Amplification of DNA sequences in human multidrug-resistant KB carcinoma cells.

Four KB carcinoma cell lines selected independently for resistance to either colchicine, adriamycin, or vinblastine were studied. All cell lines showed high levels of resistance to the selecting drug and cross-resistance to the other drugs and to actinomycin D. Double-minute chromosomes could be identified on chromosomal spreads of these multidrug-resistant KB cell lines. Amplification of specific DNA sequences was demonstrated by using the technique of in-gel renaturation. All the cell lines share common amplified sequences. There are also amplified sequences that are specific for each cell line. A revertant cell line that has reacquired drug sensitivity has lost its amplified sequences. Specific probes obtained by cloning amplified sequences from the cell line selected in vinblastine recognize amplified sequences in all the resistant lines. The presence of common amplified sequences in these cell lines is strong evidence for the importance of these regions in multiple drug resistance.

Base Sequence↗
PubMed1985

Epidermal growth factor regulates the expression of its own receptor.

The epidermal growth factor (EGF) receptor gene is the cellular homolog of the avian erythroblastosis virus erbB oncogene. Control of EGF receptor expression determines cellular responsiveness to EGF and might play an important role in neoplastic development. Using RNA blot hybridization, we have found that exposure of human KB carcinoma cells to EGF results in elevated levels of EGF receptor mRNA. The phorbol ester 4 beta-phorbol 12-myristate 13-acetate also stimulates EGF receptor RNA accumulation. Immunoprecipitation of metabolically labeled (30 min) EGF receptor protein revealed that synthesis of new EGF receptor follows the increase in receptor RNA. Addition of cycloheximide together with EGF further enhances EGF receptor RNA accumulation. Results of nuclear runoff-transcription experiments suggest that the stimulatory effects of EGF and cycloheximide are most likely due to a posttranscriptional control mechanism.

Cells, Cultured↗
PubMed1985

Multiply drug-resistant human KB carcinoma cells have decreased amounts of a 75-kDa and a 72-kDa glycoprotein.

Human KB carcinoma cells were selected in sequential steps for resistance to colchicine and found to be cross-resistant to multiple drugs, including vinblastine, adriamycin, and actinomycin D. Compared with the parental line, the multiply resistant cells have decreased amounts of two [35S]methionine-labeled proteins with apparent molecular masses of 75 and 72 kDa. These proteins reappear in a revertant, drug-sensitive cell line. Both proteins are labeled with [14C]glucosamine and are retained on a wheat germ agglutinin-agarose column, indicating that they are glycoproteins. These data suggest that in this human cell line, these two glycoproteins can serve as a marker of the multiple drug-resistance phenotype and may play a role in its etiology.

Antineoplastic Agents↗
PubMed1985

Phorbol esters induce transient internalization without degradation of unoccupied epidermal growth factor receptors.

4 beta-Phorbol 12-myristate 13-acetate (PMA) treatment of KB cells at 37 degrees C rapidly induces a 50% reduction in epidermal growth factor (EGF) binding that is maximal by 30 min. EGF binding activity returns to the original value by 1 hr and remains constant for 2 hr thereafter. Using a polyclonal antibody directed against the cytoplasmic domain of the EGF receptor (EGF-R), we examined the fate of the receptor after PMA treatment. Immunofluorescent and electron microscopic localization of the EGF-R after PMA treatment demonstrated that about 50% of the receptor became internalized into endocytic vesicles (receptosomes) and Golgi-associated structures. Unlike EGF-induced internalization, PMA-induced internalization did not cause delivery of EGF-R to lysosomes or receptor degradation. Rather, receptor reappeared on the cell surface. No stimulation of EGF-R synthesis was observed after 1 hr of PMA treatment. Loss of cell surface binding correlated with the internalization of the EGF-R observed morphologically. A possible explanation for these observations is that PMA, an activator of protein kinase C, confers a signal sufficient for EGF-R clustering and internalization but not for transport to lysosomes.

Carcinoma, Squamous Cell↗
PubMed1985

Identification of two lysosomal membrane glycoproteins.

Two murine lysosome-associated membrane proteins, LAMP-1 of 105,000-115,000 D and LAMP-2 of 100,000-110,000 D, have been identified by monoclonal antibodies that bind specifically to lysosomal membranes. Both glycoproteins were distinguished as integral membrane components solubilized by detergent solutions but not by various chaotropic agents. The lysosome localization was demonstrated by indirect immunofluorescent staining, co-localization of the antigen to sites of acridine orange uptake, and immunoelectron microscopy. Antibody binding was predominantly located at the limiting lysosomal membrane, distinctly separated from colloidal gold-labeled alpha-2-macroglobulin accumulated in the lumen during prolonged incubation. LAMP-1 and LAMP-2 also appeared to be present in low concentrations on Golgi trans-elements but were not detected in receptosomes marked by the presence of newly endocytosed alpha-2-macroglobulin, or in other cellular structures. LAMP-1 and LAMP-2 were distinguished as different molecules by two-dimensional gel analysis, 125I-tryptic peptide mapping, and sequential immunoprecipitations of 125I-labeled cell extracts. Both glycoproteins were synthesized as a precursor protein of approximately 90,000 D, and showed a marked heterogeneity of apparent molecular weight expression in different cell lines. LAMP-2 was closely related or identical to the macrophage antigen, MAC-3, as indicated by antibody adsorption and tryptic peptide mapping. It is postulated that these glycoproteins, as major protein constituents of the lysosomal membrane, have important roles in lysosomal structure and function.

Animals↗
PubMed1985

Elevated epidermal growth factor receptor gene copy number and expression in a squamous carcinoma cell line.

The human epidermal growth factor (EGF) receptor is known to be homologous to the v-erb B oncogene protein of the avian erythroblastosis virus. Overexpression of the EGF receptor gene in A431 epidermoid carcinoma cells is due to gene amplification. In this study, a variety of squamous cell carcinomas were examined and one, SCC-15, contained high levels of the EGF receptor as determined by immunoprecipitation via an EGF receptor-specific polyclonal antibody. Using a cloned EGF receptor complementary DNA as a probe, the level of EGF receptor RNA was found to be elevated four-fold in SCC-15 relative to normal cultured keratinocytes. When the same probe was used to identify EGF receptor gene fragments on a genomic DNA blot, the SCC-15 cell line was shown to possess an EGF receptor gene copy number amplified four to five times. Gene amplification results in the enhancement in the level of the EGF receptor in several carcinomas and could be responsible for the appearance of the transformed phenotype in these cells.

Carcinoma, Squamous Cell↗
PubMed1985

Characterization of immunotoxins active against ovarian cancer cell lines.

The purpose of the present study was to develop immunotoxins directed against human ovarian carcinoma cells. Four monoclonal antibodies (260F9, 454C11, 280D11, and 245E7) were chosen because they were found to bind to various ovarian carcinoma cell lines. These antibodies were covalently linked to either Pseudomonas exotoxin (PE) or ricin A chain (RTA), and the conjugates were tested against five ovarian cancer cell lines (OVCAR-2, -3, -4, -5; A1847). The ability of the immunotoxins to inhibit both protein synthesis and colony formation was evaluated. Qualitatively similar results were obtained for both types of assays. Usually, PE conjugates were more toxic than their corresponding RTA conjugates. 454C11-PE was very toxic for all ovarian carcinoma lines, whereas 454C11-RTA had low activity. Both 260F9-PE and 260F9-RTA were active in all OVCAR cell lines but not in A1847 cells. 280D11-PE was toxic for OVCAR-4; otherwise, 280D11-PE and RTA conjugates of both 280D11 and 245E7 had little activity. Specificity of immunotoxin action was shown by competition by excess antibody, nontoxicity in nontarget cells, and inactivity of an irrelevant immunotoxin. To investigate the basis of antibody-dependent differences in activity of the various immunotoxins, antibody uptake was studied in OVCAR-2 cells, and the results indicate that antibody internalization is one important factor in the activity of immunotoxins.

ADP Ribose Transferases↗
PubMed1985

Ultrastructural immunocytochemical localization of the transferrin receptor using a monoclonal antibody in human KB cells.

Using a monoclonal antibody (HB21) against the human transferrin receptor, we have localized this receptor in cultured KB human carcinoma cells by fluorescence and ultrastructural immunocytochemistry. The receptor was found diffusely distributed on the cell surface, concentrated in clathrin-coated pits of the cell surface, in intracellular endocytic vesicles (receptosomes) derived from coated pits, in tubular elements of the trans-reticular Golgi system, and in microtubule-associated membranous elements thought to be part of the constitutive exocytic system. This distribution is the same as that previously shown for labeled transferrin in these same cells (Willingham MC, Hanover JA, Dickson BB, Pastan J: Proc Natl Acad Sci USA 81:175, 1984). No significant amounts of receptor were found in lysosomes. An aggregation of membranous elements containing this receptor was found in the pericentriolar region of cells during mitosis. Together with the previous data on the immunocytochemical localization of transferrin, these results suggest that the transferrin receptor may constitutively enter and exit KB cells by endocytosis and exocytosis, carrying bound transferrin into and out of the cell for the purpose of supplying iron from the extracellular environment for cell growth.

Antibodies, Monoclonal↗
PubMed1985

Characterization of the 3,3',5-triiodo-L-thyronine-binding site on plasma membranes from human placenta.

The binding of [125I]T3 to sites on human placenta plasma membranes was characterized, and the binding site was solubilized after affinity labeling with N-bromoacetyl-[125I]T3 (BrAc[125I]T3). Two classes of T3-binding sites were detected. One class has a high affinity (Kd = 2.0nM) and a low capacity (approximately 320 fmol/mg protein); the other has a low affinity (Kd = 18.5 microM) and a high capacity (approximately 2.2 pmol/mg protein). The binding sites were found to be specific for T3 in that other thyroid hormone analogs (D-T3, rT3, D-T4, and L-T4) were less effective or ineffective in displacing the bound [125I]T3. The affinity labeling ligand BrAc[125I]T3 was found to specifically label a protein with an apparent mol wt of 65,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The BrAc[125I]T3-labeled protein was solubilized with 2 mM 3-[( 3-cholamidopropyl)dimethylammonio]1-propane sulfonate. The apparent mol wt of the labeled protein was between 140,000 and 150,000 by Sephadex-G-200 gel filtration. These data demonstrate that a high affinity binding site specific for T3 is present on plasma membranes from human placenta and that the binding site is a protein, most likely a dimer, with a native mol wt between 140,000 and 150,000.

Affinity Labels↗
PubMed1985

Anti-transferrin receptor antibody linked to Pseudomonas exotoxin as a model immunotoxin in human ovarian carcinoma cell lines.

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.

ADP Ribose Transferases↗