monensin and Carcinoma

Topics: Animals; Bacterial Toxins; Brain Neoplasms; Carcinoma; Clinical Trials as Topic; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Diphtheria Toxin; Drug Carriers; Drug Synergism; Glioma; Guinea Pigs; Humans; Immunotoxins; Injections, Intralesional; Injections, Spinal; Ligands; Medulloblastoma; Meningeal Neoplasms; Mice; Monensin; Plant Proteins; Rats; Rats, Nude; Recombinant Fusion Proteins; Tissue Distribution; Transferrin; Treatment Outcome; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

A rat medullary thyroid carcinoma cell line, CA-77, was shown to express the cholecystokinin (CCK) gene. Measurements using a library of sequence-specific radioimmunoassays before and after enzymic treatment of extracts and chromatographic fractions showed that the cells contained 1.0 pmol of alpha-carboxyamidated cholecystokinins/10(6) cells, 0.4 pmol of glycine-extended intermediates/10(6) cells and 1.0 pmol of further C-terminal-extended pro-CCK/10(6) cells. Gel chromatography and reverse-phase h.p.l.c. revealed both sulphated and nonsulphated CCK-8 in the cells. The growth medium contained in addition alpha-amidated CCK-33, glycine-extended CCK-8 and pro-CCK. Exposure to 0.1 microM-dexamethasone for 6 days increased the cellular content and secretion of all of the described CCK peptides by 2-3-fold. The increase was first noted after 3 days of treatment. Monensin inhibited the synthesis of alpha-carboxyamidated CCK and the secretion of all of the CCK forms measured. Colchicine at a low concentration (0.2 mumol/l) apparently increased the synthesis and secretion of alpha-carboxyamidated CCK, whereas higher concentrations inhibited CCK synthesis. Finally, chloroquine inhibited the alpha-carboxyamidation of CCK. We conclude that the CA-77 cell line is a useful tool for studies of the expression and post-translational processing of pro-CCK.

Topics: Animals; Carcinoma; Chloroquine; Cholecystokinin; Chromatography, Gel; Colchicine; Dexamethasone; Gene Expression; Glycine; Monensin; Peptide Fragments; Protein Precursors; Protein Processing, Post-Translational; Rats; Sincalide; Sulfates; Thyroid Neoplasms; Tumor Cells, Cultured

The distribution of interstitial collagenase in a rat mammary carcinoma model system has been studied by immunocytochemistry. Rabbit antibodies were raised against collagenase from neoplastic epithelial cells which were derived from an anaplastic, invasive, rat mammary carcinoma (BC1). Specificity of the antibodies was determined by Western blot analysis which showed reactivity with the inactive procollagenase from conditioned culture medium of BC1 cells as well as with purified, active BC1 collagenase. Anti-BC1 collagenase antibodies did not recognize BC1 collagenase entrapped by the inhibitor, rat alpha-2-macroglobulin (alpha 2M), or collagenase derived from TPA-stimulated human fibroblasts. Anti-human fibroblast collagenase antibodies did not recognize BC1 collagenase, suggesting that the human-mesenchymal and rat-epithelial enzymes are immunologically distinct molecules. Collagenase was immunolocalized intracellularly in BC1 cells cultured in the presence of monensin. Neither BC1 collagenase, alpha 2M nor enzyme-inhibitor complexes were demonstrated in or around invading tumours by immunostaining of tissue sections of rat mammary carcinomas.

Topics: alpha-Macroglobulins; Animals; Antibodies, Neoplasm; Antibody Specificity; Blotting, Western; Carcinoma; Enzyme-Linked Immunosorbent Assay; Epithelium; Female; Immunoenzyme Techniques; Mammary Neoplasms, Experimental; Microbial Collagenase; Monensin; Neoplasm Transplantation; Rabbits; Rats; Tumor Cells, Cultured

Human colon carcinoma cells synthesize a high-molecular-weight heparan sulfate proteoglycan which is localized at the cell surface. In this study we have performed a series of immunoprecipitation and pulse-chase experiments associated with various pharmacological agents that interfere with the synthesis and post-translational modification of the proteoglycan. We demonstrate that colon carcinoma cells synthesize the heparan sulfate proteoglycan from a 400-kDa precursor protein that is immunologically related to the Engelbreth-Holm-Swarm (EHS) tumor cell proteoglycan. The cells contain a large pool of precursor protein with a half-life of about 75 min. Most of the precursor protein receives heparan sulfate side chains and is then transported to the cell surface and released into the medium. A portion of the precursor pool, however, does not receive heparan sulfate chains but is secreted into the medium. The glycosylation and subsequent secretion of the 400-kDa precursor protein was inhibited by NH4Cl and even more by monensin, indicating that the transit of precursor from the rough endoplasmic reticulum to the cell surface occurred through the Golgi complex and acidic compartments. The existence of a sizable pool of precursor protein was confirmed by additional experiments using cycloheximide and xyloside. These experiments showed that the half-life of the precursor protein was also 75 min and that stimulation of heparan sulfate synthesis by xyloside was greatly enhanced (about 12-fold) after new protein core synthesis was blocked by cycloheximide. Although the structural models proposed for the EHS and colon carcinoma heparan sulfate proteoglycans differ, the observation that they are derived from a precursor protein with dimensional and immunological similarities suggests that they may be genetically related.

Topics: Ammonium Chloride; Blotting, Western; Carcinoma; Cell Line; Chondroitin Sulfate Proteoglycans; Colonic Neoplasms; Cycloheximide; Glycosaminoglycans; Glycosides; Heparitin Sulfate; Humans; Monensin; Polysaccharide-Lyases; Precipitin Tests; Protein Precursors; Protein Processing, Post-Translational; Proteoglycans; Trypsin

A monoclonal antibody R1 against the human epidermal growth factor receptor has been used to study biosynthesis in the carcinoma cell line A431. Two glycoproteins of apparent mol. wts. 95 000 and 160 000 were immunoprecipitated from cells labelled for short times with [35S]methionine or [3H]mannose. Pulse-chase studies show the 160 000 mol. wt. glycoprotein to be a precursor of the 175 000 mol. wt. receptor, but do not establish a precursor role for the 95 000 mol. wt. glycoprotein. Limited proteolysis, peptide mapping, endoglycosidase digestion and the use of monensin and tunicamycin show that the 95 000 mol. wt. glycoprotein is structurally related to the 160 000 mol. wt. glycoprotein and that both glycoproteins have approximately 22 000 - 28 000 mol. wt. of oligosaccharide side chains. Monensin blocks conversion of the 160 000 to the 175 000 mol. wt. mature receptor, a process which involves complexing several of its N-linked oligosaccharide chains. Pulse-chase studies showed that an immunoprecipitable polypeptide of 115 000 mol. wt., or 95 000 mol. wt., in the presence of monensin, was secreted into the medium at late chase times. The possible mechanisms for the origins of all the receptor-related polypeptides are discussed.

Topics: Carcinoma; Cell Line; ErbB Receptors; Glycoproteins; Humans; Molecular Weight; Monensin; Peptides; Receptors, Cell Surface; Tunicamycin

A conjugate of Pseudomonas exotoxin and epidermal growth factor (PE-EGF) inhibits proteins synthesis in KB cells, and this inhibition is increased by adenovirus. Protein synthesis inhibition is dependent on the amount of adenovirus and PE-EGF used and the time of incubation of cells with these agents. With 1 microgram of adenovirus and 0.5 micrograms of PE-EGF per ml, protein synthesis is inhibited about 80% in a 60-min experiment. Under these conditions neither adenovirus nor PE-EGF alone has any effect. In the presence of several weak bases or monensin, the enhancement of toxicity was substantially inhibited; half-maximal inhibition was achieved with 40 microM chloroquine, 10 mM ammonium chloride, 5 mM methylamine, 0.1 mM N-hexylamine and 1 microM monensin. At the concentrations employed, none of the inhibitors affected the amount of virus taken up or bound to the cell surface, and chloroquine had no effect on the amount of EGF taken up in 60 min. Chloroquine did not prevent the toxicity of the PE-EGF (5 micrograms/ml) alone. Because these compounds are known to elevate the pH in receptosomes, it seems likely that the acidification of the receptosome either enhances the lysis of the membrane by adenovirus or enhances some other step in the release of PE-EGF.

Topics: Adenoviruses, Human; Amines; Ammonium Chloride; Carcinoma; Cell Line; Chloroquine; Drug Synergism; Epidermal Growth Factor; Exotoxins; Humans; Hydrogen-Ion Concentration; Kinetics; Methylamines; Monensin; Mouth Neoplasms; Protein Biosynthesis; Pseudomonas; Receptors, Virus