sodium-dodecyl-sulfate and Carcinoma--Hepatocellular

The authors examined the expression of transforming growth factor-beta receptor (TGF-beta r) types I and II and the mannose 6-phosphate/insulin-like growth factor-II receptor (M6-P/IGF-IIr) in human hepatocellular carcinoma (HCC).. Transforming growth factor-beta (TGF-beta) is part of a superfamily of peptide-signaling molecules that play an important role in modulating cell growth. It is secreted as a latent complex and therefore, must be activated to elicit a biological response. Bioactivation of the TGF-beta complex is facilitated by binding to the M6-P/IGF-IIr. Once activated, TGF-beta exerts its effects by binding to specific cell membrane TGF-beta receptors. The loss of responsiveness of hepatocytes to TGF-beta has been implicated in hepatocarcinogenesis and could result from a loss in the expression of either the TGF-beta receptors or the M6-P/IGF-IIr.. Human hepatocellular carcinomas and surrounding normal tissue were collected from operating room samples and snap-frozen in liquid nitrogen (n = 13). Tissues from two tumors were fixed in Omni-fix for sectioning and immunohistochemistry staining for the M6-P/IGF-IIr and TGF-beta 1. RNA was extracted from both normal and malignant liver tissue and analyzed using an RNase protection assay. SDS-PAGE of purified membrane hybridized with 125I-TGF-beta 1 and 125I-IGF-II was used to determine the TGF-beta type I (TGF-betarI) and type II (TGF-beta rII) receptors and M6-P/IGF-IIr protein levels, respectively. Gels were quantitated by phosphorimager, and a paired t test was used for statistical analysis.. In HCC, a 60% (p < 0.01) and 49% (p < 0.02) reduction in the mRNA levels for T beta rI and T beta rII, respectively, relative to the receptor levels in surrounding normal liver, was shown. A similar decrease in the receptor protein levels also was observed. The M6-P/IGF-IIr mRNA and protein levels were reduced in 7 of 11 hepatocellular carcinomas. Immunohistochemical staining demonstrated an absence of intracellular TGF-beta 1 and reduced M6-P/IGF-IIr in the hepatocellular carcinoma cells.. These results demonstrate that human HCCs have a significantly reduced expression of both the TGF-beta rI- and TGF-beta rII-signaling receptors for TGF-beta. This may provide a selective growth advantage to the HCC by allowing them to escape the mito-inhibitory effects of activated TGF-beta. Furthermore, in the subset of HCC in which the expression of the M6-P/IGF-IIr is downregulated, the bioactivation of TGF-beta also may be impaired.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Hepatocellular; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Female; Gene Expression Regulation, Neoplastic; Humans; Liver; Liver Neoplasms; Male; Middle Aged; Receptor, IGF Type 2; Receptors, Transforming Growth Factor beta; RNA, Messenger; Sodium Dodecyl Sulfate

To evaluate the role of glutathione S-transferase (GST) isoenzymes in induced resistance of hepatocytes to aflatoxin B1 (AFB1), we compared DNA protective activities of different hepatic cytosol preparations and purified GSTs from normal rats, rats exposed to different polychlorinated biphenyls (PCBs), and rats with carcinogen-induced hepatocellular neoplasms, with cytosols or purified GSTs from mouse, rainbow trout, and human livers. These comparisons were performed in an in vitro assay for [3H]AFB1-DNA binding after activation by rat liver microsomes. Cytosol and S-hexylglutathione-affinity-purified GST preparations from livers of mice consistently had strong protective activity against AFB1-DNA binding. The majority of this activity was dependent on the presence of reduced glutathione (GSH) but some GSH-independent protection was observed in mouse hepatic cytosol, but not in purified GST preparations. We found that all of the GSH-dependent DNA-protective activity in mouse liver eluted as a single GST isoenzyme by hydroxyapatite chromatography. Preparations of cytosol and purified GSTs from normal rat liver, rainbow trout liver, and human liver had much less AFB1-specific DNA protective activity than GSTs found in mouse liver preparations. Cytosol from rats with carcinogen-generated liver neoplasms and livers induced with 3,3',4,4'-tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl had more GST activity toward CDNB than cytosol from normal rat liver. When equivalent units of GST activity (CDNB) were compared, there was little difference observed between the DNA-protective activities of PCB-induced and normal rat liver cytosols, yet cytosol from rat liver neoplasms was more protective. Purified GST-P (7-7), the GST isoenzyme most induced in carcinogen-generated rat liver neoplasms, was not protective when added at protein concentrations found to be protective for total GSTs isolated from these neoplasms. These studies demonstrate that the resistance of mouse liver to AFB1 can be explained primarily by a single constitutive GST isoenzyme (YaYa or 4-4) with a relatively high activity toward DNA-binding metabolites of AFB1. GST isoenzymes with such high specific DNA protective activity against AFB1 metabolites were not evident in human, rat, or rainbow trout liver or in PCB-induced or neoplastic rat liver preparations.

Topics: Aflatoxin B1; Aflatoxins; Animals; Carcinogens; Carcinoma, Hepatocellular; Cytosol; DNA; Electrophoresis, Polyacrylamide Gel; Glutathione Transferase; Humans; Isoenzymes; Liver; Liver Neoplasms; Liver Neoplasms, Experimental; Male; Mice; Microsomes, Liver; Polychlorinated Biphenyls; Rats; Sodium Dodecyl Sulfate; Trout

Human Hep G2 hepatoma and HT 1080 fibrosarcoma cells were cultured in large scale under conditions which allowed enhanced secretion of plasminogen activator inhibitor-1 (PAI-1). A modified urokinase was obtained by reacting urokinase with phenylmethylsulfonyl fluoride followed by alkali treatment. The resulting product, called anhydrourokinase, was found to reversibly bind the PAI-1 when immobilized on cyanogen bromide-activated Sepharose 4B beads. Using this affinity absorbent, we have purified PAI-1 from the cell-conditioned media. A number of differences have been observed during Hep G2 and HT 1080 PAI purification. 1) The PAI activity in Hep G2 medium concentrate is more stable, and the concentrate depleted of active PAI-1 showed spontaneous regeneration of PAI-1 activity. In contrast, the PAI activity in HT 1080 medium concentrate declines rapidly on standing. 2) Hep G2 PAI-1 invariably copurified with an adhesive protein, vitronectin or its NH2-terminal fragment, while pure HT 1080 PAI-1 alone was obtained by affinity purification on anhydrourokinase-Sepharose 4B. 3) Based on specific activity measurement and complex formation analysis using a sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis technique, the purified Hep G2 PAI-1 appears completely active while the HT 1080 PAI-1 is only one-fourth as active. SDS was found to exert dual effects on purified PAI-1s. SDS treatment partially inactivated a fully active Hep G2 PAI-1 and a moderately active HT 1080 PAI-1 but partially activated an HT 1080 PAI-1 whose activity had previously been allowed to decay to a very low level. Purified vitronectin was found to enhance and stabilize the PAI-1 activity of the partially active HT 1080 PAI-1. It is concluded that fully active PAI-1 in association with vitronectin can be isolated by anhydrourokinase-Sepharose 4B chromatography and that vitronectin is a binding protein for PAI-1 which activates and stabilizes PAI-1.

Topics: Carcinoma, Hepatocellular; Chromatography, Affinity; Culture Media; Drug Stability; Electrophoresis, Polyacrylamide Gel; Enzymes, Immobilized; Fibrosarcoma; Glycoproteins; Humans; Liver Neoplasms; Molecular Weight; Phenylmethylsulfonyl Fluoride; Plasminogen Activators; Plasminogen Inactivators; Sodium Dodecyl Sulfate; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator; Vitronectin

Catalytic activity of tissue-type plasminogen activator (t-PA) in plasma is regulated in part by formation of complexes with specific inhibitors as well as by hepatic clearance. Potential interaction of these two regulatory mechanisms was examined in the human hepatoma cell line Hep G2. These cells secrete plasminogen activator inhibitor type-1 (PAI-1) and initiate catabolism of exogenous t-PA by receptor-mediated endocytosis. Specific binding of 125I-t-PA to cells at 4 degrees C results in dose-dependent formation of a 95-kDa species recognized by monospecific anti-PAI-1 and anti-t-PA antibodies and stable in the presence of low (0.2%) concentrations of sodium dodecyl sulfate (SDS). Specific binding of 125I-t-PA and formation of the 95-kDa SDS-stable species are inhibited in a concentration-dependent manner following preincubation of cells with anti-PAI-1 antibodies. High and low molecular weight forms of urokinase plasminogen activator (u-PA) capable of forming specific complexes with PAI-1 complete for 125I-t-PA binding sites. However, the proenzyme form of u-PA (scu-PA), incapable of forming complexes with PAI-1, does not compete for 125I-t-PA binding sites. The role of the serine protease active site of t-PA in mediating both interaction with PAI-1 and specific binding was examined using 125I-t-PA that had been functionally inactivated with D-phenylalanyl-L-propyl-L-arginyl-chloromethyl ketone (PPACK). 125I-t-PA-PPACK, despite a 6-fold lower affinity than active 125I-t-PA, exhibited specific binding to cells without detectable formation of SDS-stable complexes with PAI-1. Both surface-bound 125I-t-PA and 125I-t-PA-PPACK are internalized and degraded by cells at 37 degrees C. 125I-t-PA is internalized as a stable complex with PAI-1, whereas 125I-t-PA-PPACK is internalized with similar kinetics but without the presence of an SDS-stable complex. Thus, PAI-1 appears capable of modulating t-PA catabolism in the human hepatocyte.

Topics: Antibodies, Monoclonal; Binding Sites; Binding, Competitive; Carcinoma, Hepatocellular; Cell Membrane; Endocytosis; Glycoproteins; Humans; Liver Neoplasms; Macromolecular Substances; Molecular Weight; Plasminogen Inactivators; Protein Binding; Sodium Dodecyl Sulfate; Tissue Plasminogen Activator; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

We examined 19 hepatoma tissues for alkaline phosphatase isoenzyme and found that six have both the Kasahara isoenzyme and an alkaline phosphatase with a unique electrophoretic mobility, in addition to the liver-type enzyme. From two of six carcinoma tissues, the abnormal enzyme was partly purified and subjected to a detailed analysis, which clarified that the abnormal enzyme resembled a fetal intestinal alkaline phosphatase in most of its enzymic and immunologic properties and also in properties that reflect enzyme structure. This fetal intestinal-type alkaline phosphatase was not found in 24 specimens of normal liver from adults. The relevance of fetal intestinal-type alkaline phosphatase to Kasahara isoenzyme and adult intestinal alkaline phosphatase is discussed. The fetal and adult intestinal alkaline phosphatases differ in electrophoretic mobility, heat stability, and reactivity with concanavalin A. The adult-type enzyme has two components; only the electrophoretically slower, neuraminidase-resistant one is described here.

Topics: Adult; Alkaline Phosphatase; Amino Acids; Carcinoma, Hepatocellular; Drug Stability; Female; Fetus; Humans; Immunodiffusion; Intestines; Isoenzymes; Liver Neoplasms; Pregnancy; Sodium Dodecyl Sulfate

The feeding of carcinogenic 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) in the early stages results in a change in the protein composition of the nuclear ribonucleoprotein particles of the rat liver. These particles are associated with newly synthesized RNA and it is assumed that they are involved in the processing and in the transport of this RNA. After 6 weeks of feeding of this azocarcinogen, the amount of one of the main polypeptides (apparent molecular weight 42 000) is decreased and after 10 weeks of feeding the particles are devoid of this polypeptide completely. Feeding of the non-carcinogenic p-aminoazobenzene (AB) is without any effect. The loss of this polypeptide is not characteristic for the malignant transformation. In the nuclear ribonucleoprotein particles isolated from hepatoma which has been induced by 3'-MeDAB this polypeptide is present in even higher proportion to other polypeptides than it is in particles isolated from liver cells of control animals. The 3'-MeDAB binds to the proteins of the liver nuclear ribonucleoprotein particles and interferes with the RNA processing. It is proposed that the changes in the composition of the protein moiety of the particles reflect changes in the population of liver cells leading finally to the selection of hepatoma cells which are resistant to the toxic effect of 3'-MeDAB on RNA processing.

Topics: Animals; Carcinoma, Hepatocellular; Cell Nucleus; Cell Transformation, Neoplastic; Electrophoresis, Polyacrylamide Gel; Liver; Liver Neoplasms; Methyldimethylaminoazobenzene; Neoplasm Proteins; Nucleoproteins; p-Dimethylaminoazobenzene; Rats; Ribonucleoproteins; Sodium Dodecyl Sulfate; Spectrum Analysis

Phosphoenolpyruvate carboxykinase (GTP) was induced by a combination of dibutyryl cyclic AMP, theophyline and dexamethasone in Reuber H35 hepatoma cells under conditions where an amino acid in the medium was replaced by an appropriate analogue. 2. With canavanine replacing arginine or with 5-fluorotryptophan or 6-fluorotryptophan replacing tryptophan the induced enzyme had a lower catalytic activity-relative to antibody reactivity. 3. These aberrant enzyme molecules were heat-labile in vitro. 4. Measurements of enzyme degradation in vivo indicated that the canavanine-containing enzyme and the 6-fluorotryptophan-containing enzyme were degraded more rapidly than the enzyme containing all natural amino acids.

Topics: Amino Acids; Antibodies; Bucladesine; Canavanine; Carcinoma, Hepatocellular; Cell Line; Dexamethasone; Electrophoresis, Polyacrylamide Gel; Enzyme Induction; Guanosine Triphosphate; Liver Neoplasms; Neoplasms, Experimental; Phosphoenolpyruvate Carboxykinase (GTP); Sodium Dodecyl Sulfate; Theophylline; Tryptophan

Using precipitating antibodies to ACI rat liver ferritin and to sodium-dodecyl-sulfate-dissociated protein subunits of ACI rat liver ferritin, we have demonstrated the presence of ferritin-positive sites and subunit-positive sites in situ in several rat hepatoma cell lines by immunofluorescence. Hepatoma cells from three transplantable rat hepatomas (Reuber H-139, Reuber H-35, and Morris 5123) were explanted and propagated. Rabbit antibodies specific for either protein subunits of ferritin or ferritin were prepared by affinity chromatography or by dissociation of antibody-antigen complexes with 0.1 M acetic acid followed by differential ultracentrifugation. Explants of Reuber H-139, Reuber H-35, and Morris 5123 hepatoma cells, grown either in ordinary McCoy's 5a medium or in such medium enriched with iron (0.002% Fe), gave positive immunofluorescence for subunits as well as ferritin. Exposure of a clonal strain of Morris 5123 hepatoma cells to iron-enriched culture medium for varying lengths of time of up to 24 hours resulted in progressive increase in the quantity of ferritin-specific immunofluorescent cytoplasmic material, which was at first present diffusely, and later in clumps. By contrast, during the initial 24-hour period, subunit-specific immunofluorescence remained at relatively low intensity, with diffuse distribution through the cytoplasma. Our findings indicate a) the presence, in the cytoplasm, of the three kinds of hepatoma cells, of unassembled or only partly assembled subunits of fragments of subunits as well as of ferritin, and b) rapid assembly of the protein subunits into apoferritin and ferritin after administration of iron, so that the concentration of subunits in the cytoplasm was not significantly increased.

Topics: Animals; Antibodies; Antibody Specificity; Apoferritins; Carcinoma, Hepatocellular; Cells, Cultured; Chromatography, Affinity; Culture Media; Female; Ferritins; Fluorescent Antibody Technique; Immunoelectrophoresis; Immunologic Techniques; In Vitro Techniques; Iron; Liver Neoplasms; Neoplasms, Experimental; Rabbits; Rats; Rats, Inbred ACI; Sodium Dodecyl Sulfate; Time Factors; Ultracentrifugation

Topics: Alkaline Phosphatase; Animals; Carcinoma, Hepatocellular; Electrophoresis, Paper; Electrophoresis, Polyacrylamide Gel; Liver; Liver Neoplasms; Macromolecular Substances; Male; Neoplasms, Experimental; Organophosphorus Compounds; Phosphates; Phosphoproteins; Phosphorus Radioisotopes; Rats; Serine; Sodium Dodecyl Sulfate; Tyrosine Transaminase

Topics: Adenine Nucleotides; Animals; Base Sequence; Carcinoma, Hepatocellular; Chromatography, Gel; Electrophoresis, Starch Gel; Liver; Liver Neoplasms; Male; Neoplasms, Experimental; Phosphorus Radioisotopes; Polynucleotides; Polyribosomes; Rats; RNA, Neoplasm; RNA, Ribosomal; Sodium Dodecyl Sulfate

Topics: Animals; Carcinoma, Hepatocellular; Cell Fractionation; Chromatin; Chromosomes; DNA; Electrophoresis, Polyacrylamide Gel; Histones; Liver; Liver Neoplasms; Molecular Weight; Neoplasm Transplantation; Neoplasms, Experimental; Nucleoproteins; Rats; Sodium Dodecyl Sulfate

Topics: Amino Acids; Animals; Antigen-Antibody Reactions; Carcinoma, Hepatocellular; Cells, Cultured; Drug Stability; Electrophoresis, Polyacrylamide Gel; Electrophoresis, Starch Gel; Glyoxylates; Hot Temperature; Immunodiffusion; Immunoelectrophoresis; L-Lactate Dehydrogenase; Liver; Liver Neoplasms; Male; Molecular Weight; Neoplasms, Experimental; Rabbits; Sodium Dodecyl Sulfate; Spectrophotometry, Ultraviolet

Topics: Animals; Bacterial Proteins; Carbon Radioisotopes; Carcinoma, Hepatocellular; Costs and Cost Analysis; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Evaluation Studies as Topic; Indicators and Reagents; Liver Neoplasms; Methods; Microchemistry; Molecular Weight; Neoplasm Proteins; Neoplasms, Experimental; Rats; Ribosomes; Sodium Dodecyl Sulfate; Species Specificity; Sulfur Radioisotopes; Time Factors

The replacement of thymidine by 5-bromodeoxyuridine in DNA leads to a greatly enhanced stability of chromatin from hepatoma tissue culture or embryonic rat pancreas, as measured by thermal chromatography on hydroxylapatite. The increased stability is directly correlated with the degree of bromodeoxyuridine substitution. On the other hand, the incorporation of bromodeoxyuridine into DNA results in a modest stabilization of purified DNA. Substitution of nucleotide also alters slightly the hyperchromicity profile generated during the thermal denaturation of purified DNA and chromatin. The observed changes can best be explained by an altered interaction between the bromodeoxyuridine-DNA and other chromatin components, presumably proteins. These results suggests that the selective effects of bromodeoxyuridine on cytodifferentiation may be due to an increased affinity of regulatory proteins for bromodeoxyuridine-DNA.

Topics: Animals; Bromodeoxyuridine; Carbon Radioisotopes; Carcinoma, Hepatocellular; Cells, Cultured; Chromatin; Chromatography; DNA; Hot Temperature; Liver Neoplasms; Nucleic Acid Denaturation; Pancreas; Protein Binding; Rats; Sodium Dodecyl Sulfate

Topics: Amino Acid Sequence; Animals; Carcinoma, Hepatocellular; Electrophoresis, Polyacrylamide Gel; Genes; Genotype; Liver; Liver Neoplasms; Molecular Weight; Muscles; Neoplasms, Experimental; Peptides; Phosphotransferases; Rats; Sodium Dodecyl Sulfate; Ultracentrifugation

Topics: Animals; Binding Sites; Carbon Radioisotopes; Carcinogens; Carcinoma, Hepatocellular; Cell Nucleus; Electrophoresis, Polyacrylamide Gel; Ethylmaleimide; Isoelectric Focusing; Liver; Liver Neoplasms; Male; Molecular Weight; Nitrosamines; Nucleoproteins; Protein Binding; Rats; Sodium Dodecyl Sulfate; Time Factors; Urea

Topics: Animals; Carcinoma, Hepatocellular; Cell Nucleus; Electrophoresis, Polyacrylamide Gel; Liver; Liver Neoplasms; Mercaptoethanol; Microscopy, Electron; Molecular Weight; Neoplasm Proteins; Neoplasms, Experimental; Nucleoproteins; p-Dimethylaminoazobenzene; Peptides; Phosphotungstic Acid; Rats; Sodium Dodecyl Sulfate; Staining and Labeling; Urea; Vibration

Topics: Animals; Bleomycin; Carcinoma, Hepatocellular; Cells, Cultured; Centrifugation, Density Gradient; Cycloheximide; Dactinomycin; DNA Repair; DNA, Neoplasm; DNA, Single-Stranded; Drug Resistance; Liver Neoplasms; Neoplasms, Experimental; Rats; Sodium Dodecyl Sulfate; Thymidine; Tritium

A two-dimensional polyacrylamide gel electrophoresis technique is presented for separation of acid-extracted proteins of isolated nucleoli of normal rat liver and Novikoff hepatoma ascites cells. About 100 distinct protein spots were resolved. Comparison of the patterns for normal liver and Novikoff hepatoma revealed nine spots with markedly different intensities in the two patterns. Two spots were found that were unique to the normal liver pattern, and one spot was found that was unique to the hepatoma pattern.

Topics: Acetates; Animals; Buffers; Carcinoma, Hepatocellular; Cell Nucleolus; Electrophoresis, Polyacrylamide Gel; Histones; Liver; Liver Neoplasms; Methods; Neoplasm Proteins; Neoplasms, Experimental; Proteins; Rats; Sodium Dodecyl Sulfate

Topics: Animals; Carbon Isotopes; Carcinoma, Hepatocellular; Centrifugation, Density Gradient; Cholesterol; Citrates; Diphosphates; Electrophoresis, Polyacrylamide Gel; Half-Life; Leucine; Liver; Liver Neoplasms; Male; Methods; Microsomes, Liver; Molecular Weight; Neoplasm Proteins; Neoplasms, Experimental; Phospholipids; Polytetrafluoroethylene; Proteins; Rats; Sodium Dodecyl Sulfate; Urea

Topics: Animals; Carbon Radioisotopes; Carcinoma, Hepatocellular; Cell Fractionation; Electrophoresis, Polyacrylamide Gel; Female; Leucine; Liver Neoplasms; Male; Mitochondria, Liver; Neoplasm Proteins; Neoplasms, Experimental; Protein Biosynthesis; Rats; Sodium Dodecyl Sulfate

Topics: Acrylamides; Animals; Antibody Formation; Blood Preservation; Carcinoma, Hepatocellular; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis; Electrophoresis, Starch Gel; Female; Fetal Proteins; Gestational Age; Humans; Immunodiffusion; Immunoelectrophoresis; Iodine Isotopes; Isoelectric Focusing; Liver Neoplasms; Macromolecular Substances; Molecular Weight; Neuraminidase; Pregnancy; Rabbits; Radioimmunoassay; Sodium Dodecyl Sulfate