guanosine-triphosphate and Liver-Neoplasms

Lipoylated enzymes such as the E2 component of pyruvate dehydrogenase complex (PDC-E2) are targets for autoreactive immune responses in primary biliary cirrhosis, with lipoic acid itself forming a component of the dominant auto-epitopes. A candidate mechanism for the initiation of tolerance breakdown in this disease is immune recognition of neo-antigens formed by xenobiotic substitution of normal proteins. Importantly, sensitization with proteins artificially substituted with the lipoic acid analogue xenobiotic 6-bromohexanoic acid (6BH) can induce an immune response that cross-reacts with PDC-E2. This study investigated the potential of recombinant lipoylation enzymes lipoate activating enzyme and lipoyl-AMP(GMP):N-lysine lipoyl transferase to aberrantly incorporate xenobiotics into PDC-E2. It was found that these enzymes could incorporate lipoic acid analogues including octanoic and hexanoic acids and the xenobiotic 6BH into PDC-E2. The efficiency of incorporation of these analogues showed a variable dependence on activation by adenosine triphosphate (ATP) or guanosine triphosphate (GTP), with ATP favoring the incorporation of hexanoic acid and 6BH whereas GTP enhanced substitution by octanoic acid. Importantly, competition studies showed that the relative incorporation of both 6BH and lipoic acid could be regulated by the balance between ATP and GTP, with the formation of 6BH-substituted PDC-E2 predominating in an ATP-rich environment.. Using a well-defined system in vitro we have shown that an important xenobiotic can be incorporated into PDC in place of lipoic acid by the exogenous lipoylation system; the relative levels of lipoic acid and xenobiotic incorporation may be determined by the balance between ATP and GTP. These observations suggest a clear mechanism for the generation of an auto-immunogenic neo-antigen of relevance for the pathogenesis of primary biliary cirrhosis.

Topics: Adenosine Triphosphate; Animals; Autoimmunity; Caproates; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Endometrium; Epithelial Cells; Escherichia coli; Female; Guanosine Triphosphate; HeLa Cells; Humans; Jurkat Cells; Lipoylation; Liver Cirrhosis, Biliary; Liver Neoplasms; Plasmids; Pyruvate Dehydrogenase Complex; Thioctic Acid; Xenobiotics

The phosphorylation of insulin-like growth factor binding protein-I (IGFBP-1) alters its binding affinity for insulin-like growth factor I (IGF-I) and thus regulates the bioavailability of IGF-I for binding to the IGF-I receptor. The kinase(s) responsible for the phosphorylation of IGFBP-1 has not been identified. This study was designed to characterize the IGFBP-1 kinase activity in HepG2 human hepatoma cells, a cell line that secretes IGFBP-1 primarily as phosphorylated isoforms. IGFBP-1 kinase activity was partially purified from detergent extracts of the cells by phosphocellulose chromatography and gel filtration. Two kinases of approximate M(r) 150,000 (peak I kinase) and M(r) 50,000 (peak II kinase) were identified. Each kinase phosphorylated IGFBP-1 at serine residues that were phosphorylated by intact HepG2 cells. The kinases were distinct based on their differential sensitivity to inhibition by heparin (IC50 = 2.5 and 16.5 micrograms/ml, peak I and II kinase, respectively) and inhibition by the isoquinoline sulfonamide CKI-7 (IC50 = 50 microM and 100 microM, peak I and II kinase, respectively). In addition, a tenfold molar excess of nonradioactive GTP relative to [gamma-32P]ATP lowered the incorporation of 32P into IGFBP-1 by 80% when the reaction was catalyzed by the peak I kinase, whereas GTP had no effect on the reaction catalyzed by the peak II kinase. In the presence of polylysine, IGFBP-1 was radiolabeled by the partially purified kinase activity when [gamma-32P]GTP served as the phosphate donor indicating the presence of casein kinase II activity. Furthermore, IGFBP-1 was phosphorylated by purified casein kinase I and casein kinase II at sites phosphorylated by the peak I and II kinases. Our data suggest that at least two kinases could be responsible for the phosphorylation of IGFBP-1 in intact HepG2 cells and that the kinases are related to the casein kinase family of protein kinases.

Topics: Amino Acid Sequence; Carcinoma, Hepatocellular; Casein Kinase II; Casein Kinases; Enzyme Inhibitors; Guanosine Triphosphate; Heparin; Humans; Insulin-Like Growth Factor Binding Protein 1; Isoquinolines; Liver Neoplasms; Molecular Sequence Data; Molecular Weight; Peptide Mapping; Phosphorylation; Phosphoserine; Protein Kinases; Protein Serine-Threonine Kinases; Tumor Cells, Cultured

The fusion capacity of rough endoplasmic reticulum membranes isolated from dissected liver tumor nodules of aflatoxin-treated rats was determined by cell free assay to be greater than that of homologous membranes from control liver. In a first attempt to understand the reason for this difference we compared the content of ras-related proteins in rough microsomal fractions and other cell fractions of both dissected tumor nodules and control liver. Using [alpha-32P]GTP blot overlay and densitometric analysis, homogenate, Golgi and rough endoplasmic reticulum fractions from dissected tumor nodules were observed to contain increased amounts of [alpha-32P]GTP binding to ras-related proteins when compared to homologous control fractions. Western blot analysis indicated that ras content was also increased in the tumor fractions. [alpha-32P]GTP-blot overlay using double-dimensional SDS-polyacrylamide gel electrophoresis confirmed quantitative differences in the amount of [alpha-32P]GTP binding to ras-related proteins between fractions from tumor and control tissues and indicated a surprising number of such proteins in each fraction. The data suggest that the changes in ras-related proteins could, in part, account for the enhanced GTP-dependent fusion capacity observed for the tumor-derived membranes.

Topics: Aflatoxin B1; Animals; Carcinoma, Hepatocellular; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum, Rough; Golgi Apparatus; Guanosine Triphosphate; Intracellular Membranes; Liver Neoplasms; Male; Membrane Fusion; Microsomes, Liver; Protein Binding; ras Proteins; Rats; Rats, Inbred F344; Subcellular Fractions

The liver was the organ most frequently involved in 50 patients with primary melanomas of the choroid or ciliary body, who were treated with enucleation or cobalt plaque radiotherapy and who subsequently developed systemic metastasis. Forty-seven (94%) of the patients had clinical or laboratory evidence of liver metastasis. The median survival time following treatment of the primary choroidal melanoma was 28 months (range, 7.7 to 123.1 months). Results of pretreatment studies, including serum liver enzyme levels, liver scans, or both, were abnormal in six (12%) of the patients studied. As a group, the eight patients who died within one year following ocular surgery had a significantly higher prevalence of pretreatment laboratory abnormalities. Extraocular extension of the choroidal melanoma was also present in four (50%) of these eight patients. Conversely, no patient who survived longer than the median survival period (28 months) had abnormal pretreatment laboratory findings.

Topics: Adult; Aged; Alkaline Phosphatase; Aspartate Aminotransferases; Female; Guanosine Triphosphate; Humans; L-Lactate Dehydrogenase; Liver Neoplasms; Male; Melanoma; Middle Aged; Radiography; Uveal Neoplasms

A Met-tRNAf binding factor (IF-2) from the microsomal fraction of rat liver and rat hepatoma ascites cells was partially purified by ammonium sulphate fractionation, DEAE-cellulose and phosphocellulose chromatography. The factor binds [3H]Met-tRNAf only in the presence of either GTP or GMPPCP. Maximal binding takes place at 37 degrees C and in the absence of Mg++. The factor is specific for Met-tRNAf and does not bind Phe-tRNA from rat liver or from E. coli. The ternary complex [Met-tRNAf . IF-2 . GTP1 binds to 40 S ribosomal subunits from rat liver in the absence of mRNA or poly(A, G, U) without GTP hydrolysis. GDP as well as aurintricarboxylic acid inhibit the ternary complex formation. Both factors are rapidly inactivated by N-ethylmaleimide treatment and by preincubation at 45 degrees C. Heat inactivation is partially prevented by GTP and GDP. With regard to the functional properties there are no significant differences between IF-2 from normal liver and hepatoma cells. On the other hand heat denaturation compared to the rat liver factor, which may be due to differences in contaminating proteins.

Topics: Animals; Carcinoma, Hepatocellular; Ethylmaleimide; Guanosine Diphosphate; Guanosine Triphosphate; Hot Temperature; In Vitro Techniques; Liver Neoplasms; Microsomes, Liver; Peptide Initiation Factors; Rats; RNA, Transfer

1. Adenylate cyclase in plasma membranes from rat liver was stimulated by prostaglandin E1, and to a lesser extent by prostaglandin E2. Prostaglandin F1alpha and A1 did not stimulate the cyclase. The prostaglandin E1-mediated activation was found to require GTP when the substrate ATP concentration was reduced from 3 mM to 0.3 mM in the reaction mixture. Adenylate cyclase of the plasma membranes from rat ascites hepatomas AH-130 and AH-7974 was not stimulated by prostaglandin E1 in the presence or the absence of GTP, although the basal activity of adenylate cyclase as well as its stimulation by GTP alone were similar to normal liver plasma membranes. 2. Liver plasma membranes were found to have two specific binders for [3H] prostaglandin E1 with dissociation constants of 17.6-10(-9) M and 13.6-10(8) M (37 degrees C) and one specific binder for [3H]prostaglandin F2alpha with a dissociation constant of 2.31-10(8) M (37 degrees C). The specific binders for prostaglandin E1 could not be detected in the hepatoma plasma membranes. 3. Binding of [3H] prostaglandin E1 to the liver plasma membranes was exchange by, GTP dGPT, GDP, ATP and GMP-P(N)P, but not by GMP, CGMP, DTTP, UTP or CTP. The increase in the binding of [3H] prostaglandin E1 was found to be due to the increased affinity of the specific binders to prostaglandin F2alpha was not affected by GTP. 4. GTP alone was found to increase V of adenylate cyclase of liver plasma membranes, while GTP plus prostaglandin E1 was found to decrease Km of adenylate cyclase in addition to the increase of V to a further extent.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Animals; Binding Sites; Carcinoma, Hepatocellular; Cell Membrane; Guanosine Triphosphate; Kinetics; Liver; Liver Neoplasms; Male; Prostaglandins A; Prostaglandins E; Prostaglandins F; Rats; Receptors, Cell Surface; Receptors, Prostaglandin

Topics: Adenylyl Cyclases; Animals; Carcinoma, Hepatocellular; Catecholamines; Enzyme Activation; Epinephrine; Female; Fluorides; Glucagon; Guanosine Triphosphate; Liver; Liver Neoplasms; Neoplasms, Experimental; Rats; Receptors, Adrenergic

Adenylosuccinate synthetase has been partially purified from Novikoff ascites tumor cells. The properties of the protein are quite different from the enzyme from rat liver in that the Km for asparate is higher and the K1 for the feedback inhibitor AMP is also higher. The antibiotic hadacidin has a preferential inhibitory effect on the tumor enzyme. These results suggest that the Novikoff ascites tumor enzyme is less sensitive to normal feedback controls but may be more sensitive to specific antitumor drugs.

Topics: Adenosine Monophosphate; Animals; Anti-Bacterial Agents; Aspartic Acid; Carcinoma, Hepatocellular; Feedback; Guanosine Triphosphate; Inosine Nucleotides; Kinetics; Ligases; Liver Neoplasms; Neoplasms, Experimental; Rats

(1) The apparent [3H]epinephrine binding parameters of plasma membranes from rat liver and ascites hepatomas such as AH-7974, AH-371A and AH-130, as measured by equilibrium dialysis and/or Millipore filtration, were almost similar to each other. The epinephrine binding sites in the plasma membranes were heterogenous (alpha, beta-receptors and non specific sites), but the pattern of these binding sites in the liver membranes appeared almost similar to that in the hepatoma membranes. 2. The beta-receptor seemed to be specifically involved in the epinephrine-mediated activation of adenylate cyclase of the liver membranes. In spite of the presence of almost similar beta-receptors and adenylate cyclase, the adenylate cyclase of hepatoma membranes was found to be less sensitive to the epinephrine-mediated activation. 3. GTP alone was found to activate adenylate cyclase of liver and hepatoma membranes to some extents when the concentration of ATP was lower (0.3 mM). When GTP was added with epinephrine, a marked, synergistic activation of adenylate cyclase was observed in liver plasma membranes, but not in hepatoma ones. 4. The synergistic activation of adenylate cyclase by epinephrine plus GTP showed a characteristic kinetic feature, reaching a maximal peak within 1 min or so after mixing. 5. Binding of [3H]epinephrine to liver membranes proceeded monophasically in the absence of GTP, while it proceeded biphasically in the presence of GTP, showing the retardation of binding at some earlier stages. GTP added at the time of binding equilibrium induced the temporary release of bound epinephrine from the beta-receptors. The GTP-induced temporary release of bound epinephrine, occurring within 4-5 min after the addition of GTP, was less marked in the hepatoma membranes as compared with the liver membranes. 6. Possible impairment of the GTP-dependent coupling mechanism in the receptor-adenylate cyclase system of hepatoma plasma membranes was suggested.

Topics: Adenylyl Cyclases; Animals; Carcinoma, Hepatocellular; Cell Membrane; Enzyme Activation; Epinephrine; Guanosine Triphosphate; Kinetics; Liver; Liver Neoplasms; Male; Neoplasms, Experimental; Propranolol; Rats; Receptors, Cell Surface

Examination of nucleolar RNA from cultured Novikoff hepatoma cells treated for 3 hr with 5 x 10-4 M 5-azacytidine shows that significant amounts of analog-substituted 45S RNA are processed to the 32S RNA species, but 28S RNA formation is completely inhibited. Under these conditions of analog treatment 37% of the cytidine residues in the 45S RNA is replaced by 5-azacytidine. During coelectrophoresis of nucleolar RNA from 5-azacytidine-treated and control cells, the analog-substituted 45S RNA and 32S RNA display reduced mobilities compared to the control 45S RNA and 32S RNA. Coelectrophoresis of analog-substituted and control RNA after formaldehyde denaturation shows no differences in electrophoretic mobility between the two RNA samples, suggesting that 5-azacytidine incorporation may alter the secondary structure of the 45S RNA and the 32S RNA. 5-Azacytidine at 5 x 10-4 M severely inhibits protein synthesis in Novikoff cells by 3 hr. After this length of treatment, however, CsCl buoyant density analysis reveals no difference in density of either the 80S or 55S preribosomal ribonucleoprotein particles when compared to normal particles. Also 5-azacytidine treatment does not appear to cause major changes in the polyacrylamide gel electrophoresis patterns of the proteins in the 80S and 55S preribosomal particles. These results together with previous findings suggest that 5-azacytidine's inhibition of rRNA processing is possibly related to its alteration of the structure of the ribosomal precursor RNAs and is not a consequence of a general inhibition of ribosomal protein formation.

Topics: Animals; Azacitidine; Carcinoma, Hepatocellular; Cell Nucleolus; Cells, Cultured; Guanosine Triphosphate; Liver Neoplasms; Neoplasms, Experimental; Rats; Ribosomes; RNA, Neoplasm; RNA, Ribosomal

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

Topics: Adenosine Triphosphate; Animals; Binding Sites; Calcium; Carcinoma, Hepatocellular; Cell Nucleus; Centrifugation, Density Gradient; Chromatin; Chromatography, Gel; Chromatography, Ion Exchange; Cytosine Nucleotides; DNA Nucleotidyltransferases; Escherichia coli; Guanosine Triphosphate; Liver Neoplasms; Magnesium; Manganese; Molecular Weight; Neoplasms, Experimental; Rats; Sulfhydryl Reagents; Templates, Genetic; Thymine Nucleotides; Tritium

Topics: Animals; Cell-Free System; Chickens; Escherichia coli; Guanosine Triphosphate; Kinetics; Leukemia, Experimental; Leukemia, Myeloid, Acute; Liver; Liver Neoplasms; Phenylalanine; Protein Biosynthesis; Ribosomes; RNA, Messenger; RNA, Transfer; Tritium

Topics: Adenosine Triphosphate; Animals; Carcinoma, Hepatocellular; Cell Nucleus; Chromatography, DEAE-Cellulose; Cytoplasm; Cytosine Nucleotides; DNA Nucleotidyltransferases; Endoplasmic Reticulum; Guanosine Triphosphate; Liver; Liver Neoplasms; Liver Regeneration; Male; Microsomes, Liver; Molecular Weight; Polynucleotides; Rats; Ribosomes; Templates, Genetic; Thymine Nucleotides

Topics: Adenosine Triphosphate; Ammonium Sulfate; Animals; Buffers; Carcinoma, Hepatocellular; Cell Nucleus; Centrifugation, Density Gradient; Chromatography, DEAE-Cellulose; Chromatography, Gel; Cytoplasm; Cytosine Nucleotides; Deoxyribonucleases; DNA; DNA Nucleotidyltransferases; Guanosine Triphosphate; Hydrogen-Ion Concentration; Liver; Liver Neoplasms; Male; Neoplasms, Experimental; Rats; Sodium Chloride; Thymine Nucleotides; Tritium

An enzyme that polymerizes adenylate residues from adenosine triphosphate was prepared from rat liver mitochondria and compared to similar preparations from the mitochondria of three hepatomas. Enzyme activity in the hepatomas was only 1 to 2 percent of that in normal liver.

Topics: Adenosine Triphosphate; Animals; Carcinoma, Hepatocellular; Cytosine Nucleotides; Guanosine Triphosphate; Liver Neoplasms; Mitochondria, Liver; Neoplasms, Experimental; Nucleotidyltransferases; Rats; Ribonucleases; RNA Nucleotidyltransferases; Tritium; Uracil Nucleotides

Topics: Adenosine Triphosphate; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Bleomycin; Bone Marrow; Carcinoma, Hepatocellular; Cytosine Nucleotides; Dactinomycin; Deoxyribonucleotides; DNA Nucleotidyltransferases; DNA, Neoplasm; Fluorouracil; Guanosine Triphosphate; Liver; Liver Neoplasms; Liver Regeneration; Mitomycins; Neoplasms, Experimental; Rats; Rifampin; Thymidine; Tritium