cyclic-gmp and Liver-Neoplasms

Nitric oxide (NO) is a lipophillic, highly diffusible, and short-lived physiological messenger which regulates a variety of physiopathological responses. NO may exert its cellular action through cGMP-dependent and cGMP-independent pathways which includes different postranslational modifications. The effect of NO in cancer depends on the activity and localization of NOS isoforms, concentration and duration of NO exposure, cellular sensitivity, and hypoxia/re-oxygenation process. NO regulates critical factors such as the hypoxia inducible factor-1 (HIF-1) and p53 generally leading to growth arrest, apoptosis or adaptation. NO sensitizes hepatoma cells to chemotherapeutic compounds probably through increased p53 and cell death receptor expressions.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cyclic GMP; Humans; Hypoxia-Inducible Factor 1; Liver Neoplasms; Nitric Oxide; Nitric Oxide Synthase; Tumor Suppressor Protein p53

Topics: Animals; Arterial Occlusive Diseases; Arteries; Arteriovenous Fistula; Carcinoma, Hepatocellular; Cardiomegaly; Chemical Phenomena; Chemistry; Cyclic GMP; Humans; Hydrogen-Ion Concentration; Liver Neoplasms; Metals; Protein Kinases; Proteins; Substrate Specificity; Veins

Topics: alpha-Fetoproteins; Animals; Carcinoma, Hepatocellular; Cell Division; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Liver Neoplasms; Neoplasms, Experimental; Polyamines; Rats; Thymidine; Trace Elements

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenylyl Cyclases; Carcinoma, Hepatocellular; Cell Division; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Cyclic AMP; Cyclic GMP; Cytosine Nucleotides; DNA; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; Growth Substances; Liver; Liver Neoplasms; Lymphocyte Activation; Lymphocytes; Melanoma; Mitogens; Neoplasms; Skin; Thymus Gland

βig-h3 is a TGF-β (transforming growth factor β)-induced ECM (extracellular matrix) protein that induces the secretion of MMPs (matrix metalloproteinases). However, the mechanism of induction is yet to be established. In this study, siRNAs (small interfering RNAs) targeted against βig-h3 were transfected into SMMC-7721 cells [a HCC (human hepatocellular carcinoma) cell line] to knockdown the expression of βig-h3. We found that NiCl2, a potent blocker of extracellular Ca2+ entry, reduced βig-h3-induced secretion of MMP-2 and -9. Further investigation suggested that reduction in the levels of βig-h3 decreased the secretion of MMP-2 and -9 that was enhanced by an increase in the concentration of extracellular Ca2+. SNAP (S-nitroso-N-acetylpenicillamine), a NO (nitric oxide) donor, and 8-Br-cGMP (8-bromo-cGMP) inhibited thapsigargin-induced Ca2+ entry and MMP secretion in the invasive potential of human SMMC-7721 cells. Further, the inhibitory effects of 8-Br-cGMP and SNAP could be significantly enhanced by down-regulating βig-h3. βig-h3 attenuates the negative regulation of NO/cGMP-sensitive store-operated Ca2+ entry. Our findings suggest that the expression of βig-h3 might play an important role in the regulation of store-operated Ca2+ entry to increase the invasive potential of HCC cells.

Topics: Calcium; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclic GMP; Extracellular Matrix Proteins; Fluorescent Antibody Technique; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Nickel; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; S-Nitroso-N-Acetylpenicillamine; Thapsigargin; Transfection; Transforming Growth Factor beta

It is well known that glucose is a major energy source in tumors and that mitochondria are specialized organelles required for energy metabolism. Previous studies have revealed that nitric oxide (NO) protects against glucose depletion-induced cytotoxicity in mouse liver cells and in rat hepatocytes, but the detailed mechanism is not well understood. Therefore, we investigated the involvement of mitochondria in the NO protective effect in human hepatoma HepG2 cells. In this study, we showed that glucose depletion resulted in a time-dependent decrease in intracellular NO and in the protein expression of NO synthases. This glucose depletion-induced decrease in NO was blocked by NO donors. Next, we showed that the cytoprotective effect of NO is via a cyclic guanosine 3',5'-monophosphate-dependent pathway. Additionally, SNP blocked a glucose depletion-induced decrease in mitochondrial mass, mitochondrial DNA copies, and ATP level in HepG2 cells. Moreover, glucose depletion decreased the expression of various mitochondrial proteins, including cytochrome c, complex I (NADH dehydrogenase), complex III (cytochrome c reductase), and heat shock protein 60; these glucose depletion-induced effects were blocked by SNP. Furthermore, we found that rotenone and antimycin A (mitochondria complex I and III inhibitors, respectively) blocked SNP cytoprotection against glucose depletion-induced cytotoxicity. Taken together, our results indicated that the mitochondria serve as an important cellular mediator of NO during protection against glucose deprivation-induced damage.

Topics: Carcinoma, Hepatocellular; Cell Line; Cell Survival; Cyclic GMP; DNA Primers; DNA, Mitochondrial; Gene Expression Regulation, Neoplastic; Glucose; Humans; Kinetics; Liver Neoplasms; Mitochondria, Liver; Nitric Oxide; Polymorphism, Single Nucleotide

Prostacyclin, the major cyclooxygenase-derived product of arachidonic acid formed in the vasculature, mediates its potent anti-thrombotic and anti-proliferative effects through its G protein-coupled receptor (GPCR) termed the IP. Unlike many GPCRs, agonist-induced internalization of the IP occurs in an arrestin/GPCR kinase-independent manner. However, deletion of the IP COOH-terminal region prevented internalization suggesting that protein interactions at this region are involved in IP regulation. Using the COOH-terminal region of IP as bait we identified the delta subunit of cGMP phosphodiesterase 6 (PDE6delta) as a novel hIP-interacting protein in two independent yeast two-hybrid screens. Interaction of IP and PDE6delta was confirmed by co-immunoprecipitation in HEK293 cells, and in HEPG2 cells, which endogenously express neither IP nor PDE6delta. IP isoprenylation was critical for this interaction, as PDE6delta was unable to associate with an isoprenylation-deficient mutant IP (IPSSLC). PDE6delta overexpression altered the temporal pattern of agonist-induced internalization of IP, but not IPSSLC, in HEPG2 cells, increasing initial internalization but facilitating the return of IP to the cell surface despite the continued presence of agonist. Depletion of PDE6delta using short interfering RNA abolished cicaprost-induced IP internalization in human aortic smooth muscle cells. Recycling of IP, but not IPSSLC, upon agonist removal was facilitated by overexpression of PDE6delta. Thus PDE6delta interacts specifically with IP to modulate receptor trafficking.

Topics: Aorta; Carcinoma, Hepatocellular; Cells, Cultured; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Endocytosis; Humans; Kidney; Liver Neoplasms; Mutagenesis, Site-Directed; Mutation; Myocytes, Smooth Muscle; Phosphoric Diester Hydrolases; Protein Prenylation; Receptors, Prostaglandin; Saccharomyces cerevisiae; Two-Hybrid System Techniques

This study delineates the antiproliferative activities and in vivo efficacy of YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole] in human hepatocellular carcinoma cells. YC-1 inhibited the growth of HA22T and Hep3B cells in a concentration-dependent manner without significant cytotoxicity. YC-1 induced G(1) phase arrest in the cell cycle, as detected by an increase in the proportion of cells in the G(1) phase using FAC-Scan flow cytometric analysis. It was further shown that cGMP, p42/p44 mitogen-activated protein kinase, or AKT kinase-mediated signaling pathways did not contribute to the YC-1-induced effect. Of note, YC-1 induced a dramatic increase in the expression of cyclin-dependent kinase (CDK)-inhibitory protein, p21(CIP1/WAP1), and a modest increase in p27(KIP1). The association of p21(CIP1/WAP1) with CDK2 was markedly increased in cells responsive to YC-1. YC-1 did not modify the expression of cyclin D1, cyclin E, CDK2, or CDK4. In a corollary in vivo study, YC-1 induced dose-dependent inhibition of tumor growth in mice inoculated with HA22T cells. Immunohistochemical analysis revealed an inverse relationship between the staining of p21(CIP1/WAF) and the staining of Ki-67, a cell proliferation marker. Based on the results reported herein, we suggest that YC-1 induces cell cycle arrest and inhibits tumor growth both in vitro and in vivo via the up-regulation of p21(CIP1/WAP1) expression in HA22T cells. Because of this, YC-1 is a potential antitumor agent worthy of further investigation.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Proliferation; Cyclic GMP; Cyclin-Dependent Kinase Inhibitor p27; DNA-Binding Proteins; G1 Phase; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Indazoles; Liver Neoplasms; Mitogen-Activated Protein Kinase 1; Nuclear Proteins; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Resting Phase, Cell Cycle; Transcription Factors; Tumor Suppressor Proteins

Hepatoma is one of the most frequently occurring cancers worldwide. However, effective chemotherapeutic agents for this disease have not been developed. Acyclic retinoid, a novel synthetic retinoid, can reduce the incidence of postsurgical recurrence of hepatoma and improve the survival rate. OSI-461, a potent derivative of exisulind, can increase intracellular levels of cyclic GMP, which leads to activation of protein kinase G and induction of apoptosis in cancer cells. In the present study, we examined the combined effects of acyclic retinoid plus OSI-461 in the HepG2 human hepatoma cell line. We found that the combination of as little as 1.0 micromol/L acyclic retinoid and 0.01 micromol/L OSI-461 exerted synergistic inhibition of the growth of HepG2 cells. Combined treatment with low concentrations of these two agents also acted synergistically to induce apoptosis in HepG2 cells through induction of Bax and Apaf-1, reduction of Bcl-2 and Bcl-xL, and activation of caspase-3, -8, and -9. OSI-461 enhanced the G0-G1 arrest caused by acyclic retinoid, and the combination of these agents caused a synergistic decrease in the levels of expression of cyclin D1 protein and mRNA, inhibited cyclin D1 promoter activity, decreased the level of hyperphosphorylated forms of the Rb protein, induced increased cellular levels of the p21(CIP1) protein and mRNA, and stimulated p21(CIP1) promoter activity. Moreover, OSI-461 enhanced the ability of acyclic retinoid to induce increased cellular levels of retinoic acid receptor beta and to stimulate retinoic acid response element-chloramphenicol acetyltransferase activity. A hypothetical model involving concerted effects on p21(CIP1) and retinoic acid receptor beta expression is proposed to explain these synergistic effects. Our results suggest that the combination of acyclic retinoid plus OSI-461 might be an effective regimen for the chemoprevention and chemotherapy of human hepatoma and possibly other malignancies.

Topics: Antineoplastic Agents; Apoptosis; beta Catenin; Blotting, Western; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Chloramphenicol O-Acetyltransferase; Cyclic GMP; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cytoskeletal Proteins; Dose-Response Relationship, Drug; Drug Synergism; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Intracellular Space; Liver Neoplasms; Microfilament Proteins; Models, Biological; Phosphoproteins; Phosphorylation; Promoter Regions, Genetic; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Response Elements; Resting Phase, Cell Cycle; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulindac; Trans-Activators; Transfection; Tretinoin; Tumor Suppressor Protein p53

Methemoglobin (metHb) has been reported to be present in areas surrounding solid tumors. The effects of human metHb on the growth of one human hepatocellular carcinoma cell line and one human glioma cell line that simply replicate in Ham's nutrient mixture F12 (F12) were investigated. MetHb, depending on its concentration, stimulated or inhibited the in vitro growth of both cancer cell lines. The stimulatory or inhibitory effect was due to the release of hemin from metHb, which was recognized by its characteristic light absorption spectrum. The possibility of metHb or hemin acting initially through a 3', 5'-cyclic guanosine monophosphate- (cGMP-) or prostaglandin E2- (PGE2-) mediated pathway to enhance cell growth was excluded. Ferric iron derived from the catabolic degradation of hemin increased cell growth, whereas biliverdin (Bv) and its reduction product, bilirubin (Br), decreased cell growth. Hemoglobin oxidized to metHb in conditions found in tumors showing neovascularization and hemorrhage may contribute significantly to increased proliferation of cancerous cells.

Topics: Adult; Aged; Bilirubin; Biliverdine; Carcinoma, Hepatocellular; Cell Division; Cyclic GMP; Dinoprostone; Dose-Response Relationship, Drug; Female; Glioma; Globins; Hemin; Humans; Liver Neoplasms; Methemoglobin; Pituitary Neoplasms; Tumor Cells, Cultured

Pancreastatin, a chromogranin A-derived peptide, has a counter-regulatory effect on insulin action. We have previously characterized pancreastatin receptor and signalling in rat liver and HTC hepatoma cells. A G alpha(q/11)-PLC-beta pathway leads to an increase in [Ca2+]i, PKC and mitogen activated protein kinase (MAPK) activation. These data suggested that pancreastatin might have a role in growth and proliferation, similar to other calcium-mobilizing hormones.. DNA and protein synthesis were measured as [3H]-thymidine and [3H]-leucine incorporation. Nitric oxide (NO) was determined by the Griess method and cGMP production was quantified by enzyme-linked immunoassay.. Contrary to the expected results, we have found that pancreastatin inhibits protein and DNA synthesis in HTC hepatoma cells. On the other hand, when the activity of NO synthase was inhibited by N-monomethyl-L-arginine (NMLA), the inhibitory effect of pancreastatin on DNA and protein synthesis was not only reverted, but a dose-dependent stimulatory effect was observed, probably due to MAPK activation, since it was prevented by PD98059. These data strongly suggested the role of NO in the inhibitory effect of pancreastatin on protein and DNA synthesis, which is overcoming the effect on MAPK activation. Moreover, pancreastatin dose-dependently increased NO production in parallel to cyclic guanosine monophosphate (cGMP). Both effects were prevented by NMLA. Finally, an indirect effect of pancreastatin through the induction of apoptosis was ruled out.. Therefore, the NO and the cGMP produced by the NO-activated guanylate cyclase may mediate the dose-dependent inhibitory effect of pancreastatin on growth and proliferation in HTC hepatoma cells.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Chromogranin A; Chromogranins; Cyclic GMP; DNA; Dose-Response Relationship, Drug; Enzyme Inhibitors; Liver Neoplasms; Nitric Oxide; omega-N-Methylarginine; Pancreatic Hormones; Protein Synthesis Inhibitors; Rats; Tumor Cells, Cultured

This study aimed to investigate cGMP-regulated store-operated Ca(2+)entry in human 7721 hepatoma cells. [Ca(2+)](i)was measured using Fura2/AM. After incubation of the cells with 4 microm thapsigargin, Ca(2+)entry was evoked by application of 1 mMm Ca(2+)to extracellular solution and was blocked by 3 m m Ni(2+), indicating the presence of store-operated Ca(2+)entry in human 7721 hepatoma cell line. Application of 8-Br-cGMP reduced the [Ca(2+)](i)in hepatoma 7721 cells by 80%. These data demonstrated for the first time that store-operated Ca(2+)entry pathway is present in human hepatoma cells, which is regulated by cGMP.

Topics: Calcium; Calcium Channels; Calcium-Transporting ATPases; Carcinoma, Hepatocellular; Cyclic GMP; Enzyme Inhibitors; Humans; Liver Neoplasms; Nickel; Thapsigargin; Tumor Cells, Cultured

The present study examined the effect of hepatoma-associated antigen HAb18G (homologous to CD147) expression on the NO/cGMP-regulated Ca(2+) mobilization and metastatic process of human hepatoma cells. HAb18G/CD147 cDNA was transfected into human 7721 hepatoma cells to obtain a cell line stably expressing HAb18G/CD147, T7721, as demonstrated by Northern blot and immunocytochemical studies. 8-Bromo-cGMP (cGMP) inhibited the thapsigargin-induced Ca(2+) entry in a concentration-dependent manner in 7721 cells. The cGMP-induced inhibition was abolished by an inhibitor of protein kinase G, KT5823 (1 microm). However, expression of HAb18G/CD147 in T7721 cells decreased the inhibitory response to cGMP. A similar concentration-dependent inhibitory effect on the Ca(2+) entry was observed in 7721 cells in response to a NO donor, (+/-)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca(2+) entry was significantly reduced in HAb18G/CD147-expressing T7721 cells, indicating a role for HAb18G/CD147 in NO/cGMP-regulated Ca(2+) entry. Experiments investigating metastatic potentials demonstrated that HAb18G/CD147-expressing T7721 cells attached to the Matrigel-coated culture plates and invaded through Matrigel-coated permeable filters at the rate significantly greater than that observed in 7721 cells. Both the attachment and invasion rates could be suppressed by SNAP, and the inhibitory effect of SNAP could be reversed by NO inhibitor, N(G)-nitro-l-arginine methyl ester. The sensitivity of the attachment and invasion rates to cGMP was significantly reduced in T7721 cells as compared with 7721 cells when cells were pretreated with thapsigargin. The difference in the sensitivity between the two cells could be abolished by a Ca(2+) channel blocker, Ni(2+) (3 mm). These results suggest that HAb18G/CD147 enhances metastatic potentials in human hepatoma cells by disrupting the regulation of store-operated Ca(2+) entry by NO/cGMP.

Topics: Alkaloids; Antigens, CD; Antigens, Neoplasm; Antigens, Surface; Avian Proteins; Basigin; Blood Proteins; Blotting, Northern; Calcium; Carbazoles; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line; Cell Movement; Collagen; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; DNA, Complementary; Dose-Response Relationship, Drug; Drug Combinations; Enzyme Inhibitors; Humans; Immunohistochemistry; Indoles; Laminin; Liver Neoplasms; Membrane Glycoproteins; Neoplasm Metastasis; NG-Nitroarginine Methyl Ester; Nickel; Nitric Oxide; Penicillamine; Proteoglycans; Signal Transduction; Thapsigargin; Time Factors; Transfection; Tumor Cells, Cultured

Nitric oxide (NO) is believed to play an important, but as yet undefined, role in regulating hypoxia inducible gene expression. Recently, we have reported evidence suggesting that the human insulin-like growth factor-binding protein-1 (IGFBP-1) gene is directly regulated by hypoxia through the hypoxia-inducible factor-1 pathway. The goal of the current study was to investigate NO regulation of hypoxic induction of IGFBP-1 gene expression using HepG2 cells, a model system of hepatic gene expression. We report that a NO generator, sodium nitroprusside, significantly diminishes hypoxic activation of IGFBP-1 protein and messenger ribonucleic acid expression. Furthermore, these effects are independent of guanylate cyclase/ cGMP signaling, as two different inhibitors, LY 83583, a specific inhibitor of guanylate cyclase, and KT 5823, a protein kinase G inhibitor, had no effect on IGFBP-1 induction by hypoxia. Hypoxic induction of a reporter gene containing four tandemly ligated hypoxia response elements was completely blocked by sodium nitroprusside, but not by 8-bromo-cGMP, an analog ofcGMP. These results suggest that NO blocks hypoxic induction of IGFBP-1 by a guanylate cyclase/ cGMP-independent pathway, possibly at the level of oxygen sensing. The impaired hypoxia regulation of IGFBP-1 by nitric oxide may play a key role in the hyperinduction of IGFBP-1 observed in pathophysiological conditions such as fetal hypoxia and preeclampsia where dysregulation of NO has been observed.

Topics: Alkaloids; Aminoquinolines; Base Sequence; Binding Sites; Carbazoles; Carcinoma, Hepatocellular; Cell Hypoxia; Conserved Sequence; Cyclic GMP; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation; Guanylate Cyclase; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Indoles; Insulin-Like Growth Factor Binding Protein 1; Kinetics; Liver; Liver Neoplasms; Luciferases; Molecular Sequence Data; Nitric Oxide; Nitroprusside; Nuclear Proteins; Protein Biosynthesis; Recombinant Fusion Proteins; Signal Transduction; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

To evaluate the pattern of plasma cyclic adenosine 3',5'-monophosphate, cyclic guanosine 3',5'-monophosphate, atrial natriuretic factor and glucagon levels in different stages of chronic liver diseases, we measured these variables in 20 normal subjects, 25 patients with genetic hemochromatosis, associated with liver cirrhosis in 19 cases and not in six, eight patients with compensated and 15 with decompensated alcoholic or posthepatitic cirrhosis, and 12 with hepatocellular carcinoma. All variables were within the normal range in non-cirrhotic hemochromatotic patients. Cyclic adenosine 3',5'-monophosphate levels were within the normal range (9.5-15.7 nmol/l) in hemochromatotic cirrhotics and elevated in other patients. Cyclic guanosine 3',5'-monophosphate, atrial natriuretic factor and glucagon were above the normal ranges (1.92-5.91 nmol/l, 8.8-62.7 ng/l, and 39-165 ng/l, respectively) in most patients with cirrhosis both with and without hemochromatosis and in most individuals with hepatocellular carcinoma. Cyclic guanosine 3',5'-monophosphate correlated with atrial natriuretic factor in the former groups but not in the latter. These findings indicate that glucagon and atrial natriuretic factor hypersecretion is an early event in cirrhosis, regardless of its etiology. In hepatocellular carcinoma, the underlying cirrhosis may account for most hormonal and metabolic changes although cyclic guanosine 3',5'-monophosphate increases could also be due to the neoplastic process per se.

Topics: Adult; Aged; Atrial Natriuretic Factor; Carcinoma, Hepatocellular; Cyclic AMP; Cyclic GMP; Female; Glucagon; Hemochromatosis; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged

Expression of the asialoglycoprotein receptor by the human hepatocellular carcinoma cell line HuH-7 in response to intracellular cGMP concentrations was previously shown to be regulated at the translational level. In a cell-free system, initiation of asialoglycoprotein receptor mRNA translation was dependent on the presence of the 7-methylguanylate cap site and was independent of 8-bromo-cGMP levels in which the cells were grown prior to RNA isolation. Stable transfection of COS-7 cells with deletion constructs of the asialoglycoprotein receptor H2b subunit localized the cGMP-responsive cis-acting element to the mRNA 5'-untranslated region (UTR). Addition of biotin (an activator of guanylate cyclase) induced the expression of beta-galactosidase present as a chimeric plasmid containing the H2b 187-nucleotide 5'-UTR. An RNA gel retardation assay identified a 37-nucleotide cognate sequence within this 187-nucleotide region. Titration of the 5'-UTR with a cytosolic fraction isolated from HuH-7 grown in the presence or absence of 8-bromo-cGMP or biotin provided direct evidence for an RNA-binding protein responsive to intracellular levels of cGMP. Based on these findings, it seems reasonable to propose that reduction of intracellular levels of cGMP by biotin deprivation results in a negative trans-acting factor associating with the 5'-UTR of asialoglycoprotein receptor mRNAs, thereby inhibiting translation.

Topics: Animals; Asialoglycoprotein Receptor; Asialoglycoproteins; Biotin; Carcinoma, Hepatocellular; COS Cells; Cyclic GMP; Guanosine; Humans; Liver Neoplasms; Protein Biosynthesis; Receptors, Cell Surface; Restriction Mapping; RNA, Messenger; Transfection; Tumor Cells, Cultured

We have previously reported an interaction of nitric oxide (NO) and cyclic 3',5'-guanosine monophosphate (cGMP) in erythropoietin (Epo) production. Further studies have been carried out to clarify the role of NO in the hypoxic regulation of Epo production in Epo producing human hepatocellular carcinoma (Hep3B) cells, which produce Epo in response to physiological stimuli. Our reverse transcriptase-polymerase chain reaction (RT-PCR) technique revealed the expression of iNOS mRNA in Hep3B cells after incubation under hypoxic (1% O2) conditions for 6 hr. Hypoxia also significantly increased medium levels of nitrite in Hep3B cells. In order to investigate the role of NO in Epo production in Hep3B cells under normoxic (20% O2) conditions, we have studied the effects of interferon-gamma (IFN-gamma) on Epo production. IFN-gamma is known to induce iNOS and enhance the production of NO. IFN-gamma produced significant increases in medium levels of Epo and nitrite. IFN-gamma also significantly increased cGMP levels in Hep3B cells. Furthermore, NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, significantly decreased IFN-gamma induced elevations in medium levels of Epo and nitrite as well as cGMP levels in Hep3B cells. These results provide further support for an important role of the NO/cGMP system in hypoxic regulation of Epo production in Hep3B cells.

Topics: Base Sequence; Carcinoma, Hepatocellular; Cell Hypoxia; Cyclic GMP; DNA Primers; Enzyme Inhibitors; Erythropoietin; Humans; Interferon-gamma; Liver Neoplasms; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Polymerase Chain Reaction; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

Many inflammatory diseases are associated with a hypoproliferative anaemia. Patients with this anaemia often present with serum erythropoietin (EPO) concentrations that are too low for the degree of their anaemia. Proinflammatory cytokines, in addition to their inhibitory effects on proliferation of erythroid progenitors, could contribute to the pathogenesis of this anaemia by reducing EPO production. Because several cytokines stimulate nitric oxide (NO) synthase we propose that nitric oxide might mediate the suppression of EPO production during inflammation. In order to test this hypothesis we investigated the effects of NO donors on 24-h hypoxia-induced EPO production in the hepatocellular carcinoma cell line HepG2. Following application of the NO donors sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP), EPO production was dose-dependently reduced: compared to the untreated control EPO production was lowered by 89% with SNP (1000 microM), by 66% with SIN-1 (1000 microM), and by 72% with SNAP (500 microM). In contrast, 8-bromo-cGMP did not inhibit EPO formation. Since pyrogallol (300 microM) and H2O2 (250 microM) showed a comparable suppression of EPO synthesis, we propose that NO might affect EPO production either by a similar direct influence on the cellular redox state or via increasing the cellular content of reactive oxygen species.

Topics: Carcinoma, Hepatocellular; Cyclic GMP; Cytokines; Erythropoietin; Humans; Hydrogen Peroxide; Liver Neoplasms; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Penicillamine; Reactive Oxygen Species; S-Nitroso-N-Acetylpenicillamine; Tumor Cells, Cultured

The effects of 8-bromoguanosine 3':5'-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (gj) in rat Cx43-transfected cells by 24.0 +/- 3.7% (mean +/- SEM, n = 5), whereas gj was not affected in human Cx43-transfected cells by the same treatment. The relaxation of gj in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (gammaj) of about 20, 40-45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the gammaj distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [32P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [32P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased gj, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.

Topics: Amino Acids; Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Line; Cyclic GMP; DNA, Complementary; Gap Junctions; GAP-43 Protein; Humans; Ion Channels; Liver Neoplasms; Membrane Glycoproteins; Membrane Potentials; Nerve Tissue Proteins; Patch-Clamp Techniques; Phosphorylation; Precipitin Tests; Protein Kinases; Rats; Transfection

Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Base Sequence; Carcinoma, Hepatocellular; Cell Communication; Connexin 26; Connexins; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; DNA Primers; Enzyme Inhibitors; Gap Junctions; Gene Expression; Homeostasis; Humans; Liver Neoplasms; Membrane Potentials; Molecular Sequence Data; Phosphorylation; Polymerase Chain Reaction; Protein Kinase C; Recombinant Proteins; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured

The hepatic receptor for asialoglycoproteins was found to be modulated by the glucose concentration in the medium of the human hepatoma cell line HepG2. The surface binding of asialoorosomucoid, a well-documented ligand for this receptor, increased from 20 ng/mg of cellular protein to about 40 ng/mg as the glucose concentration was increased from 10 to 50 mg/dl. The up-modulating effect of glucose was mimicked by pyruvate, a product of glucose metabolism, and abolished by both 2-deoxyglucose, an inhibitor of glucose metabolism, and by cycloheximide, an inhibitor of protein synthesis. Scatchard plot analysis indicated a rise in the number of binding sites and a twofold increase in binding affinity. In contrast, the binding of antibody remained unchanged with respect to alterations in glucose concentration, an indication that the actual number of receptors remained constant in face of an increased number of binding sites. Specificity of the glucose effect was shown by the binding of insulin and transferrin to their respective receptors, which was unaffected by the high glucose concentration that increased asialoorosomucoid binding. The repression of receptor binding seen with cells grown in biotin-deprived medium was reversed by increasing the glucose concentration of the medium. In this case, binding was restored to a level sixfold to sevenfold higher than that of the control cells grown in dialyzed serum. The stimulatory effect of glucose was shown to be independent of and significantly greater than that of cyclic GMP, a known regulator of receptor expression of biotin-deficient HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Asialoglycoprotein Receptor; Asialoglycoproteins; Binding Sites; Carcinoma, Hepatocellular; Culture Media; Cyclic GMP; Cycloheximide; Deoxyglucose; Glucose; Humans; Ligands; Liver Neoplasms; Precipitin Tests; Pyruvates; Pyruvic Acid; Receptors, Cell Surface; Tumor Cells, Cultured; Up-Regulation

To investigate whether atrial natriuretic factor regulates the growth of hepatocytes and to determine the receptor subtype involved in such modulation, we studied the effect of atrial natriuretic factor 103-126 and clearance receptor binding analogs of atrial natriuretic factor, (des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 and des-(C105,121) atrial natriuretic factor 104-126) on growth of human hepatoblastoma cells. Atrial natriuretic factor 103-126 and des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 inhibited thymidine incorporation into human hepatoblastoma cells cultured in the presence of bovine serum albumin and epidermal growth factor but not in cells cultured in bovine serum albumin alone. Moreover, atrial natriuretic factor 103-126, des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 and des-(C105,121) atrial natriuretic factor 104-126, in a concentration-dependent manner, inhibited thymidine incorporation and cell proliferation. As monitored by the ability of des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121 to displace 125I-labeled atrial natriuretic factor, epidermal growth factor increased the expression of cell surface clearance receptors. Epidermal growth factor also transiently increased the cellular content of atrial natriuretic factor clearance receptor messenger RNA without altering the levels of guanylyl cyclase-linked atrial natriuretic factor receptor messenger RNA levels. Maximal increase in atrial natriuretic factor clearance receptor messenger RNA coincided with the maximal increase in des-(Q116, S117, G118, L119, G120) atrial natriuretic factor 102-121-displaceable 125I-atrial natriuretic factor binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; Atrial Natriuretic Factor; Carcinoma, Hepatocellular; Cell Division; Cyclic AMP; Cyclic GMP; DNA Replication; Epidermal Growth Factor; Kinetics; Liver Neoplasms; Peptide Fragments; Receptors, Atrial Natriuretic Factor; RNA, Messenger; RNA, Neoplasm; Thymidine; Tritium; Tumor Cells, Cultured

Post-transcriptional regulation of the asialoglyco-protein receptor (ASGR) in the HepG2 cell line can be mediated by the presence of biotin in the culture medium. To determine if the induction by biotin of intracellular cGMP affects ASGR expression, HepG2 were grown in biotin-depleted medium with the cell-permeant 8-bromo-cGMP (8-Br-cGMP). Both cell-surface and total ASGR binding of iodinated asialoorosomucoid (125I-ASOR) was increased from 30 to 95% of control levels by the addition of increasing concentrations of 8-Br-cGMP. The rate of ASGR-mediated endocytosis of 125I-ASOR also increased with increasing concentrations of 8-Br-cGMP. Estimates of the steady state levels of ASGR by transblot analysis utilizing both antisera to affinity-purified ASGR and to isoform-specific antibodies prepared against synthetic peptides confirmed that the increase in 125I-ASOR binding was due to an increase in ASGR expression. Metabolic labeling of biotin-deprived HepG2 with [35S] cysteine and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitants revealed an increase of radiolabeled ASGR within 30 min of the addition of 8-Br-cGMP. Induction of cGMP by atrial natriuretic factor also increased the metabolic labeling of ASGR. ASGR expression in a second hepatocellular carcinoma cell line, HuH-7, responded in a similar fashion to the addition of 8-Br-cGMP. In contrast to 8-Br-cGMP, exposure to 8-bromo-cAMP results in a reduction of ASGR expression even in the presence of biotin-containing medium. The antagonistic roles of cGMP and cAMP suggest a balance between cyclic nucleotides is required for the maintenance of differentiated functions by the hepatocyte.

Topics: Amino Acid Sequence; Asialoglycoprotein Receptor; Asialoglycoproteins; Atrial Natriuretic Factor; Carcinoma, Hepatocellular; Cell Line; Cyclic GMP; Endocytosis; Humans; Kinetics; Liver Neoplasms; Molecular Sequence Data; Oligopeptides; Receptors, Immunologic; Second Messenger Systems

Methyl, n-butyl 2'-TBDMS-N2-DMF-guanosine 3',5'-cyclic phosphate and 2'-TBDMS-N2-DMF-guanosine 3',5'-cyclic diethyl-phosphoramidate were synthesized by reaction of protected guanosine with trivalent phosphorus reagents in the presence of tetrazole followed by oxidation. The reaction occurred stereospecifically. Protected guanosine 3',5'-cyclic phosphotriesters and N, N'-diethyl-phosphoramidate were shown to have inhibitory activity on the synthesis of DNA and RNA in mouse liver tumor cell. Diastereoisomers of n-butyl N2-substituted guanosine 3',5'-cyclic phosphate have been shown to activate adenylate cyclase in vitro.

Topics: Adenylyl Cyclases; Animals; Cyclic GMP; DNA, Neoplasm; Liver Neoplasms; Mice; Osteosarcoma; Rats; RNA, Neoplasm

Topics: Adult; Animals; Cyclic GMP; Hepatitis; Humans; Liver Cirrhosis; Liver Diseases; Liver Neoplasms; Liver Regeneration; Male; Rats; Rats, Inbred Strains; Receptors, Muscarinic

To clarify the factor(s) responsible for changes in the plasma cyclic GMP concentration in liver diseases, we measured the plasma levels of cyclic GMP, along with cyclic AMP, in various clinical stages of chronic liver diseases and acute hepatitis. The level of cyclic GMP was found to increase significantly in the early stage of acute hepatitis, in the decompensated stage of liver cirrhosis, and in malignant diseases. In the former two states, it is postulated that decreased hepatic mass is responsible for the changes in the plasma cyclic GMP concentration. The retention rate of indocyanin green (ICGR15) was highly correlated with the plasma cyclic GMP level. The result suggests that the determination of plasma cyclic GMP is useful as an index of the reserve function of the liver in disease states.

Topics: Carcinoma, Hepatocellular; Cyclic AMP; Cyclic GMP; Hepatitis; Humans; Indocyanine Green; Liver Cirrhosis; Liver Diseases; Liver Neoplasms; Nucleotides, Cyclic

Urinary excretion of cyclic GMP (cGMP) and the plasma level of cyclic AMP (cAMP) were determined in patients with liver diseases. The urinary excretion of cGMP, expressed on the basis of creatinine excreted per day, was at significantly higher levels not only in primary hepatoma but also in liver cirrhosis, while the plasma level of cAMP was higher only in liver cirrhosis. Thus, the ratio of urinary cGMP excretion to plasma cAMP level in primary hepatoma was significantly higher than that in liver cirrhosis. In cirrhotic patients studied by catheterization, the level of cGMP in the hepatic vein was significantly lower than that in the superior mesenteric or portal vein, indicating the uptake of cGMP by the liver. Since cGMP excretion correlated with KICG both in liver cirrhosis and primary hepatoma, the increased cGMP excretion appeared to be explained by a reduced uptake of cGMP by the liver.

Topics: Adolescent; Adult; Aged; Carcinoma, Hepatocellular; Cyclic AMP; Cyclic GMP; Female; Hepatitis; Humans; Liver Neoplasms; Male; Middle Aged; Precancerous Conditions

To investigate the prediction that urinary cGMP (UcGMP) and cAMP (UcAMP) excretion is altered in a manner consistent with unregulated cell growth in hepatocellular carcinoma (HCC), we studied 31 patients with this disease, 25 without apparent disease, 16 with various hepatic diseases, and 16 with nonhepatic neoplasms. Results were expressed as UcGMP excretion per 100 ml glomerular filtration because reduced creatinine excretion in patients with muscle wasting or renal dysfunction may spuriously elevate UcGMP. UcGMP excretion was elevated in 80% of patients with HCC, 75% of patients with hepatic disease and 68% of patients with other neoplasms. Mean values for UcAMP excretion did not differ significantly from normal values. Plasma and ascitic fluid cGMP concentrations in HCC and hepatic diseases were raised. These results support the hypothesis of a shift in cyclic nucleotide metabolism toward cGMP in malignant diseases. However, UcGMP measurement does not detect progression of cirrhosis to HCC.

Topics: Adolescent; Adult; Aged; Body Fluids; Carcinoma, Hepatocellular; Child; Cyclic AMP; Cyclic GMP; Female; Humans; Liver; Liver Cirrhosis; Liver Diseases; Liver Neoplasms; Male; Metabolic Clearance Rate; Middle Aged

Cyclic nucleotide phosphodiesterase activity has been measured in muscle biopsies taken from healthy controls and from cancer patients. In both groups the muscles were clinically and morphologically normal. The phosphodiesterase activity was significantly increased in muscles from cancer patients using both cyclic AMP and cyclic GMP as substrate. These findings are in line with previous reports indicating that malignancy may interfere with metabolism of the host muscular tissues, and suggest the possibility that the observed biochemical changes might be an aspect of an early muscle neurogenic involvement.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Carcinoma; Cyclic AMP; Cyclic GMP; Humans; Liver Neoplasms; Lung Neoplasms; Muscles; Phosphoric Diester Hydrolases; Rectal Neoplasms; Stomach Neoplasms

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adenoma, Bile Duct; Animals; Cyclic AMP; Cyclic GMP; Female; Guanylate Cyclase; Liver; Liver Neoplasms; p-Dimethylaminoazobenzene; Polyethylene Glycols; Rats

Ethionine-induced hepatomas are characterized by high adenylate cyclase activity and cyclic adenosine 3',5'-monophosphate content relative to those of surrounding liver or liver from pair-fed control rats. The present study examined the properties of the guanylate cyclase-cyclic guanosine 3',5'-monophosphate (cGMP) system of these tissues. cGMP levels of the ethionine-induced hepatomas, determined in both specimens quick-forzen in situ and after in vitro incubation of tissue slices, were approximately 2 times higher than those of surrounding liver or controls. Higher cGMP in the tumors was associated with an increase in whole homogenate, soluble, and particulate guanylate cyclase activities, as well as an increase in soluble cGMP-phosphodiesterase activity. 3-Isobutyl-1-methylxanthine, a potent inhibitor of cGMP-phosphodiesterase activity, potentiated the differences in cGMP between slices of the hepatomas and surrounding liver or control, suggesting that the higher steady-state cGMP content of the tumors reflected enhanced basal cGMP synthesis which was partially offset by increased nucleotide degradation. In the hepatomas, a greater proportion of the total guanylate cyclase activity was located in the particulate cell fraction (31%) as compared to the subcellular distribution of enzyme activity in either surrounding liver or controls (15% of total in the particulate fraction). Carbamylcholine, which increased cGMP 3-fold in surrounding liver and controls, failed to alter cGMP levels inslices of hepatoma. Further, the relative changes in both cGMP accumulation and guanylate cyclase activity of the tumors in response to NaN3, NH2OH, and NaNO2 were blunted compared to surrounding liver or controls, although in each instance a response was clearly evident. Ethionine-induced hepatomas are thus characterized by: (a) significant increases in cGMP content and in guanylate cyclase and cGMP-phosphodiesterase activities, (b) a change in the subcellular distribution of guanylate cyclase, and (c) altered responsiveness of the guanylate cyclase-cGMP system to several agonists.

Topics: Adenylyl Cyclases; Animals; Azides; Carcinoma, Hepatocellular; Cell Division; Cyclic AMP; Cyclic GMP; Ethionine; Guanylate Cyclase; Hydroxylamines; Liver Neoplasms; Male; Neoplasms, Experimental; Nitrates; Rats

Concanavalin A added to intact cells at 37 degrees caused rapid and reversible inactivation of a soluble enzyme, tyrosine aminotransferase, in two lines of rat hepatoma tissue culture cells grown in monolayer culture. This temperature-dependent process was independent of de novo protein and RNA synthesis and independent of increased uptake of Ca2+ and Mg2+ or glucose. The inactivation could be reversed by adding alpha-methyl-D-mannopyranoside a competing sugar for concanavalin A binding. Other lectins known to bind to different sugars did not bring about the inactivation of tyrosine aminotransferase. Addition of concanavalin A did not result in the inactivation of another soluble enzyme, lactic dehydrogenase. The maintenance of tyrosine aminotransferase in an inactive form after the binding of concanavalin A to the cells required the continued presence of concanavalin A. This effect of concanavalin A could not be mimicked either by dibutyryl cyclic adenosine or guanosine monophosphoric acid. Incubation of cell extracts with concanavalin A did not result in inactivation nor did mixing of extracts from concanavalin A-treated cells with extracts from untreated cells. On the basis of these results we conclude that the following are the essential requirements for concanavalin A to bring about the inactivation of tyrosine aminotransferase: (a) the binding of native concanavalin A to the cells; (b) integrity of certain structural elements of the cells.

Topics: Alanine Transaminase; Animals; Carcinoma, Hepatocellular; Cell Line; Concanavalin A; Cyclic AMP; Cyclic GMP; Enzyme Induction; Fluorouracil; Kinetics; L-Lactate Dehydrogenase; Liver Neoplasms; Rats; Theophylline; Tyrosine Transaminase

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Adenylyl Cyclases; Animals; Carcinoma, Hepatocellular; Cell Division; Cyclic AMP; Cyclic GMP; Epinephrine; Glucagon; Guanylate Cyclase; Kinetics; Liver; Liver Neoplasms; Lyases; Male; Neoplasm Proteins; Neoplasms, Experimental; Nucleotides, Cyclic; Phosphoric Diester Hydrolases; Rats

Intracellular and extracellular levels of 3':5'-cyclic GMP and 3':5'-cyclic AMP were studied in synchronized Novikoff rat hepatoma cells. Intracellular levels of cyclic GMP increased spontaneously from 2-fold (without colcemid) to 10-fold (with colcemid), in proportion to the number of cells in mitosis. As cells entered mitosis, cellular cyclic AMP declined simultaneously with the rise in cyclic GMP. These reciprocal changes in cyclic nucleotide levels were reversed as cells passed out of metaphase and through anaphase. Maximum cyclic AMP and minimum cyclic GMP concentrations occurred during G-1. Less marked reciprocal fluctuations in both cyclic nucleotides were also found in S-phase and early G-2, where the ratio of cyclic AMP to cyclic GMP concentrations first fell and then increased. These changes in cyclic nucleotide ratios were closely correlated with major cell-cycle transitions at the boundaries between G-1/S-phase, S-phase/G-2, G-2/prophase, and metaphase/anaphase. Most, but not all, of the extracellular cyclic nucleotides were extruded when cells traversed mitosis. Colcemid or vinblastine completely prevented the appearance of extracellular cyclic AMP but augmented the appearance of extracellular cyclic GMP in parallel with the accumulation of mitotic cells. These results reflected changes in intracellular cyclic nucleotides and indicated that increased intracellular turnover of cyclic GMP and cyclic AMP occurred before and after metaphase, respectively. Elevated cyclic GMP levels during mitosis and S-phase are consistent with potential modulatory roles for this cyclic nucleotide in proliferation.

Topics: Bleomycin; Carcinoma, Hepatocellular; Cell Division; Cell Line; Cyclic AMP; Cyclic GMP; Demecolcine; Liver Neoplasms; Mitosis; Neoplasms, Experimental; Vinblastine

Topics: Animals; Calcium; Cyclic AMP; Cyclic GMP; Liver; Liver Neoplasms; Magnesium; Male; Neoplasms, Experimental; Rats; Thioacetamide

Topics: Carcinoma, Hepatocellular; Cyclic GMP; Female; Humans; Liver Neoplasms; Male; Sex Factors

Cyclic guanosine 3':5'-monophosphate (cyclic GMP) and cyclic adenosine 3':5-monophosphate were measured in the urine of normal rats and those bearing transplantable liver and kidney tumors. The level of cyclic GMP ranged from 1.4 to 1.6 mumoles/g urinary creatinine in several strains of rats without tumors. Rats bearing Morris hepatomas 20, 21, 9618A and 9633F and kidney tumor MK2 had urine levels of cyclic GMP from 1.3 to 3.6 mumoles/g creatinine. Rats bearing the fast growing hepatomas 9618A2 and 3924A and Morris kidney tumor MK3 had urinary values of 5.6. 41.9, and 32.7 mumoles cyclic GMP per g creatinine, respectively. Urine levels of cyclic adenosine 3':5'-monophosphate ranged from 10.1 to 19.7 mumoles/g creatinine in all normal and tumor-bearing rats and were not significantly different in any of the groups examined.

Topics: Animals; Carcinoma, Hepatocellular; Creatinine; Cyclic AMP; Cyclic GMP; Kidney Neoplasms; Liver Neoplasms; Neoplasms, Experimental; Rats; Rats, Inbred ACI

Topics: Animals; Binding Sites; Binding, Competitive; Carcinoma, Hepatocellular; Cyclic AMP; Cyclic GMP; Endoplasmic Reticulum; In Vitro Techniques; Kinetics; Liver; Liver Neoplasms; Male; Membranes; Nucleotides, Cyclic; Protein Binding; Protein Kinases; Rats

There is evidence than adenosine 3',5'-monophosphate (cAMP) and guanosine 3',5'-monophosphate (cGMP) may have antagonistic actions on cell growth, with cAMP inhibiting and cGMP stimulating this process. However, reductions in cAMP and increases in cGMP are not charactersitic of all neoplastic tissues. Thus, benign and malignant tissues from hepatoma-bearing rats exposed to the hepatic carcinogen DL-ethionine have elevated rather than depressed cAMP, compared to control liver, and parenteral administration of this drug increases hepatic cAMP within hours. In the present study, the effects of ethionine ingestion on the hepatic content and metabolism of both cAMP and cGMP were examined sequentially in rats at 2 and then 6 wk intervals, from the initiation of drug administration until the development of hepatomas. After 2 wk, cAMP content of quick-frozen liver from rats receiving ethionine (E) was significantly increased (826 +/- 91 pmole/g wet weight) above that of liver from pair-fed controls (C, 415 +/- 44), whether calculated by tissue wet weight, protein, or DNA content. In benign tissue from E, higher cAMP was still evident after in vitro incubations of slices with 2 mM 1-methyl-3-iso-butylxanthine (MIX) and was associated with enhanced adenylate cyclase and unchanged high or low Km cAMP-phosphodiesterase activities. These findings are compatible with accelerated cAMP generation in liver from E. Protein kinase activity ratios were significantly increased in frozen liver from E (0.52 +/- 0.04 versus 0.36 +/- 0.03 in C), and the percent glycogen synthetase in the I form was clearly reduced (19% +/- 2% in E versus 47% +/- 5% in c). incubation of hepatic slices from E or C with MIX and/or 10 muM glucagon further increased cAMP and protein kinase activity ratios, data which imply higher effective, as well as total, cellular cAMP in E. Changes in cAMP metabolism and action observed at 2 wk persisted throughout the 38-wk period of drug ingestion. Adenylate cyclase activity, cAMP content, and protein kinase activity ratios of ethionine-induced hepatomas exceeded those of both the surrounding liver from tumor-bearing rats and that of control liver, but alterations in these parameters were qualitatively similar in both tissues from E. By contrast, while cGMP in quick-frozen surrounding liver from tumor-bearing rats (36 +/- 4 pmole/g wet weight) did not differ from that of control liver (30 +/- 3), cGMP in the hepatomas was increased. This change was evident in bo

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenosine Triphosphate; Adenylyl Cyclases; Animals; Carcinoma, Hepatocellular; Cyclic AMP; Cyclic GMP; DNA; Ethionine; Glycogen Synthase; Liver; Liver Neoplasms; Male; Protein Kinases; Proteins; Rats

Topics: Animals; Binding Sites; Carcinoma, Hepatocellular; Cyclic AMP; Cyclic GMP; Cytosol; Liver; Liver Neoplasms; Neoplasms, Experimental; Rats; Receptors, Drug

Urinary excretion of cyclic guanosine monophosphate (GMP) increased in rats bearing Morris hepatoma 3924A, and a correlation coefficient of .842 was observed comparing nucleotide excretion and tumor size. Irradiation of tumor or 5-fluorouracil administration delayed the increases in urinary cyclic GMP and tumor size. Surgical removal of tumors resulted in a rapid decline in cyclic GMP excretion to baseline levels. Cyclic adenosine monophosphate excretion was not altered by implantation, irradiation, or excision of tumor.

Topics: Animals; Carcinoma, Hepatocellular; Cyclic AMP; Cyclic GMP; Female; Fluorouracil; Liver Neoplasms; Neoplasms, Experimental; Rats; Rats, Inbred ACI; X-Rays

A variety of 6- and 8-substituted analogs of cAMP (cyclic adenosine 3:5-monophosphate) have been tested for their ability to increase activity of tyrosine aminotransferase (EC 2.6.1.5) in cultured Reuber H35 hepatoma cells. Some analogs, particularly the 8-thio-substituted ones, produced effects approximately equivalent to those generated by N-6, O2'-dibutyryl cAMP. In contrast, cAMP and its O-2-monobutyryl derivative were relatively ineffective even at very high concentrations, whereas three other analogs actually depressed the activity of the aminotransferase. Changes in enzyme activity generated by the various analogs were paralleled closely by changes in the relative rate of aminotransferase synthesis. An excellent correlation was found to exist between the ability of any given analog to influence the activity of tyrosine aminotransferase and that of phosphoenolpyruvate carboxykinase (EC 4.1.1.32). A similar correlation was found to exist between the ability of various analogs to evelate the activity of these enzymes and to inhibit reversibly the growth of H35 cells. Only one of five inhibitors of cAMP phosphodiesterase activity tested produce any increase in aminotransferase activity when added alone. All of the 6- and 8-substituted analogs tested, including noniducers, stimulated f1 histone phosphorylation in crude rat liver extracts with approximately equal potencies. On the other hand, dibutyryl cAMP was only a weak activator of protein kinase in vitro, even though it is a potent enzyme inducer. A possible resolution of this apparent discrepancy has been provided by preliminary analyses of site-specific f1 histone phosphorylation in whole cells. Only compounds active as aminotransferase inducers are capable of stimulating phosphorylation of the serine-37 residue of endogenous f1 histone (3- to 10-fold).

Topics: Animals; Butyrates; Carbon Radioisotopes; Carcinoma, Hepatocellular; Cell Line; Cyclic AMP; Cyclic GMP; Enzyme Activation; Inosine Nucleotides; Kinetics; Liver Neoplasms; Neoplasms, Experimental; Nucleotides, Cyclic; Phosphodiesterase Inhibitors; Phosphoenolpyruvate Carboxykinase (GTP); Phosphorus Radioisotopes; Protein Kinases; Rats; Structure-Activity Relationship; Theophylline; Time Factors; Tritium; Tyrosine Transaminase

Topics: Adenosine Monophosphate; Animals; Carcinoma, Hepatocellular; Cell Nucleus; Chromatography, Ion Exchange; Cyclic GMP; Cytosine Nucleotides; Electrophoresis, Polyacrylamide Gel; Liver Neoplasms; Molecular Weight; Neoplasms, Experimental; Phosphorus Radioisotopes; Rats; Ribonucleotides; RNA; Uracil Nucleotides

Topics: Adenosine Triphosphate; Animals; Carcinoma, Hepatocellular; Cell Line; Cell-Free System; Chromatography, Thin Layer; Cyclic AMP; Cyclic GMP; Hot Temperature; Liver Neoplasms; Lymphoma; Pentosephosphates; Phosphorus Radioisotopes; Phosphotransferases; Protein Kinases; Rats; Ribose; Tritium

Topics: Animals; Binding Sites; Carcinoma, Hepatocellular; Cell Fractionation; Cell Line; Cyclic AMP; Cyclic GMP; Drug Stability; Hot Temperature; Hydrogen-Ion Concentration; Kinetics; Liver; Liver Neoplasms; Male; Mathematics; Neoplasm Proteins; Neoplasms, Experimental; Protein Binding; Proteins; Rats; Ribonucleotides; Structure-Activity Relationship; Subcellular Fractions; Tritium; Ultrafiltration

Topics: Animals; Carcinoma, Hepatocellular; Chromatography, Ion Exchange; Cyclic AMP; Cyclic GMP; Liver; Liver Neoplasms; Male; Neoplasm Transplantation; Rats; Tritium