guanosine-5--o-(3-thiotriphosphate) and Carcinoma--Hepatocellular

To make clear whether CD147 (EMMPRIN) expression in pathological tumor samples with a fine-needle aspiration biopsy is useful for pathological diagnosis of early hepatocellular carcinoma (HCC).. Twenty-two patients (15 men and 7 women; median age 68 years, range 56-81 years) underwent a liver tissue biopsy in order to make a diagnosis of HCC. Paraffin-embedded liver biopsy tissue samples from 22 patients were stained with anti-CD147 antibody, murine monoclonal antibody 12C3 (MAb12C3) for immunohistochemical analysis. An immunohistochemical analysis of CD147 was performed and the degree of staining compared between tumor and non-tumor tissue. In addition, the degree of staining within tumor tissue was compared according to a number of clinicopathological variables.. The degree of staining of CD147 was significantly higher in tumor tissues than non-tumor tissues, even in tumors less than 15 mm in diameter. The expression of this protein was significantly elevated in HCC tissue specimens from patients with a low value of serum AST and gamma-GTP.. CD147 serves potentially as a pathological target for cancer detection of early HCC.

Topics: Aged; Aged, 80 and over; Aspartate Aminotransferases; Basigin; Biomarkers, Tumor; Biopsy, Needle; Carcinoma, Hepatocellular; Female; Gene Expression Regulation, Neoplastic; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Liver; Liver Neoplasms; Male; Middle Aged

p2y5 is an orphan G protein-coupled receptor that is closely related to the fourth lysophosphatidic acid (LPA) receptor, LPA4. Here we report that p2y5 is a novel LPA receptor coupling to the G13-Rho signaling pathway. "LPA receptor-null" RH7777 and B103 cells exogenously expressing p2y5 showed [3H]LPA binding, LPA-induced [35S]guanosine 5'-3-O-(thio)triphosphate binding, Rho-dependent alternation of cellular morphology, and Gs/13 chimeric protein-mediated cAMP accumulation. LPA-induced contraction of human umbilical vein endothelial cells was suppressed by small interfering RNA knockdown of endogenously expressed p2y5. We also found that 2-acyl-LPA had higher activity to p2y5 than 1-acyl-LPA. A recent study has suggested that p2y5 is an LPA receptor essential for human hair growth. We confirmed that p2y5 is a functional LPA receptor and propose to designate this receptor LPA6.

Topics: Animals; Blotting, Western; Calcium; Carcinoma, Hepatocellular; Cell Membrane; Cells, Cultured; Cloning, Molecular; Cyclic AMP; Endothelium, Vascular; GTP-Binding Protein alpha Subunits, G12-G13; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Liver Neoplasms, Experimental; Lysophospholipids; Neuroblastoma; Radioligand Assay; Rats; Receptors, Purinergic P2; Umbilical Veins

Circulating hormones produce rapid changes in the Cl(-) permeability of liver cells through activation of plasma membrane receptors coupled to heterotrimeric G-proteins. The resulting effects on intracellular pH, membrane potential, and Cl(-) content are important contributors to the overall metabolic response. Consequently, the purpose of these studies was to evaluate the mechanisms responsible for G-protein-mediated changes in membrane Cl(-) permeability using HTC hepatoma cells as a model. Using patch clamp techniques, intracellular dialysis with 0.3 mm guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) increased membrane conductance from 10 to 260 picosiemens/picofarads due to activation of Ca(2+)-dependent Cl(-) currents that were outwardly rectifying and exhibited slow activation at depolarizing potentials. These effects were mimicked by intracellular AlF(4)(-) (0.03 mm) and inhibited by pertussis toxin (PTX), consistent with current activation through Galpha(i). Studies using defined agonists and inhibitors indicate that Cl(-) channel activation by GTPgammaS occurs through an indomethacin-sensitive pathway involving sequential activation of phospholipase C, mobilization of Ca(2+) from inositol 1,4,5-trisphosphate-sensitive stores, and stimulation of phospholipase A(2) and cyclooxygenase (COX). Accordingly, the conductance responses to GTPgammaS or to intracellular Ca(2+) were inhibited by COX inhibitors. These results indicate that PTX-sensitive G-proteins regulate the Cl(-) permeability of HTC cells through Ca(2+)-dependent stimulation of COX activity. Thus, receptor-mediated activation of Galpha(i) may be essential for hormonal regulation of liver transport and metabolism through COX-dependent opening of a distinct population of plasma membrane Cl(-) channels.

Topics: Adenosine Triphosphate; Adrenergic alpha-Agonists; Animals; Arachidonic Acid; Calcium; Carcinoma, Hepatocellular; Cell Line; Cell Membrane; Chloride Channels; Electric Conductivity; Electrophysiology; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Indomethacin; Inositol 1,4,5-Trisphosphate; Liver; Models, Biological; Norepinephrine; Patch-Clamp Techniques; Pertussis Toxin; Phospholipases A; Prostaglandin-Endoperoxide Synthases; Protein Binding; Rats; Time Factors; Virulence Factors, Bordetella

The polypeptide ligand angiogenin, a potent inducer of angiogenesis, localizes in the nucleus/nucleolus subsequent to endocytosis by relevant cell types. This study examines the kinetic properties of the nucleolar targeting signal (NTS) of angiogenin (IMRRRGL(35)) at the single cell level. We show that the NTS is sufficient to target green fluorescent protein (GFP), but not beta-galactosidase, to the nucleolus of rat hepatoma cells. Mutation of Arg(33) to Ala within the NTS abolishes targeting activity. Nuclear/nucleolar import conferred by the NTS of angiogenin is reduced by cytosolic factors as well as ATP and is independent of importins and Ran. The NTS also confers the ability to bind to nuclear/nucleolar components which is inhibited by ATP hydrolysis; nonhydrolysable GTP analogs prevent nuclear accumulation in the absence of an intact nuclear envelope through an apparent cytoplasmic retention mechanism. Since the lectin wheat germ agglutinin does not inhibit transport, we postulate a mechanism for angiogenin nuclear/nucleolar import involving passive diffusion of angiogenin through the nuclear pore and NTS-mediated nuclear/nucleolar retention, and with cytoplasmic retention modulating the process. This pathway is clearly distinct from that of conventional signal-mediated nuclear protein import.

Topics: Active Transport, Cell Nucleus; Adenosine Triphosphate; Amino Acid Motifs; Animals; beta-Galactosidase; Carcinoma, Hepatocellular; Cell Nucleolus; Cell Nucleus; Cholic Acids; Cytosol; Detergents; Genes, Reporter; Green Fluorescent Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Liver Neoplasms, Experimental; Luminescent Proteins; Mutagenesis, Site-Directed; Protein Binding; ran GTP-Binding Protein; Rats; Recombinant Fusion Proteins; Ribonuclease, Pancreatic; Tumor Cells, Cultured; Wheat Germ Agglutinins

Alterations in the expression and activity of guanine nucleotide regulatory proteins (G proteins) have been linked to the growth of several human tumors. We hypothesized that the expression and activity of G proteins are altered in human hepatocellular carcinoma (HCC). The expression of Gi and Gs proteins was determined in six human tumors and six normal controls (adjacent nonneoplastic liver) by Western blotting using specific antisera raised against the alpha subunit of G proteins Gi1, Gi1-2, Gi3, and Gs. Differences in G-protein expression were quantified by densitometry and expressed as percentage change from normal controls. The expression of Gi alpha1 was significantly increased in 80% of tumors (Gi alpha1, 284% +/- 77%; P < .05 percent of normal tissue), whereas Gi alpha1-2 and Gi alpha3 expression was increased in 67% of tumors (Gi alpha1-2, 218% +/- 21%; Gi alpha3, 154% +/- 6%; P < .05 percent of normal tissue). The functional activity of Gi alpha proteins as determined by pertussis toxin-catalyzed adenosine diphosphate (ADP)-ribosylation was also significantly increased in these tumors. In contrast, Gs alpha-protein expression was significantly reduced in all tumors examined (74% +/- 8% of normal tissue, P < .05). The functional activity of Gs alpha, as determined by adenylyl cyclase (AC) activity, was significantly decreased in tumor as compared to normal liver under both basal and agonist stimulated (guanosine triphosphate gamma S and forskolin) conditions. In summary, these data show for the first time a significant alteration in G-protein expression and functional activity in human HCC tissue. These alterations indicate a down-regulation of the AC-linked enzyme effector system in HCC that may be of critical importance to the formation and progression of human hepatocellular carcinoma.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclases; Aged; Blotting, Western; Carcinoma, Hepatocellular; Colforsin; Densitometry; Female; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gs; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Liver; Liver Neoplasms; Male; Middle Aged; Pertussis Toxin; Reference Values; Virulence Factors, Bordetella

We developed a sensitive fluorometric assay to study in vitro fusion between early endosomes isolated from the human hepatoma, Hep G2. Biochemical characterization of this assay showed that fusion between endosomal vesicles was dependent on physiologic temperature, cytosol, and ATP. Fusion was inhibited by pretreatment of vesicles and cytosol with either 1 mM N-ethylmaleimide or 20 microM GTP gamma S. Neither 3 mM ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid nor 1 mM CaCl2 significantly affected fusion. In addition, ATP gamma S neither inhibited fusion at 50 microM nor supported fusion at 5 mM. To further our understanding of the factors regulating fusion, inhibitors of endoprotease activity and phosphotyrosine phosphatase activity were assayed for their effect on fusion. The dipeptide inhibitor of endoprotease activity, Cbz-gly-phe-amide, inhibited fusion 70% at 3 mM whereas a dipeptide analogue, Cbz-gly-gly-amide, was without effect. Furthermore, orthovanadate, an inhibitor of phosphotyrosine phosphatase activity, stimulated fusion twofold at 0.5 mM. These results suggest that both tyrosine dephosphorylation and endoprotease activity contribute to the regulation of endosome fusion.

Topics: Animals; Carcinoma, Hepatocellular; Cell Fusion; Dipeptides; Endopeptidases; Enzyme-Linked Immunosorbent Assay; Ethylmaleimide; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Liver Neoplasms; Membrane Fusion; Mice; Organelles; Phosphoprotein Phosphatases; Protease Inhibitors; Rats; Tumor Cells, Cultured