calcimycin and Carcinoma--Hepatocellular

To investigate the regulation of activin receptor-interacting protein 2 (ARIP2) expression and its possible relationships with collagen type IV (collagen IV) in mouse hepatoma cell line Hepal-6 cells.. The ARIP2 mRNA expression kinetics in Hepal-6 cells was detected by RT-PCR, and its regulation factors were analyzed by treatment with signal transduction activators such as phorbol 12-myristate 13-acetate (PMA), forskolin and A23187. After pcDNA3-ARIP2 was transfected into Hepal-6 cells, the effects of ARIP2 overexpression on activin type II receptor (ActRII) and collagen IV expression were evaluated.. The expression levels of ARIP2 mRNA in Hapel-6 cells were elevated in time-dependent manner 12 h after treatment with activin A and endotoxin LPS, but not changed evidently in the early stage of stimulation (2 or 4 h). The ARIP2 mRNA expression was increased after stimulated with signal transduction activators such as PMA and forskolin in Hepal-6 cells, whereas decreased after treatment with A23187 (25.3% +/- 5.7% vs 48.1% +/- 3.6%, P < 0.01). ARIP2 overexpression could remarkably suppress the expression of ActRIIA mRNA in dose-dependent manner, but has no effect on ActRIIB in Hepal-6 cells induced by activin A. Furthermore, we have found that overexpression of ARIP2 could inhibit collagen IV mRNA and protein expressions induced by activin A in Hapel-6 cells.. These findings suggest that ARIP2 expression can be influenced by various factors. ARIP2 may participate in the negative feedback regulation of signal transduction in the late stage by affecting the expression of ActRIIA and play an important role in regulation of development of liver fibrosis induced by activin.

Topics: Activin Receptors, Type II; Activins; Adaptor Proteins, Signal Transducing; Adenylyl Cyclases; Animals; Calcimycin; Calcium; Carcinoma, Hepatocellular; Cell Line, Tumor; Colforsin; Collagen Type IV; Enzyme Activators; Gene Expression Regulation, Neoplastic; Ionophores; Kinetics; Lipopolysaccharides; Liver Neoplasms; Membrane Proteins; Mice; Protein Kinase C; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Transfection

We present evidence that increases in intracellular calcium, induced by treatment with calcium ionophore A23187 or the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin, dephosphorylated histone H3 at serine10 (histone H3-Ser10) in a dose-dependent manner in human hepatoma HepG2 cells. Inhibition of p42/44MAPK, pp90RSK, or p38MAPK did not affect the ability of A23187 to dephosphorylate histone H3-Ser10. This response is significantly blocked by okadaic acid, indicating a requirement for protein phosphatase 2A (PP2A). A23187 increased the activity of PP2A towards phosphorylated histone H3-Ser10. Furthermore, pretreatment with calphostin C, a selective protein kinase C (PKC) inhibitor, blocked A23187-dependent dephosphorylation of histone H3-Ser10, and coimmunoprecipitation analysis showed PP2A association with the PKCbetaII isoform. Unlike untreated cells, coimmunoprecipitated complex from A23187-treated cells showed greater dephosphorylation of histone H3-Ser10 in a PP2A-dependent manner. Inhibition of PP2A increased phosphorylation at Ser660 that determines calcium sensitivity and activity of PKCbetaII isoform, thus supporting a role for intracomplex regulation. Finally, chromatin immunoprecipitation assays following exposure to A23187 and okadaic acid revealed regulatory role of histone H3-Ser10 phosphorylation in selective gene induction. Altogether, our findings suggest a novel role for calcium in modulating histone H3-Ser10 phosphorylation level and led us to propose a model emphasizing PP2A activation, occurring downstream following perturbations in calcium homeostasis, as key event in dephosphorylating histone H3-Ser10 in mammalian cells.

Topics: Calcimycin; Calcium; Carcinoma, Hepatocellular; Cell Line, Tumor; Histones; Humans; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Phosphoserine; Promoter Regions, Genetic; Protein Binding; Protein Kinase C; Protein Kinase C beta; Protein Phosphatase 2; Receptors, LDL; Signal Transduction; Thapsigargin; Transcription, Genetic

A novel approach to reducing organ toxicity of anticancer agents is the application of nontoxic glucuronide prodrugs from which the active drug is released by human beta-glucuronidase, an enzyme present at high levels in many tumors. In view of high interindividual variability in beta-glucuronidase expression, regulation of this enzyme is an essential factor modulating bioactivation of glucuronide prodrugs. However, data on regulation of human beta-glucuronidase expression are not available. Preliminary evidence from animal experiments points to a role of intracellular calcium in regulation of beta-glucuronidase activity. Therefore, we investigated regulation of beta-glucuronidase by the calcium ionophore A23187 and the calcium ATPase inhibitor thapsigargin in the human hepatoma cell line HepG2. The enzyme was characterized on activity, protein, and mRNA levels by cleavage of 4-methylumbelliferyl-beta-D-glucuronide, Western blotting, Northern blotting, and nuclear run-on transcription. Incubation of HepG2 cells with A23187 and thapsigargin, respectively, revealed a time and concentration dependent down-regulation of beta-glucuronidase activity to about 50% of the control level. This effect could also be demonstrated in several other cell lines (e.g., HL-60, ECV 304, 32M1, Caco-2/TC7). Effects on protein and mRNA levels paralleled those obtained on enzymatic activity. In line with these data, A23187 and thapsigargin decreased beta-glucuronidase transcriptional rate. Our data demonstrate regulation of human beta-glucuronidase by xenobiotics. Down-regulation of beta-glucuronidase by A23187 and thapsigargin is at least partly mediated by a transcriptional mechanism. Based on our findings, we speculate that beta-glucuronidase activity and hence bioactivation of glucuronide prodrugs in humans can be modulated by exogenous factors.

Topics: Blotting, Northern; Blotting, Western; Calcimycin; Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Glucuronidase; Humans; Ionophores; Lung Neoplasms; RNA, Messenger; Thapsigargin; Time Factors; Transcription, Genetic; Tumor Cells, Cultured

Some investigators have reported previously that phorbol esters inhibit in vitro erythropoietin production stimulated by hypoxia; whereas others have reported that phorbol esters enhanced Epo production during exposure to hypoxia. We have demonstrated in the present experiments that hypoxia significantly increased diacylglycerol levels in cultured human hepatocellular carcinoma (Hep3B) cells. 1-oleoyl-2-acetyl-ras-glycerol (OAG) and N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9), two well-known protein kinase C activators, significantly increased medium levels of erythropoietin as well as erythropoietin messenger RNA levels in normoxic Hep3B cells. A potent protein kinase C inhibitor, chelerythrine chloride, significantly decreased hypoxia-induced increases in medium levels of erythropoietin as well as erythropoietin messenger RNA levels in Hep3B cells. A cis-unsaturated free fatty acid, oleic acid, significantly enhanced OAG-induced medium levels of erythropoietin in normoxic Hep3B cells, whereas a phospholipase A2 inhibitor, mepacrine, significantly decreased hypoxia-induced erythropoietin production in Hep3B cells. These results provide strong support for a positive role for protein kinase C in the hypoxic regulation of erythropoietin production.

Topics: Alkaloids; Benzophenanthridines; Calcimycin; Carcinoma, Hepatocellular; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Erythropoietin; Humans; Ionophores; Liver Neoplasms; Models, Chemical; Oleic Acid; Phenanthridines; Phospholipases A; Phospholipases A2; Protein Kinase C; Quinacrine; Thapsigargin; Tumor Cells, Cultured

The human hepatoma cell line, Hep 3B, produces biologically active erythropoietin (Epo) in response to normal physiologic stimuli and thus provides a model system for the study of Epo regulation. The addition of phorbol 12-myristate 13-acetate (PMA) to Hep 3B cells subsequently grown under hypoxic conditions resulted in a dose-dependent inhibition of hypoxia-induced Epo production by as much as 95 +/- 1% with half-maximal inhibition at 8 ng/mL. By Northern blot analysis, Epo mRNA levels were correspondingly decreased after treatment with PMA. Direct measurement of both membrane and cytosolic protein kinase C activity in Hep 3B cells following treatment with PMA demonstrated a biphasic response as a function of time. Membrane-associated protein kinase C activity initially increased but subsequently decreased to baseline levels by 12 hours. The PMA-induced inhibition of hypoxia-induced Epo production was shown to occur as early as 3 hours after PMA addition, suggesting that the initial activation, rather than the subsequent decrease in protein kinase C activity, is of primary importance. The relative specificity of the PMA-induced inhibition of Epo production was demonstrated by 1) the finding that overall protein and RNA synthesis were not similarly decreased as measured by 3H-leucine and 3H-uridine pulse labeling studies and 2) the observation that the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, failed to have any effect on hypoxia-induced Epo production. In addition, the synthetic analog of diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG) and the calcium ionophore, A23187, inhibited hypoxia-induced Epo production up to 85 +/- 3% and 82 +/- 4%, respectively, in a dose-dependent manner. Taken together, these findings suggest that hypoxia-induced Epo production may be negatively regulated by activators of a protein kinase C-mediated pathway.

Topics: Blotting, Northern; Calcimycin; Carcinoma, Hepatocellular; Diglycerides; DNA; Dose-Response Relationship, Drug; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Leucine; Liver Neoplasms; Protein Kinase C; Radioimmunoassay; RNA; Tetradecanoylphorbol Acetate; Tritium; Tumor Cells, Cultured; Uridine

Addition of 2 mM dithiothreitol (DTT) to HepG2 human hepatoma cells blocks secretion of newly made albumin and causes it to accumulate in the endoplasmic reticulum in the reduced state. Subsequent incubation of the cells in the absence of DTT allows the reduced albumin to form apparently normal disulfide bonds and to be secreted normally. Similarly, DTT treatment causes all newly made subunits of the H1 subunit of the asialoglycoprotein receptor to be retained in the endoplasmic reticulum in the reduced state; following removal of DTT H1 subunits form disulfide bonds via a normal endoplasmic reticulum folding intermediate and mature to the Golgi. Thus, apparently normal formation of disulfide bonds on these two proteins can occur post-translationally, as has been shown previously for the influenza HA hemagglutinin (Braakman, I., Helenius, J., and Helenius, A. (1992) EMBO J. 11, 1717-1722; Braakman, I., Helenius, J., and Helenius, A. (1992) Nature 356, 260-262). alpha 1-Antitrypsin contains no disulfide bonds; in the continuous presence of DTT it acquires a normal complement of complex oligosaccharides and is secreted at an only slightly reduced rate. Thus, the secretory pathway functions efficiently even when it is reduced by DTT. Endoplasmic reticulum retention of albumin and H1 during treatment with DTT presumably occurs because, without disulfide bonds, these proteins cannot fold properly, not because of any defect in the overall processes of vesicular transport and protein secretion.

Topics: alpha 1-Antitrypsin; Asialoglycoprotein Receptor; Asialoglycoproteins; Autoradiography; Calcimycin; Carcinoma, Hepatocellular; Centrifugation, Density Gradient; Cysteine; Disulfides; Dithiothreitol; Endoplasmic Reticulum; Humans; Liver Neoplasms; Macromolecular Substances; Methionine; Neoplasm Proteins; Oxidation-Reduction; Receptors, Immunologic; Ribonucleases; Serum Albumin; Subcellular Fractions; Sulfur Radioisotopes; Transferrin; Tumor Cells, Cultured

The effects of calcium depletion on the proteolytic cleavage and secretion of plasma protein precursors were investigated in primary cultured rat hepatocytes and HepG2 cells. When the cells were incubated with A23187, the calcium-specific ionophore, in a medium lacking CaCl2, precursors of serum albumin and the third and fourth components of complement, C3 and C4, respectively, were found to be released into the medium. The addition of ionomycin or EGTA to the medium inhibited the processing of pro-C3 as well. Blocking the secretory pathway either at the mixed endoplasmic reticulum/Golgi in the presence of brefeldin A or at the endoplasmic reticulum/tubular-vesicular structure at a reduced temperature caused accumulation of pro-C3 within hepatocytes or HepG2 cells, indicating that the cleavage of the precursor occurs at a later stage of the secretory pathway. Once the blockade was released by incubating the cells either in the brefeldin A-free medium or at 37 degrees C, the secretion of plasma proteins resumed, irrespective of the presence of A23187. However, the processing of pro-C3 was almost completely inhibited in the presence of A23187, with only the precursor being released into the medium, implying that a decline in Ca2+ levels within the cell modulates the activity of a Golgi endoprotease responsible for the cleavage of pro-C3. When incubated with isolated Golgi membranes, pro-C3 secreted from Ca(2+)-depleted cells was cleaved in vitro into their subunits in the presence of Ca2+ but not in its absence, pointing to the involvement of a Ca(2+)-dependent Golgi endoprotease in the processing of pro-C3. These results collectively suggest that calcium depletion blocks the proteolytic cleavages of plasma protein precursors presumably by exhausting a Ca2+ pool available to the Ca(2+)-dependent processing enzyme(s) located at the Golgi and/or trans-Golgi network.

Topics: Animals; Calcimycin; Calcium; Carcinoma, Hepatocellular; Cell Line; Cells, Cultured; Complement C3; Complement C4; Endopeptidases; Golgi Apparatus; Humans; Ionomycin; Kinetics; Liver; Liver Neoplasms; Prealbumin; Protein Precursors; Protein Processing, Post-Translational; Rats

This study investigated the intracellular signal transduction regulating the appearance of HLA class I antigens on Huh 6 cells induced by interferon-gamma. The expression was blocked by a protein kinase C inhibitor, H-7, but not by a calmodulin antagonist, W-7, nor by a protein kinase A inhibitor, H-8, at low dose. The antigen expression was induced by a direct activator of protein kinase C, phorbol myristate acetate, but not by calcium ionophore A23187 nor an analog of cAMP, dbcAMP. Therefore, we concluded that protein kinase C is involved in the expression of HLA class I antigens on Huh 6 cells induced by interferon-gamma but Ca(2+)-calmodulin and cAMP are not.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Calcimycin; Calmodulin; Carcinoma, Hepatocellular; Cell Line; Cyclic CMP; Histocompatibility Antigens Class I; Humans; Interferon-gamma; Isoquinolines; Liver Neoplasms; Piperazines; Signal Transduction; Sulfonamides; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

In the cultured human hepatoma HepG2, Ca2+ ionophores block secretion of different secretary proteins to different extents, alpha 1-antitrypsin secretion being more sensitive to A23187 and ionomycin than is alpha 1-antichymotrypsin, and albumin secretion the least of the three proteins studied. As judged by subcellular fractionation experiments and by treatment of pulse chase labeled protein with endoglycosidase H, A23187 and ionomycin cause newly made secretory proteins to remain within the rough endoplasmic reticulum (ER). Experiments in which A23187 is added at different times during a pulse or chase show that secretion of newly made alpha 1-antitrypsin becomes resistant to the ionophore, on average, 15 min after synthesis; this is about 20 min before it reaches the trans-Golgi, and while it is still within the rough ER. We speculate that a high concentration of Ca2+ within the ER may be essential for certain secretory proteins to fold properly, that folding is inhibited when ER Ca2+ levels are lowered by ionophore treatment, and that unfolded proteins, particularly alpha 1-antitrypsin, cannot exit the rough ER. Treatment of murine 3T3 fibroblasts or human hepatoma HepG2 cells with the Ca2+ ionophores A23187 or ionomycin also induces a severalfold accumulation of the ER lumenal protein Bip (Grp78). These findings disagree with a recent report that Ca2+ ionophores cause secretion of Bip and other resident ER proteins, but is consistent with other reports that A23187 causes accumulation of mRNAs for Bip and other ER lumenal proteins.

Topics: Acetylglucosaminidase; Albumins; alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Amino Acid Sequence; Animals; Blotting, Western; Calcimycin; Calcium; Carcinoma, Hepatocellular; Carrier Proteins; Cell Fractionation; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Fibroblasts; Heat-Shock Proteins; Humans; Ionomycin; Liver Neoplasms; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Mice; Molecular Chaperones; Molecular Sequence Data; Molecular Weight; Proteins; Tumor Cells, Cultured

Induction of C-reactive protein (CRP) by conditioned medium from lipopolysaccharide-stimulated human monocytes in two human hepatoma-cell lines, Hep 3B and NPLC/PRF/5, was potentiated 3-6-fold by the methylxanthine caffeine. The induction observed in the presence of conditioned medium plus caffeine was as much as 180-fold, comparable with that seen after many stimuli in vivo. This potentiation was accompanied by an increase in the levels of CRP mRNA. By contrast, no potentiating effect on CRP induction by conditioned medium was found when we tested theophylline, forskolin, 8-bromo cyclic AMP or two Ca2+ ionophores, namely ionomycin and A23187. None of the above compounds, including caffeine, when tested alone, had any detectable effect on the synthesis and secretion of CRP. Our previous study [Ganapathi, May, Schultz, Brabenec, Weinstein, Sehgal & Kushner (1988) Biochem. Biophys. Res. Commun. 157, 271-277], employing defined cytokines, had shown that induction of CRP in Hep 3B cells requires IL(interleukin)-6 plus IL-1, whereas, in the NPLC/PRF/5 cell line, IL-6 alone is effective. Caffeine similarly potentiated induction of CRP by these defined cytokine signals in these two cell lines. Changes in synthesis of other acute-phase proteins, including serum amyloid A (SAA), alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin and albumin, induced by conditioned medium or, in some cases, by IL-6 and/or IL-1 alpha, were only minimally affected by caffeine. Thus these results indicate that the mechanism by which caffeine potentiates CRP induction by cytokines appears to be independent of increases in intracellular concentrations of the two second messengers, cyclic AMP and Ca2+; the precise nature of this mechanism is unclear at the present time. Our results also indicate that the intracellular mechanisms by which cytokines regulate synthesis of CRP may differ from those regulating synthesis of some other acute-phase proteins. The differential response of CRP and SAA to caffeine is of particular interest, since induction of both of these two major acute-phase proteins can be accomplished by identical extracellular signals.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; C-Reactive Protein; Caffeine; Calcimycin; Calcium; Carcinoma, Hepatocellular; Colforsin; Cyclic AMP; Drug Synergism; Humans; Interleukin-1; Interleukin-6; Ionomycin; Liver Neoplasms; Recombinant Proteins; Second Messenger Systems; Theophylline; Tumor Cells, Cultured

The human hepatic adenocarcinoma cell line, SK-hep-1, was found to constitutively produce Interleukin 1. Addition of the ionophore A23187 and lipopolysaccharide resulted in a 30-fold enhancement in the release of biological activity. Serum supplementation did not affect the level of production. Interleukin 1 from these cells had a molecular weight of 10-20,000 daltons on gel exclusion chromatography. Polyadenylated RNA, when fractionated on sucrose density gradients and injected into Xenopus laevis oocytes, produced high levels of biological activity in the 14-16s region. An oligonucleotide probe, complementary to the coding sequence of the Interleukin 1 cDNA isolated from human monocytes, hybridized specifically to this part of the gradient. These results demonstrate that SK-hep-1 cells are a valuable source of material for studying the polypeptide and messenger RNA of Interleukin 1.

Topics: Adenocarcinoma; Animals; Biological Assay; Calcimycin; Carcinoma, Hepatocellular; Cell Line; Centrifugation, Density Gradient; Chromatography, Gel; Female; Humans; Interleukin-1; Lipopolysaccharides; Liver Neoplasms; Mice; Molecular Weight; Oocytes; RNA, Messenger; Xenopus laevis