sq-23377 and Liver-Neoplasms

HIF-1 (hypoxia-inducible factor-1) is the main transcription factor responsible for increased gene expression in hypoxia. The oxygen-dependent regulation of HIF-1 activity occurs at multiple levels in vivo. The mechanisms regulating HIF-1alpha protein expression have been most extensively analyzed, but the ones modulating HIF-1 transcriptional activity remain unclear. Changes in the phosphorylation and/or redox status of HIF-1alpha certainly play a role. Here, we show that ionomycin could activate HIF-1 transcriptional activity in a way that is additive to the effect of hypoxia without affecting HIF-1alpha protein level and HIF-1 DNA binding capacity. In addition, a calmodulin dominant-negative mutant as well as BAPTA, an intracellular calcium chelator, inhibited the hypoxia-induced HIF-1 activation. These results indicate that elevated calcium in hypoxia could participate in HIF-1 activation. PD98059, an inhibitor of the ERK pathway, but not KN-93, an inhibitor of calmodulin kinases II and IV, also blocked HIF-1 activation by hypoxia and by ionomycin. Altogether, these results suggest that calcium and calmodulin would act upstream of ERK in the hypoxia signal transduction pathway leading to enhanced HIF-1 transcriptional activity.

Topics: Calcium; Calcium Signaling; Carcinoma, Hepatocellular; Cell Hypoxia; Cloning, Molecular; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Ionomycin; Liver Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nuclear Proteins; Plasmids; Transcription Factors; Transfection; Tumor Cells, Cultured

Gene therapy is an attractive therapy for hepatocarcinoma, and several approaches have been studied using murine leukemia virus-derived retroviruses. We compared gene transfer efficacy and transgene expression kinetics after transduction of hepatocarcinoma cell lines using enhanced green fluorescent protein (EGFP)-expressing murine leukemia virus-derived retroviral vectors and HIV-derived lentiviral vectors. First, we showed that both retroviral and lentiviral vectors efficiently transduce cycling hepatocarcinoma cell lines in vitro. However, after cell cycle arrest, transduction efficacy remained the same for lentiviral vectors but it decreased by 80% for retroviral vectors. Second, we studied EGFP expression kinetics using lentiviral vectors expressing EGFP under the control of cytomegalovirus (CMV) or phosphoglycerolkinase (PGK) promoter. We show that the CMV promoter allows a stronger EGFP expression than the PGK promoter. However, in contrast to PGK-driven EGFP expression, which persists up to 2 months after transduction, CMV-driven EGFP expression rapidly decreased with time. This phenomenon is due to promoter silencing, and EGFP expression can be restored in transduced cells by using transcription activators such as interleukin-6 or phorbol myristate acetate/ionomycin and, to a lesser extent, the demethylating agent 5'-azacytidine. Altogether, our results suggest that lentiviral vectors, which allow efficient transduction of hepatocarcinoma cell lines with a strong and a sustained expression according to the promoter used, are promising tools for gene therapy of hepatocarcinomas.

Topics: Azacitidine; Carcinoma, Hepatocellular; Cell Survival; Dexamethasone; Flow Cytometry; Genetic Vectors; Green Fluorescent Proteins; HIV; Humans; Interleukin-6; Ionomycin; Kinetics; Leukemia Virus, Murine; Liver Neoplasms; Luminescent Proteins; Recombinant Proteins; Tetradecanoylphorbol Acetate; Transduction, Genetic; Transgenes; Tumor Cells, Cultured

1. The human hepatoma cell line Hep3B is a widely used model for studies of hypoxia-related gene expression. Cytosolic free calcium concentration ([Ca2+]i) has been implicated in cellular oxygen-sensing processes. We investigated whether calcium ions have a significant impact on the production of erythropoietin (EPO) and vascular endothelial growth factor (VEGF). 2. We found that the calcium ionophore ionomycin induced a rapid and sustained increase of [Ca2+]i while thapsigargin, an inhibitor of endoplasmic reticulum calcium ATPase, only caused a 20 % elevation of [Ca2+]i within 10 min after application. However, the calcium content of intracellular stores was considerably reduced by thapsigargin after an incubation period of 24 h. 3. Variations in [Ca2+]o did not result in altered EPO or VEGF secretion rates. Ionomycin decreased EPO production while the lowering of VEGF production was not statistically significant. In the presence of extracellular Ca2+ the membrane permeant calcium chelator BAPTA-AM stimulated the production of EPO (P < 0.05) but not of VEGF while EGTA-AM, a closely related agent, affected neither EPO nor VEGF formation under these conditions. Incubation with thapsigargin resulted in decreased EPO synthesis (P < 0.05) but stimulated VEGF secretion (P < 0.05). 4. In the absence of extracellular calcium, EGTA-AM led to an accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha). This treatment significantly stimulated VEGF synthesis but also decreased EPO secretion (P < 0.05). 5. Our data suggest that the calcium transient and the cytosolic Ca2+ concentration do not play a key role in hypoxia-induced EPO and VEGF production in Hep3B cells.

Topics: Blotting, Northern; Calcium; Calcium-Transporting ATPases; Carcinoma, Hepatocellular; Cell Hypoxia; Chelating Agents; Egtazic Acid; Endothelial Growth Factors; Enzyme Inhibitors; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Ionomycin; Kinetics; Liver Neoplasms; Lymphokines; Thapsigargin; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Mobilization of Ca2+ from the endoplasmic reticulum (ER) suppresses translational initiation and inhibits post-translational processing and secretion of glycoproteins. This study explores the mechanism whereby ionomycin, a Ca2+ ionophore, and thapsigargin, an ER Ca(2+)-ATPase inhibitor, promote retention of alpha 1-antitrypsin (alpha 1-AT) bearing high mannose, endoglycosidase H (Endo H)-sensitive oligosaccharide side chains within the ER of HepG2 cells. Arrest occurred at the removal of mannose residues such that intermediates with Man7-9GlcNAc2 side chains accumulated with the Man8-9GlcNAc2 structures predominating. Maturation of alpha 1-AT bearing Man5-6GlcNAc2 side chains was unaffected. Inhibition of alpha 1-AT processing by ionomycin occurred independently of translational suppression. Forms of alpha 1-AT identical to those retained with ionomycin or thapsigargin were observed upon treatment with the alpha-1,2-mannosidase inhibitor 1-deoxymannojirimycin whereas castanospermine, an inhibitor of ER alpha-glucosidase I, produced different forms of the glycoprotein. Neither inhibitor impaired transport or secretion of alpha 1-AT. With brefeldin A, which causes redistribution of Golgi enzymes to the ER, alpha 1-AT was retained intracellularly but acquired resistance to Endo H. With ionomycin, thapsigargin, or 1-deoxymannojirimycin-treated cells, however, brefeldin A failed to promote further processing of the glycoprotein. Possible mechanisms for the suppression of alpha 1-AT processing at the alpha-1,2-mannosidase step by Ca(2+)-mobilizing agents are discussed. Excepting tunicamycin, traditional inhibitors of protein processing did not affect amino acid incorporation.

Topics: 1-Deoxynojirimycin; alpha 1-Antitrypsin; alpha-Mannosidase; Brefeldin A; Calcium; Calcium-Transporting ATPases; Carbohydrate Conformation; Carcinoma, Hepatocellular; Cyclopentanes; Endoplasmic Reticulum; Glycoside Hydrolase Inhibitors; Hexosaminidases; Humans; Indolizines; Ionomycin; Liver Neoplasms; Mannose; Mannosidases; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Oligosaccharides; Terpenes; Thapsigargin; 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

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

Interleukin 6 (IL 6) and interleukin 1 (IL-1) regulate the expression of acute phase plasma proteins in rat and human hepatoma cells. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), partially mimics the stimulatory effect of IL-6 but reduces that effect of IL-1. TPA and IL-6 act synergistically. These regulatory properties of TPA are also manifested in HepG2 cells transiently transfected with an indicator gene construct carrying the IL-1/IL-6 regulatory enhancer element of the rat alpha 1-acid glycoprotein gene. IL-6 and IL-1 act independently of TPA-inducible kinase C, and of changes in intracellular Ca2+ concentrations. However, prolonged pretreatment of HepG2 cells with TPA results in a drastically reduced cytokine response that is proportional to the loss of cell surface binding activity for the cytokine. These data suggest that hormones activating protein kinase C probably play a contributing role in stimulating the expression of acute phase plasma protein genes but they may be crucial in controlling the responsiveness of liver cells to inflammatory cytokines during subsequent stages of the hepatic acute phase reaction.

Topics: Acute-Phase Proteins; Animals; Carcinoma, Hepatocellular; Cell Line; Ethers; Humans; Interleukin-1; Interleukin-6; Interleukins; Ionomycin; Kinetics; Liver Neoplasms; Liver Neoplasms, Experimental; Rats; Recombinant Proteins; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription, Genetic