Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A membrane-bound arylamidase from well-differentiated hepatocellular carcinoma having the same electrophoretic mobility as placental membrane-bound arylamidase was found. The enzyme was found to be a sialoprotein and was activated by Co2+. Hepatoma membrane-bound arylamidase had a similar molecular weight (240 000) and kinetic properties to normal liver membrane-bound arylamidase, but differed from the liver enzyme with respect to electrophoretic mobility, heat stability and urea inactivation.
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PMID:Membrane-bound arylamidase from human hepatoma cells having the same electrophoretic mobility as placental membrane-bound arylamidase. 20 22

Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) has been purified over 100 000-fold from a whole cell extract of guinea pig liver. The enzyme yields a single stainable band when subjected to non-denaturing polyacrylamide gel electrophoresis, and this band corresponds to the DNA polymerase activity when a sister gel is sliced and assayed. The final fraction has a specific activity of 21 000 units/mg; this value can be increased significantly by addition of various components, including glycols, polyamines or any of several protein factors which can be purified from the crude extract. The DNA polymerase-beta lacks detectable exonuclease or endonuclease activity, has an alkaline pH optimum and has a requirement for all four deoxyribonucleoside triphosphates, a divalent cation and a primer-template for maximal activity. While activated DNA is the preferred primer-template, the enzyme is capable of utilizing native and denatured DNA as well as several synthetic polynucleotides as primer-templates. The latter are especially effective when manganese is the divalent cation. Magnesium, at 10 mM, is the preferred divalent cation when activated DNA is used. Manganese, and to a lesser extent cobalt, can substitute for magnesium while zinc and calcium cannot. The beta-polymerase has a half-life of 10 min at 40 degrees C and this is increased in the presence of either DNA or NaCl. The enzyme is stimulated by glycols, polyamines and NaCal or KCl, and is inhibited by several known inhibitors of DNA polymerase activity including o-phenanthroline, heparin, organic solvents and sulfhydryl blocking agents. Guinea pig liver DNA polymerase-beta is remarkably similar to the rat Novikoff hepatoma beta-polymerase with respect to its isoelectric point of 8.4 and its molecular weight of 32 000 as determined by sucrose gradient centrifugation under high or low salt conditions or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This similarity is further extended to the removal, at the final step in purification, of a protein capable of stimulating the homogeneous enzyme. Removal of this protein could explain the lower molecular weight of the guinea pig and other rodent-derived beta-polymerases, when compared to the beta-polymerases from other systems.
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PMID:Purification and properties of DNA polymerase-beta from guinea pig liver. 70 39

This study reports the effects of cyclic adenosine 3'-5' monophosphate (cAMP) and hypoxia on erythropoietin biosynthesis in an erythropoietin-producing hepatocellular carcinoma cell line (Hep3B). Erythropoietin levels in the medium and cell extracts of low-density Hep3B cells after 20-hour incubation under hypoxic conditions (1% O2) were 25.33 +/- 1.50 mU/ml/10(7) cells and 3.60 +/- 0.50 mU/10(7) cells, respectively. These levels were significantly higher than in the respective normoxic controls (medium, 2.51 +/- 0.31 mU/ml/10(7) cells; cell extracts, undetectable [less than 0.31 mU/10(7) cells]). Cobalt also produced a significant increase in medium and cell erythropoietin levels. However, hypoxia and cobalt alone failed to produce an increase in cAMP accumulation in the cell cultures. Erythropoietin levels in the medium and cell extracts from cells exposed to 8-bromo cAMP (1 x 10(-4) mol/L) and forskolin (4 x 10(-6) mol/L) in a hypoxic atmosphere were significantly (p less than 0.05) higher than in the respective hypoxic controls. In addition, forskolin produced a significant (p less than 0.05) increase in cAMP accumulation (180 +/- 11.5 pmol/10(6) cells) under hypoxic conditions compared with the hypoxic controls (cAMP, 2.27 +/- 0.33 pmol/10(6) cells). These results suggest that cAMP elevation is not required in vitro in Hep3B cells for the increase in medium and cell levels of erythropoietin after hypoxia, but may be involved indirectly in erythropoietin biosynthesis, secretion, or both in vivo through some synergistic action with hypoxia.
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PMID:Characterization of erythropoietin production in a hepatocellular carcinoma cell line. 131 42

We examined the effects of insulin-like growth factors (IGFs) and insulin on erythropoietin (EPO) production by human hepatoma cells (Hep G2). Compared with normoxia (20% O2), EPO production by Hep G2 cells during a 72-h incubation was stimulated fivefold by exposure to low oxygen tension (1% O2) and nearly threefold by exposure to cobalt chloride (100 microM). IGF-I caused a concentration-dependent attenuation of EPO formation under normoxic conditions and inhibited (maximally 50%) EPO production stimulated by either low oxygen tension or cobalt [half-maximal effect (ED50) approximately 5 nM]. The increase of EPO mRNA levels in response to hypoxia was significantly reduced by IGF-I. Similarly to IGF-I, IGF-II (ED50 approximately 8 nM) and insulin (ED50 approximately 80 nM) also inhibited EPO formation in Hep G2 cells. IGF-I (100 pM-100 nM) stimulated the incorporation of radiolabeled alanine as a measure for total protein synthesis, 3H-labeled thymidine incorporation into DNA, and glycogen synthesis at 20 and 1% O2 in a concentration-dependent fashion. IGF-I exhibited a high affinity for the IGF-I receptor (apparent Kd approximately 3 nM). Unlabeled insulin was greater than 100-fold less potent than IGF-I in competing for 125I-IGF-I binding (apparent Kd approximately 360 nM). Conversely, insulin bound to the insulin receptor with high affinity (apparent Kd approximately 0.3 nM), whereas IGF-I was less than 1% as potent in competing for 125I-insulin binding. In summary, IGFs and insulin exert a negative control function on oxygen-regulated EPO production in Hep G2 cells. The inhibitory effect of IGFs and insulin on EPO formation appears to be mediated via the IGF-I receptor.
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PMID:Insulin-like growth factors decrease oxygen-regulated erythropoietin production by human hepatoma cells (Hep G2). 132 19

Because the human hepatoma cell line Hep3B produces erythropoietin (Epo) in a regulated fashion, it can be used to investigate the cis-acting regulatory elements of the Epo gene. Comparison of primate and mouse sequences shows strong homology not only in the coding sequence but also within the 5' flanking region, the first intron, and the 3' flanking region. These portions of the Epo gene were inserted 5' and 3' to a reporter gene, human growth hormone (GH). 5A is a 1,192-base pair (bp) HindIII-Xbal fragment that extends from 378 bp 5' to the cap site through the first intron. To obviate the problem of false initiation of translation from the Epo ATG start codon, this site was changed to TAG by site-directed mutagenesis. 3A is a 255-bp Accl-BglII fragment that extends 67 bp upstream from the Epo termination codon and covers most of the 3' noncoding region of homology. The plasmid DNAs were transfected by electroporation into Hep3B cells with RSVCAT as an internal standard to correct for transfection efficiency. One aliquot of cells was exposed to 50 mumol/L CoCl2 or to 1% O2. At the end of the incubations, GH and Epo were measured in the cell media and the cell pellet was assayed for CAT. Production of GH was stimulated 1.7-fold by cobalt or hypoxia. Furthermore, addition of 3A to the GH gene, irrespective of orientation, stimulated GH production 2.6-fold with CoCl2 and 2.3-fold with hypoxia. Stable cell lines were produced by cotransfection of the above constructions, along with the selectable marker pSV-Neo. In two clones, exposure to hypoxia resulted in much more marked (16-fold) induction of GH. Stimulus of both GH and Epo production by hypoxia was partially abrogated by carbon monoxide. These results demonstrate the presence of promoter and enhancer elements within the human Epo gene that are appropriately responsive to hypoxia and cobalt.
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PMID:Regulatory elements of the erythropoietin gene. 198 94

Highly purified GH-receptor preparations from 3T3-F442A fibroblasts, whose differentiation into adipocytes is promoted by GH, have been shown to contain a tyrosine kinase capable of phosphorylating GH receptors. In the current work, characteristics of the tyrosine kinase responsible for the in vitro phosphorylation of GH receptors from cultured 3T3-F442A fibroblasts were examined, and the presence of this GH receptor-associated tyrosine kinase activity was demonstrated in multiple cell types. GH-receptor complexes from GH-treated cells were partially purified by immunoprecipitation using anti-GH antibodies and then incubated as an immune complex with [gamma 32P] ATP. Incorporation of 32P into the GH receptor from 3T3-F442A fibroblasts was apparent within 1 min at 30 C after the addition of [gamma 32P]ATP (5-10 microM). A divalent cation was requisite for the phosphorylation; Mn2+ was significantly more effective than Mg2+ and Co2+; Ba2+, Ca2+, or Zn2+ had no effect. Excess unlabeled ATP, but not cytosine triphosphate, GTP, or uridine triphosphate, abolished 32P incorporation into the GH receptor and [gamma 32P]GTP could not replace [gamma 32P]ATP as a source of 32P. At 5.5 mM Mn2+, phosphorylation exhibited a biphasic dose response to ATP, with maximal phosphorylation occurring at a concentration of 10 microM ATP. At more physiological concentrations of ATP (1 mM), phosphorylation of the GH receptor was also stimulated by lower concentrations of Mn2+ (as low as 500 nM). Optimal reaction conditions determined for the phosphorylation reaction in 3T3-F442A fibroblasts were used to demonstrate incorporation of 32P from [gamma 32P]ATP into partially purified GH receptors from cultured human IM-9 lymphocytes, murine 3T3-F442A adipocytes, rat H-35 hepatoma cells, and freshly isolated rat adipocytes. The 32P was shown to be incorporated into tyrosyl residues in receptors from the two cell types tested (IM-9 lymphocytes and rat adipocytes). Cross-linked [125I] hGH-receptor complexes solubilized from the four cell types (IM-9 lymphocytes, 3T3-F442A adipocytes, H-35 hepatoma cells, and freshly isolated rat adipocytes) bound to and could be eluted from phosphotyrosyl antibody, suggesting that tyrosyl phosphorylation of GH receptors in all of these cells occurs in vivo. The presence of tyrosine kinase activity associated with GH receptors in multiple cell types from different species is consistent with tyrosine kinase activity playing a role in the actions of GH.
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PMID:Demonstration of growth hormone (GH) receptor-associated tyrosine kinase activity in multiple GH-responsive cell types. 217 17

Erythropoietin (EPO) is the primary humoral regulator of mammalian erythropoiesis. The single-copy EPO gene is normally expressed in liver and kidney, and increased transcription is induced by anemia or cobalt chloride administration. To identify cis-acting DNA sequences responsible for regulated expression, transgenic mice were generated by microinjection of a 4-kilobase-pair (kb) (tgEPO4) or 10-kb (tgEPO10) cloned DNA fragment containing the human EPO gene, 0.7 kb of 3'-flanking sequence, and either 0.4 or 6 kb of 5'-flanking sequence, respectively. tgEPO4 mice expressed the transgene in liver, where expression was inducible by anemia or cobalt chloride, kidney, where expression was not inducible, and other tissues that do not normally express EPO. Human EPO RNA in tgEPO10 mice was detected only in liver of anemic or cobalt-treated mice. Both tgEPO4 and tgEPO10 mice were polycythemic, demonstrating that the human EPO RNA transcribed in liver is functional. These results suggest that (i) a liver inducibility element maps within 4 kb encompassing the gene, 0.4 kb of 5'-flanking sequence, and 0.7 kb of 3'-flanking sequence; (ii) a negative regulatory element is located between 0.4 and 6 kb 5' to the gene; and (iii) sequences required for inducible kidney expression are located greater than 6 kb 5' or 0.7 kb 3' to the gene. RNase protection analysis revealed that human EPO RNA in anemic transgenic mouse liver and hypoxic human hepatoma cells is initiated from several sites, only a subset of which is utilized in nonanemic transgenic liver and human fetal liver.
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PMID:Human erythropoietin gene expression in transgenic mice: multiple transcription initiation sites and cis-acting regulatory elements. 230 68

Human hepatoma (Hep G2) cells have been shown to secrete nanogram quantities of carboxypeptidase N (Grimwood, B. G., Plummer, T. H., Jr., and Tarentino, A. (1988) J. Biol. Chem. 263, 14397-14401). A second carboxypeptidase with an acidic pH optimum (pH 5.5) is also secreted at levels 2-3-fold greater than carboxypeptidase N. This enzyme was partially purified from the conditioned medium and compared with pure bovine pituitary carboxypeptidase H. The two enzymes behaved in a similar fashion in DE52 ion-exchange chromatography and on gel filtration, with the Hep G2 enzyme being slightly larger than the bovine pituitary enzyme (52-54 versus 50-52 kDa). Both enzymes hydrolyzed COOH-terminal basic amino acids from typical synthetic substrates as well as from natural leuenkephalin peptides and were identical based on pH activity profiles, inhibition by EDTA or guanidinoethyl mercaptosuccinic acid, and stimulation by Co2+ ions. Inhibition of enzyme secretion from Hep G2 cells by tunicamycin indicated that the Hep G2 enzyme was glycosylated. This finding was confirmed by a parallel deglycosylation of the Hep G2 and bovine pituitary carboxypeptidase H enzymes with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Immunoblots using mouse antiserum to bovine pituitary carboxypeptidase H revealed that the Hep G2 enzyme was immunocross-reactive with the bovine enzyme but was slightly larger in size (54 versus 52 kDa). Continuous [35S]methionine labeling and purification to near homogeneity using an affinity matrix corroborated the observations that the secreted Hep G2 carboxypeptidase H was slightly larger than bovine pituitary carboxypeptidase H. The Hep G2-secreted enzyme in pulse-chase experiments was initially detected intracellularly after a 15-min pulse as a single protein of about 54 kDa and was present in the 30-min chase medium with no evidence for pre- or postsecretion proteolytic processing. The human adrenergic cell line IMR-32 continuously labeled with [35S]methionine also secreted carboxypeptidase H of the same size as the Hep G2 enzyme.
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PMID:Carboxypeptidase H. A regulatory peptide-processing enzyme produced by human hepatoma Hep G2 cells. 254 70

Treatment of mouse hepatoma (Hepa) cells with heme or cadmium chloride in serum-free medium causes a rapid increase in the steady-state level of heme oxygenase (HO) messenger RNA. This increase is both dose- and time-dependent. Maximum accumulation of HO mRNA is observed 3 h after addition of either agent. Treatment of Hepa cells with heme or CdCl2 also stimulates the transcription of the HO gene, as judged by in vitro nuclear transcription run-on assays. The maximum rate of HO gene transcription occurs 2 h after treatment with either agent. Comparison of the relative increase in the rate of HO gene transcription with the relative increase in the level of HO mRNA demonstrates that transcriptional activation is the primary mechanism by which heme and cadmium produce the accumulation of HO mRNA in Hepa cells. Cadmium may also influence other processes involved in the expression of HO, since the time course of mRNA accumulation diverges from that of gene transcription. However, neither heme nor cadmium alters the rate of HO mRNA degradation. Cobalt chloride and heat shock, which are potent inducers of HO mRNA in rat liver and rat C6 glioma cells, respectively, have only a small effect on the level of HO mRNA in mouse hepatoma cells.
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PMID:Transcriptional activation of the heme oxygenase gene by heme and cadmium in mouse hepatoma cells. 270 93

The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. We have screened multiple renal and hepatic cell lines (including MDCK, LLC-PK1, BHK, WRL 68, CLCL, A704, CRFK, A498, ACHN, TCMK-1, LLC-MK2, CaKi-2, HepG2, and Hep3B) for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10(6) cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities less than 3.3 X 10(5) cells per cm2, there was little constitutive release of Epo in the medium (less than 30 milliunits per 10(6) cells in 24 hr). With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 microM cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.
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PMID:The regulated expression of erythropoietin by two human hepatoma cell lines. 282 72


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