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)

Glucocorticoids rapidly and completely inhibit intracellular plasminogen activator activity in rat hepatoma cells, as assayed by the solubilization of 125I-labeled fibrin. Experiments in which extracts of dexamethasone-treated cells are mixed with extracts of control cells demonstrate an inhibitor of protease activity. Plasminogen activator is found primarily in the particulate fraction of cell lysates, whereas the inhibitor is localized in the soluble fraction. Variant cells have been isolated previously that are fully resistant to the dexamethoasone inhibition of plasminogen activator activity. These variants have no demonstrable inhibitor activity, whereas plasminogen activator in these cells is fully sensitive to inhibitor from wild-type cells. Thus, the basis for hormone resistance appears to be the failure of dexamethasone to induce an inhibitor.
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PMID:Mechanism of dexamethasone inhibition of plasminogen activator in rat hepatoma cells. 10

Incubation of rat hepatoma cells (HTC) in tissue culture with glucocorticoids alters several membrane properties characteristic of transformed cells, without affecting the growth rate of these cells. Variant cell lines resistant to dexamethasone inhibition of plasminogen activator production have been isolated using an agar-fibrin overlay technique to detect plasminogen activator production by individual colonies of HTC cells. The resistance of dexamethasone is not secondary to abnormal or absent glucocorticoid receptors, but due to a lesion in a later step in hormone action specific for plasminogen activator. These variants should prove useful for the study of the mechanism of steroid action as well as for the analysis of the role of proteases in the hormonal regulation of membrane function.
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PMID:Hormonal regulation of membrane phenotype. 20 7

Glucocorticoids affect the composition and function of the plasma membrane in a variety of cell types. Cultured rat hepatoma (HTC) cells in tissue culture provide an excellent model system for analysis of such effects. In these cells, dexamethasone rapidly and dramatically inhibits the influx of amino acids sharing the A or alanine-preferring transport system. Inhibition is half-maximal within 2 h, and maximal after 6 h incubation with the hormone. The inhibition is rapidly reversed by insulin, and more slowly by removing the steroid. Microtubules and microfilaments are not apparently involved in this hormonal effect, but continuous protein synthesis is required for the glucocorticoid inhibition of transport. Dexamethasone also decreases the number of microvilli on the surface of HTC cells, increases their adhesiveness to a substratum, and dramatically decreases the production of plasminogen activator, but it does not affect the growth rate or plating efficiency of the cells. Variant cell lines stably resistant to dexamethasone inhibition of plasminogen activator production have been isolated using an agar-fibrin overlay technique to detect protease production by individual colonies of HTC cells. The hormonal resistance to inhibition of protease production is associated witha maintenance of inducibility of other glucocorticoid-regulated functions and therefore is not apparently secondary to abnormal or absent glucocorticoid receptor, but due to a lesion in a later step in hormone action specific for plasminogen activator. Combined genetic and biochemical analysis of such dexamethasone-resistant variants should facilitate study of the hormonal regulation of specific membrane phenotypes and of the role of proteases in this regulation.
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PMID:Glucocorticoids and the plasma membrane. 38 92

Glucocorticoids induce several phenotypic changes in rat hepatoma cells in tissue culture, including the inhibition of plasminogen activator activity. Variant cell lines resistant to dexamethasone inhibition of plasminogen activator activity have been isolated using an agar-fibrin overlay technique to identify colonies with fibrinolytic (plasminogen activator) activity. The variants are resistant to concentrations of dexamethasone 1,000 times that necessary to completely inhibit plasminogen activator activity in wild-type cells. The variant phenotype has been inherited in a stable manner for more than 300 generations in continuous culture in the absence of dexamethasone. These variants are unique in that the resistance is not secondary to defective or absent glucocorticoid receptors but is due to a lesion specific for regulation of plasminogen activator. Fluctuation analyses support the hypothesis that resistance to dexamethasone arises randomly and is not induced by dexamethasone. Because HTC cells are heteroploid and karyotypically highly variable, variants are thought to arise primarily by chromosomal segregation events. These variants provide a valuable tool for studying the mechanism of hormonal regulation of plasminogen activator as well as the role of proteases in hormonal regulation of membrane functions.
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PMID:Isolation of rat hepatoma cell variants selectively resistant to dexamethasone inhibition of plasminogen activator. 45 95

The binding, internalization, and degradation of tissue-type plasminogen activator (t-PA) were studied in a rat hepatoma (Novikoff) cell line. Binding of t-PA to specific saturable high affinity binding sites (Kd = 12 nM, 54,000 sites/cell) was followed by internalization and degradation and did not require a functional active site. The catabolism of t-PA was not inhibited by an excess of urokinase-type plasminogen activator (u-PA), and t-PA bound to Novikoff membranes was not complexed to PAI-1, suggesting a mechanism independent of PAI-1. Additionally, a mannose receptor is not involved since t-PA binding was not influenced by an excess of mannose, galactose, ovalbumin, or EDTA. Furthermore, the degradation of t-PA was not influenced by 10 mM 6-aminohexanoic acid, a lysine analogue. The t-PA receptor binds to and can be eluted from wheat germ agglutinin-Sepharose. Cross-linking of t-PA with partially purified receptor and ligand blot analysis, suggest that t-PA binds to two proteins, a principal one of 55 kDa and a minor one of 43 kDa. Novikoff cells are able also to bind (Kd = 1.4 nM, 25,000 sites/cell) and degrade u-PA. The binding was inhibited by pro-u-PA and the amino-terminal fragment of u-PA, but not by an excess of t-PA. The u-PA receptor, but not the t-PA receptor, was removed by treatment with phosphatidylinositol-specific phospholipase C. Our results show that the clearance receptor for t-PA on Novikoff cells is different from the mannose receptor and the PAI-1-dependent receptor described in other cells. The rat hepatoma cells are thus a good model to study the PAI-1 independent hepatocyte-specific clearance of t-PA.
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PMID:Demonstration of a specific clearance receptor for tissue-type plasminogen activator on rat Novikoff hepatoma cells. 131 32

Human tissue-type plasminogen activator (t-PA) is cleared rapidly from the circulation by hepatic receptors, one of which recognizes a site in the epidermal growth factor-like domain of the molecule. To define this site more precisely, we have used oligonucleotide-mediated mutagenesis to introduce amino acid substitutions at specific positions located in turns that connect antiparallel beta-sheets in the epidermal growth factor-like domain. Mutated t-PA proteins with amino acid substitutions of the tyrosine residue at position 67 showed markedly lower rates of endocytosis and degradation by cultured cells of the rat hepatoma (H4) line that express a specific receptor for t-PA, and their half-life in the circulation of rats was extended significantly because of a reduction in the rate of the rapid alpha-phase of clearance. The enzymatic properties and fibrinolytic activity of these mutants in vitro were not significantly different from those of wild-type t-PA. We conclude that tyrosine 67 comprises a key determinant in the clearance of t-PA by a specific hepatic receptor.
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PMID:Tyrosine 67 in the epidermal growth factor-like domain of tissue-type plasminogen activator is important for clearance by a specific hepatic receptor. 131 65

Plasma tissue-type plasminogen activator (t-PA) is cleared rapidly in vivo by the liver. Previous studies with the human hepatoma cell line HepG2 have identified a clearance system for t-PA modulated by plasminogen activator inhibitor type 1 (PAI-1). In the present study, a rat hepatoma cell line MH1C1 is shown to contain a PAI-1-independent t-PA clearance system. At 4 degrees C, binding of 125I-t-PA to MH1C1 cells was rapid, specific, and saturable. Scatchard analysis of the binding data yielded a mean estimate of 105,000 high affinity binding sites per cell (Kd = 4.1 nM). When the bound ligand was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the majority (about 90%) of the specific binding was in the form of uncomplexed 125I-t-PA. This is in contrast to HepG2 cells in which specific binding was mainly in the form of a sodium dodecyl sulfate-stable 125I-t-PA.PAI-1 complex. When availability of matrix-associated PAI-1 was blocked by preincubation with anti-PAI-1 antibody or removed by elastase treatment, specific 125I-t-PA binding to MH1C1 cells was unaffected, whereas most of the specific 125I-t-PA binding to HepG2 cells was abolished. Furthermore, when the active site of t-PA was inactivated with diisopropyl fluorophosphate, the diisopropyl fluorophosphate-t-PA specifically competed for binding of 125I-t-PA to MH1C1 cells, but failed to block specific 125I-t-PA binding to HepG2 cells. At 37 degrees C, PAI-1-independent t-PA binding to MH1C1 cells was followed by ligand uptake and degradation with kinetics similar to that seen in HepG2 cells. Chemical cross-linking of t-PA to MH1C1 cells revealed a specific t-PA binding protein with a molecular mass of about 500,000 daltons. Ligand-receptor complexes generated by chemical cross-linking were immunoprecipitable by anti-t-PA antibody but not by anti-PAI-1 antibody, further supporting the finding that binding of t-PA to MH1C1 cells is PAI-1-independent.
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PMID:Identification and partial characterization by chemical cross-linking of a binding protein for tissue-type plasminogen activator (t-PA) on rat hepatoma cells. A plasminogen activator inhibitor type 1-independent t-PA receptor. 132 1

The human hepatoma HuH-7 cell line was shown to constitutively express both a plasminogen activator (PA) and a plasminogen activator inhibitor (PAI). Four sublines of the HuH-7 cell line were analyzed and found to express differing amounts of both PA and PAI. The plasminogen activator produced by these cells was identified as urokinase based upon molecular weight, inhibition of activity with anti-UK but not anti-t-PA antibodies, adherence to an anti-UK affinity column and by Northern blotting demonstrating positive hybridization with the cDNA for UK, but not with the t-PA cDNA. The inhibitor produced by HuH-7 cells was identified as PAI-1 by molecular weight, immunoblotting techniques, adherence to an anti-PAI-1 affinity column, and by Northern blotting demonstrating positive hybridization with the cDNA for PAI-1, but not with the PAI-2 cDNA. The expression of both UK and PAI-1 by HuH-7 cells could be modulated by cytokines known to influence the acute phase response. The addition of interleukin-1 (IL-1) induced the expression of both UK and PAI-1. The increase of PAI-1 was due to an increase in amount of the PAI-1 mRNA. The presence of both interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF) also increased UK and PAI-1 levels, although not as dramatically as IL-1. The addition of IL-1 together with IL-6 produced a slight synergistic response with respect to PAI-1 expression. This suggests that PAI-1 is able to respond to mediators which aid in the induction of the acute phase response. These studies demonstrate that cells of liver origin are able to produce components of the fibrinolytic system. The synthesis of these components can be altered by inflammatory mediators and thus may be involved in hepatic regulation of fibrinolysis in both normal and diseased states.
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PMID:Human HuH-7 hepatoma cells express urokinase and plasminogen activator inhibitor-1: identification, characterization and regulation by inflammatory mediators. 137 1

We have developed a general method for screening randomly mutagenized expression libraries in mammalian cells by using fluorescence-activated cell sorting (FACS). The cDNA sequence of a secreted protein is randomly mutagenized by PCR under conditions of reduced Taq polymerase fidelity. The mutated DNA is inserted into an expression vector encoding the membrane glycophospholipid anchor sequence of decay-accelerating factor (DAF) fused to the C terminus of the secreted protein. This results in expression of the protein on the cell surface in transiently transfected mammalian cells, which can then be screened by FACS. This method was used to isolate mutants in the kringle 1 (K1) domain of tissue plasminogen activator (t-PA) that would no longer be recognized by a specific monoclonal antibody (mAb387) that inhibits binding of t-PA to its clearance receptor. DNA sequence analysis of the mutants and localization of the mutated residues on a three-dimensional model of the K1 domain identified three key discontinuous amino acid residues that are essential for mAb387 binding. Mutants with changes in any of these three residues were found to have reduced binding to the t-PA receptor on human hepatoma HepG2 cells but to retain full clot lysis activity.
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PMID:Random PCR mutagenesis screening of secreted proteins by direct expression in mammalian cells. 137 21

The binding of tissue-type plasminogen activator (t-PA) to membranes prepared from human liver was investigated, and a specific, saturable, high-affinity binding site (Kd = 3.4 nM) was identified. The binding of t-PA to liver membranes was not affected by an excess of D-mannose or D-galactose, or by active urokinase (u-PA), whereas binding of t-PA to membranes prepared from human HepG2 hepatoma cells was inhibited by u-PA. HepG2-membrane-bound t-PA was fully complexed to PA inhibitor 1 (PAI-1), whereas liver-membrane-bound t-PA was not complexed. Gel filtration on Sephacryl S300 of membrane proteins solubilized in deoxycholate revealed that high-affinity t-PA binding activity elutes at an apparent molecular mass of 40 kDa. Monoclonal antibodies specific for the growth factor and the kringle 2 domains inhibited the binding of t-PA to liver membranes and the catabolism of t-PA by rat hepatoma cells. Human liver membranes also bound u-PA; binding was inhibited by pro-u-PA, the N-terminal fragment of u-PA, but not by the 33 kDa form of u-PA or by t-PA. Our results show that human liver membranes contain a specific 40 kDa binding protein for t-PA that is different from the PAI-1-dependent receptor described on HepG2 cells and the mannose receptor isolated from human liver.
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PMID:Characterization of the binding of plasminogen activators to plasma membranes from human liver. 144 49


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