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)

Six hybridoma cell lines (AP1-AP6) secreting monoclonal antibodies (McAb) against PAI-1 were obtained by fusing the murine myeloma cell line SP2/0 with the spleen cells from Balb/c mouse immunized with recombinant PAI-1 expressed in E. coli. These antibodies were purified by SPA affinity chromatography. All McAbs recognized rPAI-1 and PAI-1 from the human hepatoma cell line HepG2. The titers of ascites were more than 10(6). The antibody-antigen affinity constants (Kaff) for anti-PAI-1 McAb measured by EIA were between 3.45 x 10(7)-1.05 x 10(10) M. AP2 and AP3 McAbs were effective in quenching the activity of PAI-1. Partial quenching of PAI-1 activity was achieved with AP4, AP5 and AP6 McAbs respectively. AP1 McAb had no effect upon PAI-1 activity. Three of the six McAbs (AP1, AP4 and AP5) bound to the PAI-1/t-PA complex, while the others did not. The PAI-1 was purified 51 folds to homogeneity from serum free medium of HepG2 with the recovery rate of 92% by one-step procedure using Sepharose 4B conjugated with anti-PAI-1 McAb (AP1, AP3 and AP4). A sandwich ELISA for the measurement of PAI-1 antigen in human plasma was developed, based on anti-PAI-1 McAb against non-overlapping epitopes. The mean value of plasma PAI-1 for the healthy donors was 24.7 +/- 7.75 ng/ml measured by ELISA.
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PMID:Preparation, characterization and application of monoclonal antibodies against PAI-1. 780 88

We have reported previously that tissue-type plasminogen activator (tPA) gene expression is regulated by glucocorticoids and cyclic nucleotides in HTC rat hepatoma cells. Incubation of HTC cells with the synthetic glucocorticoid dexamethasone (Dex) transiently increases tPA messenger RNA accumulation 2-fold, whereas incubation with 8-bromo-cAMP (cAMP) alone results in a sustained 2-fold increase. Nuclear run-on studies indicate that these effects occur at the level of gene transcription. In combination, however, Dex and cAMP act synergistically to induce tPA messenger RNA levels 10- to 15-fold; this synergistic induction is at least in part transcriptional. We now report that this synergistic induction of tPA gene transcription requires concomitant protein synthesis. Furthermore, the action of Dex must precede that of cAMP, and the action of Dex requires ongoing protein synthesis, whereas the action of cAMP has no such requirement. To further investigate the mechanism of the synergistic induction of tPA gene transcription, we cloned the tPA promoter from an HTC genomic library. We established the start site of transcription in HTC cells by primer extension and determined the nucleotide sequence of 2.3 kilobase-pairs (kb) of the 5'-flanking region, including 1.7 kb of sequence not previously reported. A 2.3-kb segment of the rat tPA promoter has been ligated to a chloramphenicol acetyltransferase reporter gene and its hormonal regulation evaluated in transient and stable transfection studies in HTC cells. Although this promoter length is sufficient to mediate the 2-fold induction in gene expression seen with cAMP alone, it is not sufficient to recapitulate the synergistic induction of endogenous tPA gene transcription seen with Dex plus cAMP in combination. We have ruled out relief of transcriptional arrest as the mechanism of the synergistic induction. Therefore, we suggest that sequences lying outside the most proximal 2.3 kb of tPA promoter mediate the synergistic interaction of Dex and cAMP.
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PMID:Synergistic induction of tissue-type plasminogen activator gene expression by glucocorticoids and cyclic nucleotides in rat HTC hepatoma cells. 807 Mar 63

The glycoprotein tissue-type plasminogen activator (t-PA) is subject to hepatic clearance in humans. Here, the interaction of t-PA with a well-differentiated hepatoma cell line (HepG2) was examined. Suspended HepG2 cells bound 125I-t-PA in a specific, saturable, and reversible fashion through a Ca(2+)-dependent, active site-independent mechanism. Binding isotherms indicated a high affinity system with a single class of saturable binding sites (Kd 39 nM; maximum binding capacity 493,000 sites per cell). Bound t-PA was rapidly degraded at 37 degrees C in a manner inhibited by lysosomotropic agents or metabolic inhibitors. Pretreatment of t-PA with monoclonal antibodies against the EGF/fibronectin finger domain, but not kringle 2 or kringle 1, reduced total binding by 86%. Binding of 125I-t-PA to HepG2 cells was inhibited by monosaccharides fucose and galactose and by the neoglycoprotein fucosyl-albumin. Enzymatic removal of alpha-fucose residues, but not alpha-galactose, high mannose, or complex oligosaccharide from 125I-t-PA, reduced specific binding by 60 +/- 5%. Binding was also inhibited by high, but not low, molecular weight urokinase, which contains an EGF-based threonine-linked alpha-fucose homologous to that of t-PA. These data suggest that EGF-associated O-linked alpha-fucose may mediate t-PA binding and degradation by HepG2 cells. This mechanism may be relevant to other proteins with analogous structures.
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PMID:alpha-Fucose-mediated binding and degradation of tissue-type plasminogen activator by HepG2 cells. 811 82

The role of plasminogen activator inhibitor type 1 (PAI-1) in the clearance of tissue-type plasminogen activator (t-PA) by hepatocyte-like cells was studied. Rat (Novikoff) hepatoma cells were able to bind and degrade t-PA in a PAI-1 independent fashion, but PAI-1 markedly increased the rate of degradation and t-PA/PAI-1 was a more efficient inhibitor of 125I-t-PA or of 125I-t-PA/PAI-1 degradation than free t-PA. Competition studies revealed that the effect of PAI-1 is unlikely to involve determinants located on the PAI-1 part of the complex: 1) an excess of free PAI had no effect on the rate of degradation of 125I-t-PA/PAI-1.2) Complexes of PAI-1 with urokinase-type PA or with a t-PA mutant lacking the finger and growth factor domains were unable to compete for the binding and degradation of free or PAI-1-complexed 125I-t-PA.3) t-PA KHRR296-299AAAA, a mutant which reacts 2 orders of magnitude slower with PAI-1 than wild type t-PA, behaved similar to wild type t-PA. The clearance via both the PAI-1-dependent and the PAI-1-independent mechanisms was inhibited by the receptor-associated protein, a general inhibitor of clearance mediated by the LDL receptor-related protein. We conclude that t-PA can be cleared by rat hepatoma cells in a PAI-1 independent fashion, but after complex formation with PAI-1, binding of t-PA to the cells is increased and clearance accelerated. Both mechanisms seem to involve the same receptor.
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PMID:The role of plasminogen activator inhibitor type 1 in the clearance of tissue-type plasminogen activator by rat hepatoma cells. 811 17

The effect of nafamostat mesilate on coagulation and fibrinolysis was investigated in a study of 22 patients with hepatocellular carcinoma who underwent a hepatic resection. The patients were divided into two groups: group 1, control (n = 11) and group 2, those with the intraoperative and postoperative use of nafamostat mesilate (0.2 to 0.4 milligram per kilogram per hour, n = 11). Nafamostat mesilate tended to suppress the coagulation expressed by thrombin-antithrombin III complex and fibrinopeptide A both during and immediately after operation. Moreover, nafamostat mesilate significantly suppressed the fibrinolysis expressed by euglobulin lysis activity both during and after operation. With regard to the initial stage of the fibrinolytic system, such as tissue-type plasminogen activator and plasminogen activator inhibitor-1, there was no difference between the groups. Therefore, the suppression of the euglobulin lysis activity may be caused by the inhibition of plasmin activity. There was no difference between the groups regarding operative blood loss. However, the rate of blood transfusion in group 2 was lower than that in group 1, and no fresh frozen plasma was required for the patients who lost over 2,000 milliliters of blood. Nafamostat mesilate can suppress euglobulin lysis activity both intraoperatively and postoperatively, and thus decrease the amount of blood transfusion needed. Therefore, at present, nafamostat mesilate seems to be one of the most useful agents for stabilizing the coagulant and fibrinolytic systems in hepatic resection.
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PMID:Effect of nafamostat mesilate on coagulation and fibrinolysis in hepatic resection. 816 88

We have recently described a PAI-1-independent pathway of tissue-type plasminogen activator (t-PA) uptake and degradation on the rat MH1C1 hepatoma cell line. Further studies have implicated the low-density-lipoprotein-receptor-related protein (LRP) as the mediator of plasminogen-activator inhibitor type-1-independent t-PA endocytosis. The LRP is a multi-functional receptor which is shared by a variety of ligands, including alpha 2-macroglobulin, apoprotein E-enriched beta-very-low-density lipoprotein, t-PA and Pseudomonas exotoxin A. In each case, binding of ligand to this receptor can be inhibited by addition of the 39 kDa LRP-receptor-associated protein. This protein, which co-purifies with the LRP receptor, is the focus of our present study. 125I-labelled 39 kDa protein binds specifically and with high affinity to a single kinetic binding species on the rat MH1C1 cell surface. Scatchard analysis reveals an equilibrium dissociation constant (Kd) of 3.3 +/- 0.9 (S.D.) nM, with 380,000 +/- 190,000 (S.D.) binding sites per cell. Cross-linking studies indicate that the specific interaction between MH1C1 cells and the 39 kDa protein is mediated by an association with the LRP receptor. The 39 kDa protein strongly inhibits binding of 125I-t-PA, with an apparent Ki value of 0.5 nM. In addition, both unlabelled t-PA and 125I-labelled 39 kDa protein can be co-bound and cross-linked to the same cell-associated LRP receptor. Endocytosis of cell-surface-associated 39 kDa protein was shown to be rapid, with internalized ligand subsequently degraded and released to the extracellular milieu. The rate of uptake and degradation of 125I-labelled 39 kDa protein at 37 degrees C was determined to be 52 fmol/min per 10(6) cells, and supports a model for active recycling of the LRP receptor.
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PMID:Interaction of a 39 kDa protein with the low-density-lipoprotein-receptor-related protein (LRP) on rat hepatoma cells. 828 86

Tissue-type plasminogen activator (t-PA) is a plasma serine protease that catalyzes the initial and rate-limiting step in the fibrinolytic cascade. t-PA is widely used as a thrombolytic agent in the treatment of acute myocardial infarction. However, its use has been impaired by its rapid hepatic clearance from the circulation following intravenous administration. Studies with both rat hepatoma MH1C1 cells (G. Bu, S. Williams, D. K. Strickland, and A. L. Schwartz, 1992. Proc. Natl. Acad. Sci. USA. 89:7427-7431) and human hepatoma HepG2 cells (G. Bu, E. A. Maksymovitch, and A. L. Schwartz. 1993. J. Biol. Chem. 28:13002-13009) have shown that binding of t-PA to its clearance receptor, the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor, is inhibited by a 39-kD protein that copurifies with this receptor. Herein we investigated whether administration of purified recombinant 39-kD protein would alter t-PA clearance in vivo. We found that intravenous administration of purified 39-kD protein to rats prolonged the plasma half-life of 125I-t-PA from 1 min to approximately 5-6 min. The plasma half-life of t-PA enzymatic activity was similarly prolonged following intravenous administration of purified 39-kD protein. In addition we found that the 39-kD protein itself was rapidly cleared from the circulation in vivo. Clearance of 125I-39-kD protein was a biphasic process with half-lives of 30 s and 9 min and the liver was the primary organ of clearance. Preadministration of excess unlabeled 39-kD protein slowed 125I-39-kD protein clearance in rats in a dose-dependent manner, suggesting that specific clearance receptors were responsible for this process. Administration of increasing doses of unlabeled 39-kD protein along with labeled 39-kD protein resulted in a decrease in the amount of labeled 39-kD protein associating with the liver and a concomitant increase in the amount of labeled 39-kD protein associating with the kidneys, indicating two clearance mechanisms exist for the 39-kD protein.
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PMID:39-kD protein inhibits tissue-type plasminogen activator clearance in vivo. 834 26

Hepatic parenchymal cells play an essential role in the clearance of circulating tissue-type plasminogen activator (t-PA) in vivo as a major pathway in the regulation of plasma fibrinolytic activity. Previous studies have identified plasminogen activator inhibitor type 1 (PAI-1)-dependent t-PA-binding sites in the human hepatoma cell line HepG2. In this study, we demonstrate that receptor-mediated binding and endocytosis of the t-PA-PAI-1 complex are largely mediated by a recently identified low density lipoprotein receptor-related protein (LRP). A 39-kDa LRP receptor-associated protein that modulates ligand binding to LRP was found to bind specifically to HepG2 cells and to inhibit approximately 70-80% of specific 125I-t-PA.PAI-1 binding. This inhibition by the 39-kDa protein was not due to inhibition of complex formation between 125I-t-PA and PAI-1; instead, the 39-kDa protein inhibited 125I-t-PA.PAI-1 binding to LRP. Polyclonal anti-LRP antibody raised against purified human LRP also inhibited 70-80% of specific 125I-t-PA.PAI-1 binding. A similar extent of inhibition by the 39-kDa protein was also observed for 125I-t-PA.PAI-1 endocytosis and degradation. Chemical cross-linking experiments demonstrated the direct interaction between 125I-t-PA.PAI-1 and LRP on HepG2 cells as anti-LRP antibody, in addition to anti-t-PA and anti-PAI-1 antibodies, was able to immunoprecipitate the 125I-t-PA.PAI-1 complex following binding of 125I-t-PA.PAI-1 to HepG2 cells and cross-linking. This interaction of the t-PA.PAI-1 complex with LRP on HepG2 cells was also observed when the unlabeled t-PA.PAI-1 complex was cross-linked to [35S]methionine-labeled HepG2 cells. In addition, the direct binding of the 39-kDa protein to LRP on HepG2 cells was demonstrated by similar cross-linking experiments. Thus, these data clearly show that LRP is the major cell-surface receptor responsible for t-PA.PAI-1 complex binding and endocytosis on human hepatoma HepG2 cells and extend the multifunctional nature of LRP as an endocytosis receptor for several structurally and functionally distinct ligands.
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PMID:Receptor-mediated endocytosis of tissue-type plasminogen activator by low density lipoprotein receptor-related protein on human hepatoma HepG2 cells. 838 67

The low density lipoprotein receptor-related protein (LRP) has been proposed to function as an endocytosis receptor for chylomicron remnants and protease-inhibitor complexes so that these particles can be cleared from the plasma or extracellular fluid. The kidney glycoprotein 330 (gp330) may have an analogous role to LRP in the kidney. A 39-kDa protein which copurifies with LRP and gp330 inhibits the binding and/or cellular uptake of ligands to these receptors and may regulate LRP and gp330 activity in vivo. Recently, LRP has been immunochemically localized to endothelial and vascular smooth muscle cells. In the present study, the biology of the 39-kDa protein was studied in cultured endothelial cells and vascular smooth muscle cells. The 39-kDa protein is synthesized by both cell types and has an average half-life of 15 hours. Immunofluorescence shows the major part of the 39-kDa protein has an intracellular localization with enrichment in the perinuclear region. Tissue-type plasminogen activator (t-PA), a plasma serine protease that binds specifically and with high affinity to LRP on hepatoma cells, also binds to endothelial cells and vascular smooth muscle cells. 125I-t-PA binding to both cell types is inhibited by the 39-kDa protein. However, only the endothelial cells are capable of rapidly internalizing and degrading 125I-t-PA. These data thus suggest that LRP may function as a clearance receptor for t-PA on endothelial cells.
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PMID:The 39-kDa protein regulates LRP activity in cultured endothelial and smooth muscle cells. 890 16

Retinoic acid stimulates the expression of tissue-type plasminogen activator (t-PA) in vascular endothelial cells in vitro and enhances t-PA levels in plasma and tissues in vivo. Compared with the in vivo situation, high retinoic acid concentrations are required to induce optimally t-PA expression in vitro. These findings led us to study retinoic acid metabolism in cultured human endothelial cells. For comparison, these studies were also performed in the human hepatoma cell line, HepG2, and key experiments were repeated with human primary hepatocytes. Both hepatocyte cultures gave very similar results. Human endothelial cells were shown to possess an active retinoic acid metabolizing capacity, which is quantitatively comparable to that of hepatocytes, but different from that of hepatocytes in several qualitative aspects. Our results demonstrate that all-trans-retinoic acid is quickly metabolized by both endothelial cells and hepatocytes. All-trans-retinoic acid induces its own metabolism in endothelial cells but not in hepatocytes. 9-cis-Retinoic acid is degraded slowly by endothelial cells, whereas hepatocytes metabolize 9-cis-retinoic acid very quickly. Furthermore, our data show that hepatocytes, but not endothelial cells, detectably isomerise all-trans-retinoic acid to 9-cis-retinoic acid and vice versa. In both endothelial cells and hepatocytes all-trans-retinoic acid metabolism was inhibitable by the cytochrome P-450 inhibitors liarozole (10 microM) and ketoconazole (10 microM), albeit to different extents and with different specificities. In the presence of the most potent retinoic acid metabolism inhibitor in endothelial cells, liarozole, at least 10-fold lower all-trans-retinoic acid concentrations were required than in the absence of the inhibitor to obtain the same induction of t-PA. In conclusion, our results clearly demonstrate that all-trans-retinoic acid and 9-cis retinoic acid are actively but differently metabolized and isomerised by human endothelial cells and hepatocytes. The rapid metabolism of retinoic acid explains the relatively high concentrations of retinoic acid required to induce t-PA in cultured endothelial cells.
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PMID:Differences in metabolism and isomerization of all-trans-retinoic acid and 9-cis-retinoic acid between human endothelial cells and hepatocytes. 926 2


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