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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Properties of insulin receptor on plasma membranes isolated from AH-66 ascites hepatoma cells and liver of tumor-bearing rats were studied. Specific binding (total binding minus nonspecific binding) of 125-I-labeled insulin to plasma membranes from AH-66 tumor cells and liver of normal and tumor-bearing rats were 33, 31, and 16% of a fixed amount of labeled insulin (1 x 10(5) cpm) added to each membrane preparation, respectively. Using Nisonoff plot, these membranes were found to possess at least two types of insulin receptors with a high affinity-low capacity and a low affinity-high capacity, as has been shown in normal liver. Total number of binding sites (high affinity plus low affinity sites, 8.4 x 10(-12) mol/mg protein) in hepatoma cells was more than that (7.0 x 10(-12) mol/mg protein) in normal rat liver. However, kinetic constants of binding in receptors of two types on tumor cells and tumor-bearing rat liver were similar to those of membrane receptors from normal rat liver. Insulin receptors of the hepatoma cells were considered to be highly specific for insulin from the results of competition with other peptide hormones. Inhibition of insulin binding with the tumor cell membranes by concanavalin-A, a competitive inhibitor for insulin receptor sites, did not differ very greatly from that of normal liver.
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PMID:Insulin receptors in AH-66 ascites hepatoma cells and liver of tumor-bearing rats. 22 Jan 27

A soluble construct consisting of a plasmid carrying the gene of the SV40 large T-antigen and an insulin-poly-L-lysine conjugate is able to selectively transfect PLC/PRF/5 human hepatoma cells which possess insulin receptors. Transfection can be efficiently competed by excess free insulin. To examine intracellular transport of the construct, it was fluorescently labeled and its accumulation on and in cells visualized by video-enhanced microscopy and quantitative confocal laser scanning microscopy. After 2 h at 37 degrees C, the labeled construct was found predominantly in intracellular acidic compartments, with a substantial portion of fluorescence localized both near and in the cell nucleus. Binding, endocytosis, and nuclear localization of the labeled conjugate could all be competed by excess free insulin, thus indicating that entry of the conjugate into cells was specifically mediated by the insulin receptor.
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PMID:Receptor-mediated endocytosis and nuclear transport of a transfecting DNA construct. 131 9

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

Studies were carried out to examine the role of the major insulin receptor tyrosine autophosphorylation sites in stimulation of S6 kinase activity. For these studies, we employed HTC rat hepatoma cells transfected with and expressing human insulin receptors. In cells transfected with and expressing a large number of normal human insulin receptors (HTC-IR cells), the sensitivity of cells to insulin to stimulate S6 kinase was increased tenfold when compared to untransfected wild type HTC cells (HTC-WT cells). However, in cells transfected with and expressing a large number of mutated human insulin receptors where the tyrosines at three major autophosphorylation sites (1158, 1162, and 1163) were mutated to phenylalanines (HTC-F3 cells), there was no change in insulin sensitivity when compared to HTC-WT cells. We next studied the effect of a human-specific monoclonal antibody to the human insulin receptor, MA-5, on S6 kinase activation. In HTC-WT cells, MA-5 did not interact with endogenous rat insulin receptors and thus did not stimulate S6 kinase. In HTC-IR cells expressing normal human insulin receptors, MA-5 stimulated S6 kinase. Interestingly, MA-5, unlike insulin, was also able to stimulate S6 kinase in HTC-F3 cells expressing mutated receptors. In order to further understand the signaling mechanisms by MA-5 and insulin, two potential intermediate protein kinases were investigated. Neither insulin nor MA-5 appears to activate either microtubule-associated protein 2 (MAP-2) kinase or protein kinase C in these cells. These studies suggest therefore that: 1) insulin and MA-5 may signal S6 kinase activation by independent mechanisms that do not employ either MAP-2 kinase or protein kinase C; and 2) under certain circumstances, S6 kinase appears to be activated by mechanisms that are independent of insulin receptor tyrosine autophosphorylation.
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PMID:Monoclonal antibody to the human insulin receptor, but not insulin, stimulates S6 kinase via human insulin receptors mutated at three major tyrosine autophosphorylation sites. 132 57

An efficient expression system in Escherichia coli for several biologically active insulin-like growth factor-I (IGF-I) fusion peptide analogues is described. These novel IGF-I fusion protein analogues have properties that make them very useful reagents in the investigation of IGF-I action. The analogues comprise an IGF-I sequence and the first 11 amino acids of methionyl porcine growth hormone (pGH) and include [Met1]-pGH(1-11)-Val-Asn-IGF-I, which contains the authentic IGF-I sequence, and two analogues, [Met1]-pGH(1-11)-Val-Asn-[Gly3]-IGF-I and [Met1]-pGH(1-11)-Val-Asn-[Arg3]-IGF-I, where Glu-3 in the human IGF-I sequence has been replaced by Gly or Arg respectively. The three peptides are referred to as Long IGF-I, Long [Gly3]-IGF-I or Long [Arg3]-IGF-I depending on the IGF-I sequence present. Production of the purified fusion peptides was aided by folding the reduced and denatured fusion peptide sequence under conditions that gave very high yields of biologically active product. Introduction of a hydrophobic N-terminal extension peptide appears to facilitate the correct folding of the IGF-I analogues compared with that obtained previously when folding normal-length IGFs. The biological activities of the IGF-I fusion peptides were compared with authentic IGF-I and the truncated analogue, des(1-3)IGF-I. In L6 rat myoblasts, all the analogues were more potent than authentic IGF-I in their abilities to stimulate protein and DNA synthesis and inhibit protein breakdown. In H35 hepatoma cells, where the IGFs act through the insulin receptor, the Long IGF-I analogues maintained a similar potency relative to IGF-I as was observed in the L6 myoblasts. The order of biological potency in cell lines secreting IGF-binding proteins (IGFBPs) into the medium was Long [Arg3]-IGF-I-des(1-3)IGF-I greater than Long [Gly3]-IGF-I greater than Long IGF-I greater than IGF-I. In chicken embryo fibroblasts, a cell line that does not secrete detectable IGFBPs into the medium, Long [Arg3]-IGF-I, was less potent than IGF-I. Investigation of receptor and IGFBP association by these analogues reinforced our previous findings that N-terminal analogues of IGF-I show increased biological potency due to changes in the degree of their IGFBP interactions.
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PMID:Novel recombinant fusion protein analogues of insulin-like growth factor (IGF)-I indicate the relative importance of IGF-binding protein and receptor binding for enhanced biological potency. 137 42

Insulin rapidly stimulates tyrosine phosphorylation of cellular proteins which migrate between 165 and 190 kDa during SDS-PAGE. These proteins, collectively called pp185, were originally found in anti-phosphotyrosine antibody (alpha PY) immunoprecipitates from insulin-stimulated Fao rat hepatoma cells. Recently, we purified and cloned IRS-1, one of the phosphoproteins that binds to alpha PY and migrates near 180 kDa following insulin stimulation of rat liver [Sun, X. J., et al. (1991) Nature 352, 73-77]. IRS-1 and pp185 undergo tyrosine phosphorylation immediately after insulin stimulation and show an insulin dose response similar to that of insulin receptor autophosphorylation. However, IRS-1 was consistently 10 kDa smaller than the apparent molecular mass of pp185. The pp185 contained some immunoblottable IRS-1; however, cell lysates depleted of IRS-1 with anti-IRS-1 antibody still contained the high molecular weight forms of pp185 (HMW-pp185). Furthermore, the tryptic phosphopeptide map of IRS-1 was distinct from that of HMW-pp185, suggesting that at least two substrates migrate in this region during SDS-PAGE. Moreover, the phosphatidylinositol 3'-kinase and its 85-kDa associated protein (p85) bound to IRS-1 in Fao cells, but weakly or not at all to HMW-pp185. Our results show that Fao cells contain at least two insulin receptor substrates, IRS-1 and HMW-pp185, which may play unique roles in insulin signal transmission.
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PMID:Insulin stimulates tyrosine phosphorylation of multiple high molecular weight substrates in Fao hepatoma cells. 138 84

We have studied the effect of insulin stimulation on phosphotyrosine phosphatase (PTPase) activity in the well-differentiated rat hepatoma cell line Fao. PTPase activity was measured using a 32P-labeled peptide corresponding to the major site of insulin receptor autophosphorylation. Of the PTPase activity in Fao cells, 14% was in the cytosolic fraction, whereas 86% was in the particulate fraction; this latter fraction also had a 4-fold higher specific activity. Purification of the particulate fraction by lectin chromatography resulted in a 50% increase in specific activity, although this glycoprotein-rich fraction contained only 1.5% of the total activity. Both the cytosolic and particulate PTPase fractions were active toward the tyrosyl-phosphorylated insulin receptor in vitro. The activity of the particulate fraction but not the cytosolic fraction was inhibited by addition of a micromolar concentration of a phosphorylated peptide corresponding to residues 1142-1153 of the human insulin receptor sequence. By contrast, addition of the nonphosphorylated peptide even at millimolar concentration was without effect. Both PTPase fractions were inhibited by Zn+ at similar concentrations, whereas the cytosolic PTPase activity was 10-fold more sensitive to vanadate inhibition. Treatment of cells with 100 nM insulin increased PTPase activity in the particulate fraction by 40% and decreased activity in the cytosolic fraction by 35%. These effects occurred within 15 min and were half-maximal at 3-4 nM insulin. When assessed as total activity, the magnitude of the changes in PTPase activity in the particulate and cytosolic fractions could not be explained on the basis of a translocation of PTPases between the two pools.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Insulin differentially regulates protein phosphotyrosine phosphatase activity in rat hepatoma cells. 142 Jan 53

We have examined the effect of cholera toxin (CT) on the insulin receptor tyrosine kinase. Incubation of intact rat hepatoma cells FaO with CT (1 microgram/ml/2h) inhibited insulin-induced receptor autophosphorylation by 30% in vivo. This effect persisted after receptor purification in vitro. CT did not alter hormone binding of the insulin receptor, indicating that the toxin affects signal transduction of insulin at the level of the receptor kinase. Experiments using chinese hamster ovary (CHO) cells transfected either with the human insulin receptor (HIR) or a mutant lacking the last 43 amino acids of the receptor beta-subunit (HIR delta CT) showed, that the carboxy-terminal tail of the insulin receptor does not play a role in the suppressive effect of the toxin on the insulin receptor kinase.
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PMID:Cholera toxin diminishes tyrosine kinase activity of the insulin receptor. 144 89

Biochemical and morphological studies compared the ATP requirements for and the internalization routes of alpha 2-macroglobulin and insulin in H35 hepatoma cells. Cellular ATP concentrations were decreased more than 94% by 1 mM 2,4-dinitrophenol or 10 mM sodium azide, potassium cyanide, or oligomycin. ATP depletion decreased total cell-associated alpha 2-macroglobulin 70-90% by inhibiting binding 67-77% and receptor-mediated internalization 90-96%. Under the same conditions, insulin binding was decreased less than 10%, and endocytosis and intracellular accumulation were not affected. Quantitative electron microscopic analysis of the distribution of occupied receptors on the surface of control and treated cells was performed using colloidal gold-labeled alpha 2-macroglobulin or insulin. alpha 2-Macroglobulin concentrated in and was internalized almost exclusively by coated pits. Insulin was rarely associated with coated pits, but was found in and internalized by noncoated invaginations. ATP depletion did not affect receptor mobility or ligand-induced aggregation of either receptor. There was an increase in the amount of alpha 2-macroglobulin found in coated pit-like structures. The coat underlying pits in ATP-depleted cells was poorly defined and may account for the inability of coated pits to form and/or internalize. These results showed that receptor-mediated internalization via coated pits was ATP dependent, whereas internalization via pinocytotic invaginations was energy independent, which explained the difference in the ATP dependency of uptake for the two ligands. These observations suggested that autophosphorylation of the insulin receptor may not be involved in either the aggregation or internalization of the insulin-receptor complex, since ATP depletion did not affect either process. This study provided evidence that specialized mechanisms exist for the internalization of insulin which may be related to some of its intracellular effects.
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PMID:Differences in adenosine triphosphate dependency of receptor-mediated endocytosis of alpha 2-macroglobulin and insulin correlate with separate routes of ligand-receptor complex internalization. 168 53

A human hepatoma cell line, HepG2, secretes a discrete insulin-like growth factor-binding protein (IGFBP-1) into serum-free medium, which is identical to the 25K mol wt BP in amniotic fluid and plasma. IGFBP-1 levels in vivo have been shown to be inversely correlated with circulating insulin concentrations. This study investigated the direct effects of insulin on IGFBP-1 production in vitro. Addition of insulin to HepG2 cultures induced a rapid dose-dependent decrease in IGFBP-1 synthesis and secretion independent of the glucose concentration in the medium. As assessed by ligand binding and specific RIA, levels of IGFBP-1 were 20-50% of control levels in 18-h conditioned medium from insulin-treated cells. Monoclonal antibody studies indicated that the suppressive effect of insulin on IGFBP-1 synthesis was mediated through specific interaction with the insulin receptor. Therefore, HepG2 cells respond to insulin by altering the synthesis and secretion of IGFBP-1 in a manner that mimics many of the changes in plasma IGFBP-1 levels observed in vivo and provide an in vitro model for studies of IGFBP-1 biosynthesis.
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PMID:Insulin regulation of insulin-like growth factor-binding protein production in cultured HepG2 cells. 169 Jul 45


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