UMLS:C0019204 - FACTA Search

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

This paper describes the selective isolation of dedifferentiated variants from a well-differentiated rat hepatoma cell line (Fao). The well-differentiated cells express the gluconeogenic enzymes, FDPase and PEPCK, and so can grow in a glucose-free medium. By using glucose-free medium in conjunction with the BudR-visible light suicide technique it was possible to isolate two different classes of dedifferentiated variants from a mutagenized population of Fao cells. The variants of the first class resemble those previously described in that they display (1) an altered cellular morphology, (2) a pleiotrophic loss of all (or most) of the hepatic functions routinely analyzed in this laboratory, and (3) an extremely low reversion frequency (less than or equal to 10-8). The variants of the second class are characterized by an unstable phenotype and uncoordinated expression of the hepatic functions. Unstable variants can give rise to stable dedifferentiated variants, suggesting that the unstable variants may actually represent an intermediate in a two-step dedifferentiation process.
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PMID:Selective isolation of stable and unstable dedifferentiated variants from a rat hepatoma cell line. 708 65

Insulin-like growth factor-binding protein-1 (IGFBP-1) modulates the action of IGFs on target cells. IGFBP-1 transcription is highly regulated by hormonal and metabolic factors. In rat H4-II-E hepatoma cells, IGFBP-1 messenger RNA is stimulated by dexamethasone, cAMP, and phorbol esters, and dominantly inhibited by insulin. To identify the cis-elements that determine transcriptional regulation by these agents, we have coupled rat IGFBP-1 promoter fragments to a luciferase reporter gene and transfected H4-II-E cells using the cationic liposome procedure. Promoter fragments whose 5'-end was at nucleotide (nt) -925 or -327 (with respect to the transcription initiation site, 1) conferred positive regulation of promoter activity by dexamethasone, cAMP, and phorbol esters. Insulin inhibited promoter activity in the presence of any of the three stimulatory agents. Stimulation by cAMP or phorbol esters was abolished when the region between nt -327 and -235 was deleted. Although this region contains potential activating protein-2 and activating protein-1 sites, the sites responsible for this regulation have not yet been identified. By contrast, stimulation by dexamethasone was retained in deletion constructs whose 5'-end was at nt -92, but was abolished by site mutagenesis of either the left or right half-sites of a potential glucocorticoid response element (GRE) located between nt -91 and -77. Surprisingly, substitution mutations in an up-stream region, -108 to -99 (M4), also decreased dexamethasone-stimulated promoter activity despite the presence of an intact GRE. We postulate that a positive factor that binds to the wild-type M4 region neutralizes factors that inhibit interaction of the glucocorticoid receptor with the GRE. The M4 region also is involved in inhibition by insulin. Insulin inhibition of dexamethasone-stimulated promoter activity was lost after deletion of nt -135 to -92 or mutation of the region between nt -108 and -99. This insulin response element is conserved in the human IGFBP-1 promoter and is homologous to the insulin response element of the phosphoenolpyruvate carboxykinase gene, which also is rapidly inhibited by insulin in H4-II-E cells. The rat IGFBP-1 promoter provides a valuable model system for studying the multihormonal regulation of transcription.
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PMID:Identification of cis-elements mediating the stimulation of rat insulin-like growth factor-binding protein-1 promoter activity by dexamethasone, cyclic adenosine 3',5'-monophosphate, and phorbol esters, and inhibition by insulin. 752 64

The rate of transcription of the hepatic phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor-binding protein 1 (IGFBP-1) genes is stimulated by glucocorticoids and inhibited by insulin. In both cases, the effect of insulin is dominant, since it suppresses both basal and glucocorticoid-stimulated PEPCK or IGFBP-1 gene transcription. Analyses of both promoters by transfection of PEPCK or IGFBP-1-chloramphenicol acetyltransferase fusion genes into rat hepatoma cells has led to the identification of insulin response sequences (IRSs) in both genes. The core IRS, T(G/A)TTTTG, is the same in both genes, but the PEPCK promoter has a single copy of this element whereas the IGFBP-1 promoter has two copies arranged as an inverted palindrome. The IGFBP-1 IRS and PEPCK IRS both bind the alpha and beta forms of hepatic nuclear factor 3 (HNF-3), although the latter does so with a sixfold-lower relative affinity. Both the PEPCK and the IGFBP-1 IRSs also function as accessory factor binding sites required for the full induction of gene transcription by glucocorticoids. A combination of transient transfection and DNA binding studies suggests that HNF-3 is the accessory factor that supports glucocorticoid-induced gene transcription. In both genes, the HNF-3 binding site overlaps the IRS core motif(s). A model in which insulin is postulated to mediate its negative effect on glucocorticoid-induced PEPCK and IGFBP-1 gene transcription indirectly by inhibiting HNF-3 action is proposed.
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PMID:Hepatic nuclear factor 3- and hormone-regulated expression of the phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein 1 genes. 753 83

Transcription of hepatocyte-specific genes requires the interaction of their regulatory regions with several nuclear factors. Among them is the hepatocyte nuclear factor 3 (HNF3) family, composed of the HNF3 alpha, HNF3 beta, and HNF3 gamma proteins, which are expressed in the liver and have very similar fork head DNA binding domains. The regulatory regions of numerous hepatocyte-specific genes contain HNF3 binding sites. We examined the role of HNF3 proteins in the liver-specific phenotype by turning off the HNF3 activity in well-differentiated mhAT3F hepatoma cells. Cells were stably transfected with a vector allowing the synthesis of an HNF3 beta fragment consisting of the fork head DNA binding domain without the transactivating amino- and carboxy-terminal domains. The truncated protein was located in the nuclei of cultured hepatoma cells and competed with endogenous HNF3 proteins for binding to cognate DNA sites. Overproduction of this truncated protein, lacking any transactivating activity, induced a dramatic decrease in the expression of liver-specific genes, including those for albumin, transthyretin, transferrin, phosphoenolpyruvate carboxykinase, and aldolase B, whereas the expression of the L-type pyruvate kinase gene, containing no HNF3 binding sites, was unaltered. Neither were the concentrations of various liver-specific transcription factors (HNF3, HNF1, HNF4, and C/EBP alpha) affected. In partial revertants, with a lower ratio of truncated to full-length endogenous HNF3 proteins, previously extinguished genes were re-expressed. Thus, the transactivating domains of HNF3 proteins are needed for the proper expression of a set of liver-specific genes but not for expression of the genes encoding transcription factors found in differentiated hepatocytes.
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PMID:Overproduction of a truncated hepatocyte nuclear factor 3 protein inhibits expression of liver-specific genes in hepatoma cells. 756 96

The role of the GTP-binding regulatory protein (G-protein) Gi alpha 2 in vivo was explored using transgenic mice in which the alpha-subunit of Gi alpha 2 was suppressed by antisense RNA. Rat hepatoma FTO-2B cells provide an ideal test system for constructs employing the expression vector pPCK-AS, designed to express antisense RNA at birth under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter. Cells transfected with the expression vector containing a sequence antisense to Gi alpha 2 (pPCK-ASGi alpha 2) displayed expression of RNA antisense to Gi alpha 2 that, like transcription of the PEPCK gene, was inducible by cyclic AMP. Expression of RNA antisense to Gi alpha 2 and suppression of the expression of Gi alpha 2, but not Gsa and Gi alpha 3, was observed in the transfected FTO-2B cells. BDF1 mice carrying the transgene displayed suppression of Gi alpha 2 in liver and fat, two targets for tissue-specific expression of the PEPCK gene. The loss of Gi alpha 2 in white adipocytes of transgenic mice resulted in 3.1-fold elevation of basal cyclic AMP accumulation. Cyclic AMP accumulation in response to stimulation by epinephrine (10 microM) was normal in adipocytes of transgenic mice, demonstrating no alteration in the stimulatory adenylylcyclase capacity in the Gi alpha 2-deficient cells. The inhibitory adenylylcyclase pathway, in sharp contrast, was severely blunted in response to challenge by the inhibitory A1-purinergic agonist, (-)R-N6-phenylisopropyladenosine. These studies illuminate a critical role of Gi alpha 2 in the inhibitory adenylylcyclase signaling pathway in vivo.
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PMID:Gi alpha 2 mediates the inhibitory regulation of adenylylcyclase in vivo: analysis in transgenic mice with Gi alpha 2 suppressed by inducible antisense RNA. 769 86

The phosphoenolpyruvate carboxykinase (PEPCK) gene is regulated at the transcriptional level by a variety of effectors in a tissue-specific fashion. In order to study the parameters involved in the tissue-specific hormonal regulation of the PEPCK gene, we have used a transient expression test in well-differentiated rat hepatoma cells as well as in dedifferentiated variants. In this test, the PEPCK promoter is induced by glucocorticoids in well-differentiated FGC4 cells, but not in H5 dedifferentiated variants, in spite of the presence in H5 cells of the glucocorticoid receptor. Study of the PEPCK promoter using electrophoretic mobility shift assays reveals binding sites for the liver-enriched transcription factors HNF1, vHNF1, HNF3, HNF4, and CAAT/enhancer binding protein members. Overexpression of the liver-enriched transcription factors absent in the dedifferentiated variants, such as HNF1 and HNF4, is not sufficient to restore glucocorticoid response of the PEPCK promoter in the variants. Moreover, systematic analysis of the PEPCK promoter reveals that the presence of a region covering a cAMP-responsive element (CRE1 at -80) and a CAAT box is necessary for full response of the PEPCK promoter to glucocorticoids in well-differentiated rat hepatoma cells. In a cotransfection test, overexpression of the regulatory subunit of protein kinase A (PKA), causing sequestering of PKA, abolishes the glucocorticoid response of the promoter in well-differentiated cells. On the other hand, in dedifferentiated variants, overexpression of the catalytic subunit of PKA restores the response to glucocorticoids. The action of PKA on the glucocorticoid response requires the presence of the CRE1 element and is promoter specific because it does not concern nonhepatic promoters such as the long terminal repeats of the mouse mammary tumor virus. These results suggest that the full response of the PEPCK promoter to glucocorticoids requires activation of another signal transduction pathway, the cAMP-mediated pathway.
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PMID:Response of the phosphoenolpyruvate carboxykinase gene to glucocorticoids depends on the integrity of the cAMP pathway. 781 33

Oxazolidinediones are a class of oral antidiabetic agents that are closely related structurally and pharmacologically to thiazolidinediones. The thiazolidinediones have been shown to partially reverse the loss in insulin-responsive glucose uptake caused by chronic treatment with dexamethasone. This study was conducted to determine certain aspects of the mechanism of thiazolidinedione and oxazolidinedione action. We selected the oxazolidinedione CP-92,768-2 (5-[2-[(5-methyl2-phenyl-4-oxazolyl)methyl]5-benzofuranyl methyl]2,4- oxazolidinedione) to determine whether these agents could reverse the dexamethasone-induced down-regulation of IRS-1, the insulin receptor substrate-1. In 3T3-L1 adipocytes, dexamethasone treatment resulted in down-regulation of IRS-1 to 60% of control values. Simultaneous treatment with CP-92,768-2 significantly increased IRS-1 to 78% of the control value (EC50, < 10 nM), although it did not completely reverse the dexamethasone effect at any concentration tested. CP-92,768-2 alone did not have any effect on IRS-1. CP-92,768-2 did not affect the stability of IRS-1 protein in the presence or absence of dexamethasone, as measured by [35S]methionine pulse-chase labeling. Dexamethasone decreased messenger RNA (mRNA) for IRS-1 after 24 h of treatment to 40% of the control value. CP-92,768-2 partially reversed this decrease in IRS-1 mRNA to 65% of the control value after 24 h of treatment, but had no effect on IRS-1 mRNA in the absence of dexamethasone. Dexamethasone down-regulated the insulin stimulation of [3H]thymidine incorporation to 68% of the control value. Dexamethasone in the presence of CP-92,768-2 down-regulated insulin stimulation of thymidine incorporation by only 9%. Dexamethasone also down-regulated the expression of phosphoenolpyruvate carboxykinase (PEPCK) protein by 50%. CP-92,768-2 partially protected PEPCK from the dexamethasone down-regulation. Conversely, the up-regulation of expression of PEPCK and IRS-1 produced by dexamethasone in KRC-7 hepatoma cells was not affected by CP-92,768-2. One contribution of oxazolidinediones to an increase in insulin responsiveness in the presence of glucocorticoids may be the up-regulation of IRS-1 in adipose cells.
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PMID:The oxazolidinedione CP-92,768-2 partially protects insulin receptor substrate-1 from dexamethasone down-regulation in 3T3-L1 adipocytes. 789 55

To study the transcriptional regulation of the liver gluconeogenic phenotype, the underdifferentiated mouse Hepa-1c1c7 (Hepa) hepatoma cell line was used. These cells mimicked the fetal liver by appreciably expressing the alpha-fetoprotein and albumin genes but not the phosphoenolpyruvate carboxykinase (PEPCK) gene. Unlike the fetal liver, however, Hepa cells failed to express the early-expressed factors hepatocyte nuclear factor 1 alpha (HNF-1 alpha) and HNF-4 and the late-expressed factor C/EBP alpha, thereby providing a suitable system for examining possible cooperation between these factors in the transcriptional regulation of the PEPCK gene. Transient transfection assays of a chimeric PEPCK-chloramphenicol acetyltransferase construct showed a residual PEPCK promoter activity in the Hepa cell line, which was slightly stimulated by cotransfection with a single transcription factor from either the C/EBP family or HNF-1 alpha but not at all affected by cotransfection of HNF-4. In contrast, cotransfection of the PEPCK construct with members from the C/EBP family plus HNF-1 alpha resulted in a synergistic stimulation of the PEPCK promoter activity. This synergistic effect depended on the presence in the PEPCK promoter region of the HNF-1 recognition sequence and on the presence of two C/EBP recognition sequences. The results demonstrate a requirement for coexistence and cooperation between early and late liver-enriched transcription factors in the transcriptional regulation of the PEPCK gene. In addition, the results suggest redundancy between members of the C/EBP family of transcription factors in the regulation of PEPCK gene expression.
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PMID:Transcriptional regulation of the phosphoenolpyruvate carboxykinase gene by cooperation between hepatic nuclear factors. 793 27

Expression of many liver-specific genes is extinguished when cultured hepatoma cells are fused with fibroblasts, but liver genes can be reexpressed in hybrid segregants that have lost fibroblast chromosomes. To map extinguisher loci involved in this process, hepatoma microcell hybrids retaining single fibroblast chromosomes have been employed. Two different, transdominant loci that affect liver gene expression have been defined in this way. To determine whether other monochromosomal extinction phenotypes could be observed, we inserted a selectable marker into many human chromosomal sites and transferred the marked human chromosomes into rat hepatoma recipient cells by microcell fusion. Nearly 200 microcell hybrid clones were isolated and screened for expression of liver-specific mRNAs. Most liver transcripts continued to be expressed. However, PEPCK mRNA was extinguished in 12 hybrid clones. Some of these hybrids contained human TSE1, the previously characterized extinguisher locus on chromosome 17, but others contained a novel extinguishing function that mapped to human chromosome 14. The implications of these findings are discussed.
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PMID:Tissue-specific extinguisher loci in the human genome: a screening study based on random marking and transfer of human chromosomes. 794 22

Oral vanadate administration has been demonstrated to normalize blood glucose levels in ob/ob and db/db mice and streptozotocin (STZ) diabetic rats. The exact mechanism of this vanadate effect is uncertain, since there are no consistent effects on the insulin receptor tyrosine kinase activity or phosphotyrosine phosphatase activity. We have therefore studied the postreceptor actions of vanadate, focusing our attention on the steady-state levels of mRNA of enzymes involved in carbohydrate metabolism. When compared with their lean (ob/+) controls, the livers of ob/ob mice exhibited an approximately 90% reduction in the levels of phosphoenolpyruvate carboxykinase (PEPCK) mRNA and twofold to fivefold higher levels of the mRNAs for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the "liver beta-cell" glucose transporter (GLUT2), and the proto-oncogene c-myc. Administration of sodium vanadate (0.25 mg/mL) in the drinking water of ob/ob mice over a 45-day period resulted in a near normalization of blood glucose and increased PEPCK mRNA levels more than ninefold. Starvation of the ob/ob mice for 24 to 48 hours also increased PEPCK mRNA levels by fourfold to 15-fold. Vanadate treatment did not alter mRNA levels of any other proteins studied and had no effect on PEPCK mRNA in ob/+ mice. However, 1 to 100 mumol/L vanadate produced a concentration-dependent increase in PEPCK mRNA levels in an H35 hepatoma cell line, an effect opposite to the suppression of PEPCK mRNA produced by insulin. In summary, hyperglycemia in the ob/ob mouse is characterized by decreased expression of PEPCK and increased expression of GAPDH mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Vanadate normalizes hyperglycemia and phosphoenolpyruvate carboxykinase mRNA levels in ob/ob mice. 796 88

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