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)

The catalytic reaction of renin, an aspartyl proteinase, with angiotensinogen is the rate-limiting step fo the renin-angiotensin system involved in the maintenance of blood pressure and electrolyte balance in mammals. We have characterized species-specific expression of the hepatic renin gene by RNase protection experiment, primer extension analysis, and promoter assay using an in vitro DNA transfection. RNase protection experiments revealed that the renin gene is expressed in rat liver, but neither in mouse nor in human. Primer extension analysis identified the putative promoter region of the rat renin gene, which contains TATAAAA sequence, a canonical regulatory DNA element. In order to test whether the upstream region of the renin gene with respect to the putative transcription initiation site is a functional promoter, we have examined the ability of the 5'-flanking sequences of the rat renin gene as well as the human and mouse genes to activate expression of a reporter gene containing the bacterial chloramphenicol acetyltransferase (CAT)-coding sequences, by transient transfection assays. In transfected HepG2 cells, a hepatoma cell line, only the rat renin promoter was capable of driving the CAT gene expression. These results suggested that the rat-specific renin gene expression in the liver could be primarily determined by its promoter specificity.
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PMID:Species-specific expression of the hepatic renin gene. 820 34

The first three exons and the promoter of rat glia-derived nexin, also called protease nexin-1 (GDN/PN-1), have been identified through analysis of rat genomic clones. A 1.6 kilobase (kb) fragment containing 105 base pairs of the first exon and 5'-flanking sequences was sequenced. The 5'-flanking sequence and the first exon were found to be GC-rich, indicating that the 5' region of the rat GDN/PN-1 gene resides within a CpG island. A TATA box-like sequence, but no CAAT box, was found. The rat GDN/PN-1 promoter contains five SP1 consensus sites, four consensus sites for the MyoD1 transcription factor, and one binding site for the transcription factors NGFI-A, NGFI-C, Krox-20, and Wilms tumor factor. The presence of these consensus sequences is consistent with the known expression pattern of GDN/PN-1. Primer extension and RNase protection assays identified one transcriptional start site. The 1.6 kb promoter fragment cloned in a reporter plasmid was found to induce firefly luciferase expression in a cell-specific manner. A positive regulatory element is localized in the region -1545 to -389. In vitro CpG methylation blocked transcription from the GDN/PN-1 promoter in rat hepatoma cells but not in C6 rat glioma cells.
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PMID:Molecular organization of the rat glia-derived nexin/protease nexin-1 promoter. 826 20

cAMP participates in the regulation of endogenous hypothalamic and placental CRF by increasing levels of both peptide secretion and mRNA expression. In previous studies we have shown that stimulation of the protein kinase A-dependent pathway by cAMP analogues or forskolin produced a dose-dependent increase in levels of CRF mRNA when the intact hCRF gene was stably transfected and expressed in the mouse corticotroph AtT20 cell line. In the present study, we explored the mechanism of the cAMP-dependent increase in CRF gene expression in the stably transfected AtT20 cell line using pharmacologic, slot-blot, and RNase mapping methodologies. Following incubation with cAMP, there was a rapid increase in CRF mRNA which was completely blocked by pre-treatment with actinomycin D, an inhibitor of transcription. Cycloheximide, an inhibitor of protein synthesis, produced an independent increase in CRF mRNA, but did not change the relative induction of CRF mRNA produced by cAMP. Solution hybridization studies using intron- and exon-specific hCRF probes demonstrated a rapid rise in nuclear CRF hnRNA, which was apparent within 15 min of cAMP incubation and preceded the rise in cytoplasmic CRF mRNA. RNase mapping studies demonstrated that CRF transcription was initiated at discrete promoter sites in CRF-AtT20 cells, and that this pattern of promoter utilization was similar to that observed in mRNA derived from sites of endogenous CRF expression, human placenta and human hepatoma NPLC cell line. Treatment with cAMP selectively increased CRF mRNA transcripts initiated at the proximal promoter site, but had little or no effect on transcripts initiated at the distal promoters. We conclude that cAMP effects on CRF gene expression occur rapidly, do not require new protein synthesis, and are initiated within the nuclear compartment, consistent with a direct effect on CRF gene transcription. This effect is mediated predominantly through the proximal promoter element, while more distal promoters are less sensitive to transcriptional activation by cAMP.
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PMID:Transcriptional regulation of human corticotropin releasing factor gene expression by cyclic adenosine 3',5'-monophosphate: differential effects at proximal and distal promoter elements. 827 45

The 5' flanking region of the human gene for the second component of complement was sequenced and analyzed functionally. RNase protection demonstrated a cluster of four initiation sites in the 5' flanking region utilized in the hepatoma cell line, HepG2. Utilization of all four initiation sites increased in response to gamma-interferon (IFN-gamma). Transient transfection analysis was used to examine cis-acting sequence motifs controlling transcription from the 5'-flanking region. We identified a 228-bp minimal promoter fragment which was able to direct basal and IFN-gamma inducible transcription from authentic initiation sites. Sequence motifs outside of this region may modulate the transcriptional regulation of the second component of complement. Although complement components are not coordinately regulated, we identified four regions of significant homology with the promoters of multiple other complement components. Three of these regions were within the minimal promoter fragment.
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PMID:Transcriptional regulation of the gene for the second component of human complement: promoter analysis. 829 89

In humans, two cDNAs have been isolated encoding beta-galactoside alpha 2,6-sialyltransferase, differing only in part of the 5' untranslated region. Primer extension data show that the two cDNAs are near full-length clones. RNase protection analysis of different cell types showed that the transcript corresponding to the alpha 2,6-sialyltransferase cDNA isolated from a B-cell library resided only in mature B cells. In contrast, the transcript corresponding to the alpha 2,6-sialyltransferase cDNA isolated from a placenta library was found in all cells tested. Our results also indicate the existence of a third alpha 2,6-sialyltransferase transcript in the hepatoma cell line HepG2. Mature B cells were found to express high amounts of alpha 2,6-sialyltransferase mRNA, compared to other cell types tested, as shown by Northern blot analysis. Moreover there was an increased expression of beta-galactoside alpha 2,6-sialyltransferase mRNA in activated B cells compared to resting B cells. In vitro transcription and translation of the cDNAs resulted in a protein of 45 kDa, but the transcripts were translated with different efficiency, suggesting a role for the 5' untranslated region in regulation of translation. We have also made an alpha 2,6-sialyltransferase construct lacking the specific 5' regions of the two cDNAs. A transcript generated from this construct was translated more efficiently in vitro than the two alpha 2,6-sialyltransferase cDNAs.
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PMID:Cell-specific expression of human beta-galactoside alpha 2,6-sialyltransferase transcripts differing in the 5' untranslated region. 847 18

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin induces the microsomal enzyme cytochrome P4501A1 by increasing the transcription rate of the CYP1A1 gene. Induction requires two basic helix-loop-helix proteins, the ligand-binding aromatic hydrocarbon receptor (AhR) and its heterodimerization partner, the AhR nuclear translocator (Arnt). The AhR/Arnt heterodimer induces transcription by binding to dioxin-responsive elements (DREs) within an enhancer upstream of the CYP1A1 gene. The basic regions of AhR and Arnt are crucial for DRE binding. We have mutated these regions in order to analyze the relationship between DRE binding (determined in vitro using an electrophoretic mobility shift assay) and induction of CYP1A1 transcription (determined in vivo by genetic complementation of AhR-defective and Arnt-defective mouse hepatoma cells, using an RNase protection assay to measure mRNA accumulation). Our findings reveal the amino acids in the basic regions of AhR/Arnt that are important for both DRE binding and induction of transcription. This information provides biological background for the interpretation of structural (e.g. crystallographic) studies of the interactions between AhR/Arnt and the DRE. Our findings also indicate that the in vitro behavior of the mutants does not consistently predict their functional activity in vivo. Thus, genetic complementation constitutes an important and stringent test for analyzing the effects of mutations on AhR/Arnt function.
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PMID:DNA binding by the heterodimeric Ah receptor. Relationship to dioxin-induced CYP1A1 transcription in vivo. 862 73

The cytoplasmic iron regulatory protein (IRP) modulates iron homeostasis by binding to iron-responsive elements (IREs) in the transferrin receptor and ferritin mRNAs to coordinately regulate transferrin receptor mRNA stability and ferritin mRNA translational efficiency, respectively. These studies demonstrate that thyroid hormone (T3) can modulate the binding activity of the IRP to an IRE in vitro and in vivo. T3 augmented an iron-induced reduction in IRP binding activity to a ferritin IRE in RNA electrophoretic mobility shift assays using cytoplasmic extracts from human liver hepatoma (HepG2) cells. Hepatic IRP binding to the ferritin IRE also diminished after in vivo administration of T3 with iron to rats. In transient transfection studies using HepG2 cells and a human ferritin IRE-chloramphenicol acetyltransferase (H-IRE-CAT) construct, T3 augmented an iron-induced increase in CAT activity by approximately 45%. RNase protection analysis showed that this increase in CAT activity was not due to a change in the steady state level of CAT mRNA. Nuclear T3-receptors may be necessary for this T3-induced response, because the effect could not be reproduced by the addition of T3 directly to cytoplasmic extracts and was absent in CV-1 cells which lack T3-receptors. We conclude that T3 can functionally regulate the IRE binding activity of the IRP. These observations provide evidence of a novel mechanism for T3 to up-regulate hepatic ferritin expression, which may in part contribute to the elevated serum ferritin levels seen in hyperthyroidism.
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PMID:Thyroid hormone modulates the interaction between iron regulatory proteins and the ferritin mRNA iron-responsive element. 866 26

Relaxation of the trabecular smooth muscle, which is necessary for penile erection, is controlled locally by neurotransmitters and vasoactive agents. The goal of this study was to identify and characterize muscarinic acetylcholine receptor (mAChR) subtypes expressed in cultured human corpus cavernosum smooth muscle cells (HCC SMC). Binding analysis with L-[benzilic-4,4'-3H(N)]quinuclidinyl benzilate ([3H]QNB) demonstrated the expression of specific muscarinic receptor binding sites in HCC SMC. Analysis of total RNA isolated from whole corpus cavernosum tissue and smooth muscle cells, by RNase protection assays, demonstrated the expression of mRNA transcripts for m1, m2, m3, and m4 mAChR subtypes in whole tissue and m2 and m4 subtypes in cultured cells. In situ hybridization with specific m2 and m4 probes further confirmed the expression of m2 and m4 mRNA transcripts in cultured cells. Carbachol (CCh), a nonselective cholinergic agonist, inhibited cAMP synthesis at low concentrations (0.1-1 microM) and stimulated cAMP synthesis at high concentrations (100 microM), in cultured HCC SMC. CCh (100 microM) further augmented forskolin (FSK), isoproterenol (ISO), and prostaglandin E1 (PGE1)-induced cAMP synthesis. These observations suggest that, in vivo, in HCC, ACh may activate m3 mAChR subtypes on endothelial cells or m2 and m4 subtypes on the SMC. Although m2 and m4 are thought to inhibit adenylate cyclase (AC), the augmentation of cAMP synthesis by high concentrations of CCh in SMC suggests an alternative mechanism of coupling to G-proteins that stimulates AC activity. These studies show that HCC tissue expresses different subtypes of mAChR (m1, m2, m3, and m4), whereas cultured HCC SMC express m2 and m4 subtypes. It is suggested that m2 and m4 receptor subtypes may play an important role in maintaining trabecular smooth muscle tone in vivo. The augmentation of FSK-, ISO, and PGE1-induced cAMP synthesis by CCh suggests possible development of a multidrug therapeutic approach to treatment of erectile dysfunction.
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PMID:Expression of functional muscarinic acetylcholine receptor subtypes in human corpus cavernosum and in cultured smooth muscle cells. 872 95

Truncated hepatitis B virus transcripts terminating downstream of a cryptic CAUAAA polyadenylation signal within the HBx open reading frame have previously been identified in tissue samples from two patients with hepatocellular carcinoma (Hilger et al., 1991, J. Virol. 65, 4284-4291). In this study an HBx expression plasmid was systematically deleted in order to elucidate the DNA sequence context which is required for the conversion of the usually inactive CAUAAA motif into a functional polyadenylation signal. Deletions were made progressively on a stretch of viral DNA which, seen on the transcript level, started downstream of the established UAUAAA polyadenylation signal and proceeded to the cryptic CAUAAA motif. The plasmid constructs obtained were used to transfect cells of the HepG2 line. The analysis of newly synthesized RNA via an RNase protection assay revealed termination downstream of the CAUAAA motif following the removal of GU-rich auxiliary sequences downstream of the poly(A) addition site of the UAUAAA signal. Similar results were obtained when an anchored oligo(dT) primer which recognizes selectively truncated RNA was used for the differential, RT/PCR-mediated amplification of 3'-ends. Thus it could be documented in two ways that inactivation or removal of the UAUAAA signal rendered the CAUAAA motif functional as a poly(A) signal. On the basis of the results obtained, we suggest that chromosomally integrated viral DNA on which the TATAAA motif is removed may constitute a template for truncated as well as for virus/cell hybrid transcripts. We also suggest the use of anchored oligo(dT) primers for the rapid identification of truncated transcripts in tissue samples of HCC patients.
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PMID:DNA sequence requirements for the activation of a CATAAA polyadenylation signal within the hepatitis B virus X reading frame: rapid detection of truncated transcripts. 880 79

Hypoxia-inducible factor 1 (HIF-1) is a DNA-binding heterodimeric protein complex originally described in the transcriptional activation of the erythropoietin gene by hypoxia. This protein complex is composed of two subunits, HIF-1alpha and -1beta (aryl hydrocarbon receptor nuclear translocator, ARNT). In this study, we used ARNT-deficient cells, derived from the mouse hepatoma cell line Hepa1c1c7, to further characterize HIF-1 complex formation and its relationship with gene activation by hypoxia and desferrioxamine (Df). Gel shift assays revealed that ARNT is absolutely required for the formation of the HIF-1 DNA-binding complex. Results from RNase protection assays and Northern blots showed that the lack of functional HIF-1 complex completely abrogated the response to hypoxia of vascular endothelial growth factor (VEGF) and the glycolytic enzymes aldolase A (ALDA) and phosphoglycerate kinase 1 (PGK-1), genes known to be upregulated by low oxygen tension. Desferrioxamine induction of VEGF and PGK-1 genes was reduced in the ARNT-deficient cells, but at difference with hypoxia, it was not completely suppressed. These results suggest that Df is able to activate gene transcription through HIF-1-independent mechanisms. Exposure to hypoxia or Df did not induce any changes in HIF-1alpha and -1beta mRNA levels, suggesting that posttranscriptional mechanisms are involved in HIF-1 complex activation.
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PMID:Absolute requirement of aryl hydrocarbon receptor nuclear translocator protein for gene activation by hypoxia. 890 Apr 15


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