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 human UGT1 gene is a single copy gene consisting of four common exons and more than 13 variable exons which span more than 200 kb of the human genome. A single variable exon is spliced to the four common exons to form the mRNA for synthesis of a single UDP-glucuronosyltransferase (UGT) isoenzyme. Treatment of humans or hepatoma cell lines with drugs such as phenobarbital causes the induction of hepatic bilirubin UGT by increased transcription from the UGT1 gene. The upstream region of UGT1*1 (bilirubin UGT) was sequenced and found to contain consensus sequences for several transcriptional regulatory elements including a 'BARBIE box'. An unusual 'TATA' promoter sequence A(TA)6TAA was also observed. The 5' region flanking the UGT1*1 exon when cloned into reporter constructs and transfected into four cells lines was capable of promoting reporter gene expression, but not when transfected into monkey kidney cell fibroblasts (COS-7 cells) indicating a cell specific expression. Sequential deletion of the 5' flanking region in the plasmid constructs did not cause any significant reduction in reporter expression. Treatment of cells transfected with these plasmid constructs with drugs did not cause a significant increase in reporter expression except with retinoic acid plus WY 14643. Introduction of an additional two base pairs (TA) into the 'TATA' box of the 5' gene sequence (as observed in Gilbert's patients) did not significantly change reporter expression levels. The regulation of the biliruibin UGT gene by drugs is not yet understood and it will be important to identify additional genetic elements possibly further than -2kb upstream of the UGT1*1 coding region, which regulate the expression of this gene.
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PMID:Regulation of the human bilirubin UDP-glucuronosyltransferase gene. 886 42

The human gene encoding insulin-like growth factor II contains four promoters (P1-P4) that are differentially activated in various tissues during development. Expression of insulin-like growth factor II in adult liver tissue is directed by P1, which is activated by liver-enriched members of the CCAAT/enhancer binding protein family of transcription factors. In the present report we show that the region around -48 relative to the transcription start site contains a high affinity Sp1 binding site. This was demonstrated by electrophoretic mobility shift assays using nuclear extracts from Hep3B hepatoma cells and with specific antibodies directed against Sp1. Competition electrophoretic mobility shift assays revealed that the Sp1 binding site of P1 and a consensus Sp1 binding site bind Sp1 with comparable efficiencies. Mutation of the Sp1 binding site results in an 85% decrease in P1 promoter activity in transient transfection assays using two different cell lines, COS-7 and Hep3B. Investigation of P1 mutants in which the spacing of the Sp1 binding site and the transcription start site was increased showed that the role of the Sp1 binding site in regulation of P1 is position dependent. Interestingly, the Sp1-responsive element cannot be exchanged by a functional TATA box. Activation of P1 by transactivators CCAAT/enhancer binding protein-beta and hepatocyte nuclear factor-3beta is strongly impaired after mutation of the Sp1 binding site. These results demonstrate that the specific presence of a binding site for the ubiquitously expressed transcription factor Sp1 is of eminent importance for efficient activation of P1 by liver-enriched transactivators.
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PMID:A functional Sp1 binding site is essential for the activity of the adult liver-specific human insulin-like growth factor II promoter. 901 71

Expression of the asialoglycoprotein receptor by the human hepatocellular carcinoma cell line HuH-7 in response to intracellular cGMP concentrations was previously shown to be regulated at the translational level. In a cell-free system, initiation of asialoglycoprotein receptor mRNA translation was dependent on the presence of the 7-methylguanylate cap site and was independent of 8-bromo-cGMP levels in which the cells were grown prior to RNA isolation. Stable transfection of COS-7 cells with deletion constructs of the asialoglycoprotein receptor H2b subunit localized the cGMP-responsive cis-acting element to the mRNA 5'-untranslated region (UTR). Addition of biotin (an activator of guanylate cyclase) induced the expression of beta-galactosidase present as a chimeric plasmid containing the H2b 187-nucleotide 5'-UTR. An RNA gel retardation assay identified a 37-nucleotide cognate sequence within this 187-nucleotide region. Titration of the 5'-UTR with a cytosolic fraction isolated from HuH-7 grown in the presence or absence of 8-bromo-cGMP or biotin provided direct evidence for an RNA-binding protein responsive to intracellular levels of cGMP. Based on these findings, it seems reasonable to propose that reduction of intracellular levels of cGMP by biotin deprivation results in a negative trans-acting factor associating with the 5'-UTR of asialoglycoprotein receptor mRNAs, thereby inhibiting translation.
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PMID:Cytoplasmic protein mRNA interaction mediates cGMP-modulated translational control of the asialoglycoprotein receptor. 908 46

1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3' (E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089) is a novel synthetic analog of 1 alpha,25-dihydroxyvitamin D [1,25-(OH)2D3] with potential for use in the treatment of hyperproliferative disorders. It has an altered side-chain structure compared to 1,25-(OH)2D3, featuring 26,27 dimethyl groups, insertion of an extra carbon atom (24a) at C-24, and two double bonds at C-22,23 and C-24,24a. In vitro metabolism of EB1089 was studied in a human keratinocyte cell model, HPK1A-ras, previously shown to metabolize 1,25-(OH)2D3. Four metabolites were formed, all of which possessed the same UV chromophore as EB1089, indicating the retention of the side-chain conjugated double bond system. Two metabolites were present in sufficient quantities to identify them as 26-hydroxy EB1089 (major product) and 26a-hydroxy EB1089 (minor product), based on mass spectral analysis and cochromatography with synthetic standards. Similar metabolites were generated in vivo and using a liver postmitochondrial fraction in vitro (Kissmeyer et al., companion paper). Studies with the human hepatoma Hep G2 gave rise to 2 isomers of 26-hydroxy EB1089. Studies using ketoconazole, a general cytochrome P450 inhibitor, implicated cytochrome P450s in the formation of the EB1089 metabolites. COS-1 transfection cell experiments using vectors containing CYP27 and CYP24 suggest that these cytochrome P450s are probably not involved in 26- or 26a-hydroxylation of EB1089. Other experiments that examined the HPK1A-ras metabolism of related analogs containing only a single side-chain double bond: 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1' (E)-en-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (MC1473; double bond at C-22,23) and 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-3'(E)-en-1'-yl)-9, 10-secopregna-5(Z),7(E),10(19)-triene (MC1611; double bond at C-24,24a) revealed that the former compound was subject to 24-hydroxylation and the latter compound was mainly 23-hydroxylated. Metabolism experiments involving EB1089, MC1473, and MC1611 in competition with [1 beta-3H]1,25-(OH)2D3 in HPK1A-ras confirmed that CYP24 is probably not involved in the metabolism of EB1089 whereas, in the case of MC1473 and MC1611, it does appear to carry out side-chain hydroxylation. Our interpretation is that the conjugated double bond system in the side-chain of EB1089 is responsible for directing the target cell hydroxylation to the distal positions, C-26 and C-26a. We conclude that EB1089 is slowly metabolized via unique in vitro metabolic pathways, and that these features may explain the relative stability of EB1089 compared to other analogs in vivo.
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PMID:Metabolism of the vitamin D analog EB1089 by cultured human cells: redirection of hydroxylation site to distal carbons of the side-chain. 911 99

PACE4 is a processing protease which processes the precursor protein to the mature protein. Currently, four PACE4 isoforms have been reported [Tsuji, A. et al. (1994) Biochem. Biophys. Res. Commun. 200, 943 950]. In this study, we have cloned cDNA encoding a novel isoform, PACE4E, by screening the human brain cerebellum cDNA library and reverse transcriptase polymerase chain reaction analysis of total RNA from human hepatoma HepG2 cells. The PACE4E cDNA encoded an amino acid sequence of 975 residues. The sequence from the amino terminus to Arg900 of PACE4E was identical to the corresponding sequence of PACE4A, but the carboxy terminal sequence (75 residues) was unique and contained a hydrophobic cluster (Leu952-Gly968). PACE4E cDNA was transiently transfected in COS-1 cells, and the expressed proteins were a 112-kDa precursor form and a 105-kDa mature form. They were secreted into the culture medium, but their secretion was retarded compared with that of PACE4A. The expression of a mutant of PACE4E truncated up to the hydrophobic cluster from the carboxy terminus resulted in a remarkable increase in secretion level, suggesting that PACE4E tends to be retained intracellularly due to interaction with the membrane through the hydrophobic cluster. On the contrary, the transient expression experiment of PACE4C showed that only 68-kDa protein (precursor form) was detected in the cell and not secreted into the medium. In addition, coexpression experiment revealed that PACE4E was able to process the precursor form of von Willebrand factor to the mature form, but PACE4C did not process it.
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PMID:A novel human PACE4 isoform, PACE4E is an active processing protease containing a hydrophobic cluster at the carboxy terminus. 919 37

A 3,4-benzopyrene-resistant mutant clone (c4) of the mouse hepatoma Hepa-1c1c7 cell line was examined for the mutation that causes the defective function of aryl-hydrocarbon receptor (AHR) nuclear translocator (Arnt). Arnt dimerizes with AHR and mediates the induction signal of aryl-hydrocarbon hydroxylase activity. The Arnt cDNAs of c4 cells were cloned by reverse-transcription/PCR to compare the sequences with that of wild-type Arnt cDNA. The Arnt cDNA of c4 cells was found to have a single point mutation, leading to replacement of Gly326 with Asp between two internal repeats in the highly conserved Per-Arnt-Sim (PAS) domain, PAS A and PAS B. The inability of [Asp326]Arnt/AHR heterodimers to enhance reporter gene transcription under the control of the CYP1A1 gene promoter and enhancer confirmed that the G326-->D substitution was a causative mutation. While fluorescence microscopy and coimmunoprecipitation experiments showed that this mutant form of Arnt was not changed from wild-type Arnt in terms of nuclear localization or heterodimer formation with AHR, the binding activity of the [Asp326]Arnt x AHR heterodimer to the xenobiotic-responsive element was reduced markedly. Determination of the turnover rate in COS-7 cells transfected with expression plasmids for mutant Arnt or normal Arnt showed that the mutant protein turned over with an accelerated rate compared with that of the normal. Moreover, the mutant protein displayed increased proteolytic digestibility in vitro with various proteases.
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PMID:A point mutation responsible for defective function of the aryl-hydrocarbon-receptor nuclear translocator in mutant Hepa-1c1c7 cells. 920 42

6-Pyruvoyl-tetrahydrobiopterin synthase (PTPS) is involved in tetrahydrobiopterin (BH4) biosynthesis, the cofactor for various enzymes including the hepatic phenylalanine hydroxylase. Inherited PTPS deficiency leads to BH4 depletion, causes hyperphenylalaninemia, and requires cofactor replacement therapy for treatment. We previously isolated the human PTPS cDNA and recently characterized its corresponding gene, PTS. Here we developed PCR-based mutation analysis with newly designed primers to detect genomic alterations and describe five mutations, four of which are novel, in the PTS gene of four Italian families with affected individuals. The mutant alleles found included three missense mutations (T67M, K129E, D136V), a previously described triplet deletion (delta V57), and a single c-3-->g transversion in the 3'-acceptor splice site of intron 1, leading to cryptic splice site usage that resulted in a 12 bp deletion (mutant allele delta (K29-S32)). Except for K129E, all mutant alleles were inactive and/or unstable proteins, as shown by recombinant expression and Western blot analysis of patients' fibroblasts. The PTPS-deficient patient with the homozygous K129E allele had transient hyperphenylalaninemia, did not depend on BH4 replacement therapy, and showed normal PTPS immunoreactivity, but no enzyme activity in primary fibroblasts and red blood cells. In contrast to its inactivity in these cells, the K129E mutant was 2-3 fold more active than wild-type PTPS when transfected into COS-1 or the human hepatoma cell line Hep G2. K129E appears thus as a mutant PTPS whose activity depends on the cell type.
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PMID:Identification of mutations causing 6-pyruvoyl-tetrahydropterin synthase deficiency in four Italian families. 922 57

The current study demonstrates that T3-activated transcription of the NADPH:cytochrome P450 oxidoreductase (P450R) gene is dependent on the thyroid hormonal status of the animal, with both transcriptional and post-transcriptional pathways being important in regulating the cellular P450R mRNA level. The region required for transcriptional activation of the P450R gene by T3 has been identified. Nuclear run-on experiments demonstrated that the effects of T3 on P450R transcription are dependent on thyroid status, with a transcriptional enhancement obtained in T3-treated hypothyroid rat liver (1.8-fold increase) but not in T3-treated euthyroid animals. Transient cotransfection of P450R promoter/chloramphenicol acetyl transferase (CAT) constructs and the thyroid hormone receptor beta1 (TR beta1) expression plasmid into rat hepatoma H4IIE cells resulted in a 2.4-fold induction of promoter activity that was both T3 and TR beta1 dependent. Analysis of promoter deletion constructs identified a P450R-thyroid response region (P450R-TRE; bases, -564 to -536) containing three imperfect direct repeats of the thyroid response motif, AGGTCA. Mutational analysis further established that T3 induction was dependent only on the upstream direct repeat, having the sequence AGGTGAgctgAGGCCA. Footprint analysis showed that all three motifs were protected by proteins present in rat liver nuclear extracts, and a direct interaction between P450R-TRE and T3 receptors TR alpha1 and TR beta1 was demonstrated by gel-shift analysis. In vitro binding studies with P450R-TRE revealed the formation of heterodimeric complexes when TR alpha1 was coincubated with either the retinoic X receptor alpha or nuclear extract from rat liver, COS, or H4IIE cells. In addition, placement of the P450R-TRE upstream of the T3-nonresponsive heterologous thymidine kinase promoter resulted in a 2.7-fold transcriptional enhancement that was both T3 and TR beta1 dependent. Previous studies have demonstrated that T3 augments P450R mRNA levels approximately 20-30-fold and approximately 12-fold, respectively, in hypothyroid and euthyroid rats. Hence, for the hypothyroid state, transcriptional and post-transcriptional events contribute to the T3-induced mRNA increases; however, the marked increase in message level in T3-treated euthyroid animals depends primarily on post-transcriptional pathways.
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PMID:Thyroid regulation of NADPH:cytochrome P450 oxidoreductase: identification of a thyroid-responsive element in the 5'-flank of the oxidoreductase gene. 922 11

Signal transducer and transcription (STAT) factors are activated by tyrosine phosphorylation in response to a variety of cytokines, growth factors, and hormones. Tyrosine phosphorylation triggers dimerization and nuclear translocation of these transcription factors. In this study, the functional role of carboxy-terminal portions of the STAT family member acute-phase response factor/Stat3 in activation, dimerization, and transactivating potential was analyzed. We demonstrate that truncation of 55 carboxy-terminal amino acids causes constitutive activation of Stat3 in COS-7 cells, as is known for the Stat3 isoform Stat3beta. By the use of deletion and point mutants, it is shown that both carboxy- and amino-terminal portions of Stat3 are involved in this phenomenon. Dimerization of Stat3 was blocked by point mutations affecting residues both in the vicinity of the tyrosine phosphorylation site (Y705) and more distant from this site, suggesting that multiple interactions are involved in dimer formation. Furthermore, by reporter gene assays we demonstrate that carboxy-terminally truncated Stat3 proteins are incapable of transactivating an interleukin-6-responsive promoter in COS-7 cells. In HepG2 hepatoma cells, however, these truncated Stat3 forms transmit signals from the interleukin-6 signal transducer gp130 equally well as does full-length Stat3. We conclude that, dependent on the cell type, different mechanisms allow Stat3 to regulate target gene transcription either with or without involvement of its putative carboxy-terminal transactivation domain.
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PMID:Mutational analysis of acute-phase response factor/Stat3 activation and dimerization. 923 24

NS5A derived from a hepatitis C virus (HCV) genotype 1b isolate has previously been shown to undergo phosphorylation on serine residues (T. Kaneko, Y. Tanji, S. Satoh, M. Hijikata, S. Asabe, K. Kimura, and K. Shimotohno, Biochem. Biophys. Res. Commun. 205:320-326, 1994). In this report, phosphorylation of NS5A derived from HCV isolates of the 1a and distantly related 2a genotypes is demonstrated. Phosphoamino acid analysis of NS5A from the 1a isolate indicated that phosphorylation occurs predominantly on serine, with a minor fraction of threonine residues also being phosphorylated. NS5A phosphorylation was observed in diverse cell types, including COS-1, BHK-21, HeLa, and the hepatoma cell line HuH-7. Phosphorylation of a glutathione S-transferase (GST)/HCV-H NS5A fusion protein was also demonstrated in an in vitro kinase assay. This activity seemed to be highest when the pH of the reaction was neutral or slightly alkaline and displayed a preference for Mn2+ over Mg2+, with an optimum concentration of approximately 10 mM Mn2+. Somewhat surprisingly, in vitro phosphorylation of NS5A was inhibited by the addition of > or = 0.25 mM Ca2+ to reaction buffer containing Mn2+ and/or Mg2+. Comparison of phosphopeptide maps of NS5A phosphorylated in vitro and in cultured cells showed that most of the phosphopeptides comigrated, suggesting that one or more kinases involved in NS5A phosphorylation in vivo and in vitro are the same. The effects of various kinase inhibitors on NS5A phosphorylation were consistent with a kinase activity belonging to the CMGC group of serine-threonine kinases. The development of an in vitro kinase assay for NS5A phosphorylation should facilitate identification of kinase(s) responsible for its phosphorylation and of phosphorylation sites which may influence the function of NS5A in HCV propagation.
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PMID:Phosphorylation of the hepatitis C virus NS5A protein in vitro and in vivo: properties of the NS5A-associated kinase. 931 91


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