Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Apolipoprotein (apo) B mRNA editing protein is an essential catalytic component of the apoB mRNA editing enzyme complex. Its cDNA has been cloned recently from rats and humans. In the presence of other proteins of the editing enzyme complex, it will deaminate nucleotide 6666 in apoB mRNA, a cytidine residue, converting it to uridine. The end result of this reaction is the production of apoB-48 in place of apoB-100. The editing protein exists as a homodimer. It imparts editing activity to HepG2, a human hepatoma cell line, causing these cells to start producing apoB-48 in addition to apoB-100. Therefore, it can be used as a therapeutic agent to reduce apoB-100 production. Preliminary experiments in our laboratory indicate that somatic gene transfer of the editing protein is highly effective in lowering plasma low density lipoproteins.
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PMID:Apolipoprotein B mRNA editing protein: a tool for dissecting lipoprotein metabolism and a potential therapeutic gene for hypercholesterolemia. 876 53

There is evidence that the overproduction of apoB-100-containing lipoproteins by the liver is the underlying event in some forms of dyslipoproteinemia. This metabolic status is associated to an increased risk of developing premature coronary artery disease CAD. The conclusions from previous studies suggested that the availability to the hepatocytes of cholesterol that is readily esterified is an important determinant for VLDL and LDL secretion. In the present study, we set out to investigate the effect of the specific stimulation and inhibition of the rate-limiting enzyme of the cholesterol esterification, acyl-CoA:cholesterol acyltransferase (ACAT, E.C. 2.3.1.26), on the lipid and on the apoB-100 secretion rate from a human hepatoma cell line (HepG2). When the specific ACAT inhibitor FCE 27677 (10-5 M) was added to the cultures, a decrease of the cellular cholesteryl ester content and at the same time a significant reduction of the neutral lipids and of the apoB-100 secretion rate were noticed. The stimulation of ACAT by 25-hydroxycholesterol (20 microgram/ml) caused a 4-fold increase of the cellular cholesteryl ester content and a 2-fold increase of the lipoprotein secretion rate. FCE 27677 (10-5 M to 10-7 M) prevented the effects elicited by the oxysterol. On the contrary, lovastatin (10-6 M) and gemfibrozil (10-6 M) had no effect. The analysis of the lipid and of the apolipoprotein composition of the lipoproteins secreted in the medium revealed that ACAT inhibition had the dual effect of both decreasing the number of apoB-100-containing lipoproteins secreted as well as their cholesteryl ester load. Altogether, these data support the idea of a close relationship between ACAT activation, leading to increased cholesteryl ester availability, and apoB-100-containing lipoprotein secretion. It is speculated that ACAT inhibitors may prove useful for the treatment of human dyslipoproteinemias caused by the hepatic overproduction of apoB-100-containing lipoproteins.
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PMID:Inhibition of acyl-CoA: cholesterol acyltransferase decreases apolipoprotein B-100-containing lipoprotein secretion from HepG2 cells. 882 97

The microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of the lipoproteins containing apolipoprotein B (apoB): very low density lipoproteins and chylomicrons. Evidence indicates that the subclasses of these lipoproteins that contain apoB-48 are assembled in a distinct two-step process; first a relatively lipid-poor primordial lipoprotein precursor is produced, and then bulk neutral lipids are added to form the core of these spherical particles. To determine if either step is mediated by MTP, a series of clonal cell lines stably expressing apoB-53 and MTP was established in non-lipoprotein-producing HeLa cells. MTP activity in these cells was approximately 30%, and apoB secretion was 7-33% of that in HepG2 cells on a molar basis. Despite having robust levels of triglyceride and phospholipid synthesis, these cell lines, as exemplified by HLMB53-59, secreted >90% of the apoB-53 on relatively lipid-poor particles in the density range of 1.063-1.21 g/ml. These results suggested that coexpression of MTP and apoB only reconstituted the first but not the second step in lipoprotein assembly. To extend this observation, additional studies were carried out in McArdle RH-7777 rat hepatoma cells, in which the second step of apoB-48 lipoprotein assembly is well defined. Treatment of these cells with the MTP photoaffinity inhibitor BMS-192951 before pulse labeling with [35S]methionine/cysteine led to an 85% block of both apoB-48 and apoB-100 but not apoAI secretion, demonstrating inhibition of the first step of lipoprotein assembly. After a 30-min [35S]methioneine/cysteine pulse labeling and 120 min of chase, all of the nascent apoB-48 was observed to have a density of high density lipoproteins (1.063-1.21 g/ml), indicating that only the first step of lipoprotein assembly had occurred. The addition of oleic acid to the cell culture media activated the second step as evidenced by the conversion of the apoB-48 high density lipoproteins to very low density lipoproteins (d < 1.006 g/ml) during an extended chase period. Inactivation of MTP after completion of the first step, but before stimulation of the second step by the addition of oleic acid, did not block this conversion. Thus, inhibition of MTP did not hinder the addition of bulk core lipid to the primordial lipoprotein precursor particles, indicating that MTP is not required for the second step of apoB-48 lipoprotein assembly.
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PMID:Inhibition of the microsomal triglyceride transfer protein blocks the first step of apolipoprotein B lipoprotein assembly but not the addition of bulk core lipids in the second step. 895 51

Apolipoprotein(a) (apo(a)), a large glycoprotein with extensive homology to plasminogen, forms a complex with apolipoprotein B100 (apoB100), which circulates in human plasma in the form of lipoprotein(a) (Lp(a)). Evidence indicates that the association of apo(a) with apoB100 occurs in the extracellular environment. We have reevaluated the possibility that apo(a)-B100 association can also occur as an intracellular event through studies with HepG2 cells stably transfected with an apo(a) minigene. Several lines of evidence support this possibility. First, continued Lp(a) production was demonstrated following incubation of transfected HepG2 cells with anti-apo(a) antisera, conditions that effectively block the fluid-phase association of apo(a) and apoB100 in vitro. Second, an apo(a)-B100 complex was detectable in Western blot analyses of transfected HepG2 lysates following immunoprecipitation with anti-apo(a) antisera. These studies incorporated precautions to eliminate cell-surface attachment of preformed apo(a)-B100 complexes to the low density lipoprotein receptor and were conducted in the presence of the lysine analog epsilon-aminocaproic acid, which precludes apo(a)-B100 association occurring during the isolation and analyses. Third, the presence of an intracellular apo(a)-B100 complex was demonstrated in lipoproteins isolated from microsomal contents. Of particular significance was the observation that this complex contained the precursor form of apo(a), which is not secreted, in addition to the mature, recombinant form. Finally, direct evidence was provided for the synthesis of a precursor form of apo(a) in a nascent intracellular complex with apoB100 following treatment of transfected HepG2 cells with brefeldin A plus N-acetyl-leucyl-leucyl-norleucinal. Taken together, these data suggest that apo(a)-B100 association can occur as an intracellular event in a human hepatoma-derived cell line, raising important implications for the regulation of Lp(a) secretion from human liver.
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PMID:Expression of a recombinant apolipoprotein(a) in HepG2 cells. Evidence for intracellular assembly of lipoprotein(a). 903 76

The initial assembly of apolipoprotein B100 (apoB) into lipoprotein particles occurs cotranslationally. To examine steps required to initiate this process, the intracellular folding and assembly of the amino-terminal 28% of apoB (apoB28) was examined using several criteria including nonreducing gel electrophoresis, sensitivity to dithiothreitol (DTT)-mediated reduction, and buoyant density gradient centrifugation. In hepatoma cells, after a 1-min pulse with radiolabeled amino acids, labeled apoB28 migrated during gel electrophoresis in the folded position and was resistant to reduction in vivo with 2 mM DTT. A similar rate and extent of folding was observed in Chinese hamster ovary cells, a microsomal triglyceride transfer protein (MTP)-negative cell line that can neither lipidate nor efficiently secrete apoB28. Amino-terminal folding of apoB28 was essential for its subsequent intracellular lipidation as apoB28 synthesized in hepatoma cells under reducing conditions remained lipid poor (d > 1.25 g/ml) and was retained intracellularly. Upon DTT removal, reduced apoB28 underwent a process of rapid (t1/2 approximately 2 min) post-translational folding followed by a slower process of MTP-dependent lipidation. As with the cotranslational assembly pathway, post-translational lipidation of apoB28 displayed a strict dependence upon amino-terminal folding. We conclude that: 1) folding of the amino-terminal disulfide bonded domain of apoB is achieved prior to the completion of translation and is independent of MTP and events associated with buoyant lipoprotein formation and 2) domain-specific folding of apoBs amino-terminal region is required to initiate MTP-dependent lipid transfer to nascent apoB in the hepatic endoplasmic reticulum.
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PMID:Folding of the amino-terminal domain of apolipoprotein B initiates microsomal triglyceride transfer protein-dependent lipid transfer to nascent very low density lipoprotein. 909 79

The microsomal triglyceride transfer protein is necessary for the assembly and secretion of lipoproteins containing apolipoprotein B. During the past year, significant progress has been made towards understanding the role of microsomal triglyceride transfer protein in lipoprotein assembly at both a cellular and molecular level. Studies carried out in a variety of heterologous expression systems, as well as the use of microsomal triglyceride transfer protein inhibitors in hepatoma cell lines, have been critical to this progress. It has been shown that microsomal triglyceride transfer protein plays a key role in the early stages of lipoprotein assembly, most likely by transferring lipid to nascent apolipoprotein B as it enters the lumen of the endoplasmic reticulum. The evidence indicates that microsomal triglyceride transfer protein does not play a major role in addition of bulk core lipid in the late stages of apolipoprotein B48 lipoprotein assembly. Thus, microsomal triglyceride transfer protein appears to control the number of apolipoprotein B lipoprotein particles secreted but not the lipid composition.
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PMID:Recent advances in elucidating the role of the microsomal triglyceride transfer protein in apolipoprotein B lipoprotein assembly. 921 Oct 60

ApolipoproteinB (apoB) mRNA editing involves a C to U deamination of the nuclear apoB mRNA and occurs in mammalian small intestine and in the liver of certain species. This reaction is mediated by a multicomponent enzyme complex that includes a catalytic subunit, apobec-1. Apobec-1 mRNA is widely expressed in the rat and mouse and is subject to tissue-specific regulation. In order to understand the basis for the species- and tissue-specific pattern of apobec-1 gene expression we have cloned and characterized the rat chromosomal apobec-1 gene. We demonstrate its structural organization and regulation in comparison to that of the mouse apobec-1 gene. The rat apobec-1 gene spans 16 kb and includes one untranslated (exon A) and five translated exons (exons 1-5). The mouse apobec-1 gene contains eight exons, of which the first three (exons A, B, C) are untranslated. Independent approaches demonstrated three distinct clusters of transcription initiation sites in both species, including exon A, the distal region of exon 1, and a separate group in the proximal region of exon 1. These transcription start sites generate three distinct mRNA species whose proportions differ in a tissue-specific fashion. Promoter-luciferase reporter constructions using regions flanking exon A and exon 1 of the rat apobec-1 gene identified two functional regions upstream of exon 1 that independently promote luciferase expression in transfected hepatoma and colon cancer cells. These data serve as a basis for an understanding of the regulation of apobec-1 gene expression, in particular the mechanisms that serve to restrict its expression to the gastrointestinal tract in higher mammals.
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PMID:Cloning and characterization of the rat apobec-1 gene: a comparative analysis of gene structure and promoter usage in rat and mouse. 921 39

Stable plasmid-driven expression of the liver-specific gene product cholesterol 7alpha-hydroxylase (7alpha-hydroxylase) was used to alter the cellular content of transcriptionally active sterol response element binding protein 1 (SREBP1). As a result of stable expression of 7alpha-hydroxylase, individual single cell clones expressed varying amounts of mature SREBP1 protein. These single cell clones provided an opportunity to identify SREBP1-regulated genes that may influence the assembly and secretion of apoB-containing lipoproteins. Our results show that in McArdle rat hepatoma cells, which normally do not express 7alpha-hydroxylase, plasmid-driven expression of 7alpha-hydroxylase results in the following: 1) a linear relationship between (i) the cellular content of mature SREBP1 and 7alpha-hydroxylase protein, (ii) the relative expression of 7alpha-hydroxylase mRNA and the mRNA's encoding the enzymes regulating fatty acid, i.e. acetyl-CoA carboxylase and sterol synthesis, i.e. HMG-CoA reductase, (iii) the relative expression of 7alpha-hydroxylase mRNA and microsomal triglyceride transfer protein mRNA, a gene product that is essential for the assembly and secretion of apoB-containing lipoproteins; 2) increased synthesis of all lipoprotein lipids (cholesterol, cholesterol esters, triglycerides, and phospholipids); and 3) increased secretion of apoB100 without any change in apoB mRNA. Cells expressing 7alpha-hydroxylase contained significantly less cholesterol (both free and esterified). The increased cellular content of mature SREBP1 and increased secretion of apoB100 were concomitantly reversed by 25-hydroxycholesterol, suggesting that the content of mature SREBP1, known to be decreased by 25-hydroxycholesterol, mediates the changes in the lipoprotein assembly and secretion pathway that are caused by 7alpha-hydroxylase. These data suggest that several steps in the assembly and secretion of apoB-containing lipoproteins by McArdle hepatoma cells may be coordinately linked through the cellular content of mature SREBP1.
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PMID:Coordinate regulation of lipogenesis, the assembly and secretion of apolipoprotein B-containing lipoproteins by sterol response element binding protein 1. 923 33

Apolipoprotein B (apoB) mRNA editing catalyzed by apoB mRNA editing catalytic subunit 1 (APOBEC-1) has been proposed to be a nuclear process. To test this hypothesis, the subcellular distribution of hemagglutinin- (HA) tagged APOBEC-1 expressed in transiently transfected hepatoma cells was determined by indirect immunofluorescence microscopy. HA-APOBEC-1 was detected in both the nucleus and cytoplasm of rat and human hepatoma cells. Mutagenesis of APOBEC-1 demonstrated that the N-terminal 56 amino acids (1-56) were necessary for the nuclear distribution of APOBEC-1, but this region did not contain a functional nuclear localization signal (NLS). However, we identified a 24-amino acid domain in the C terminus of APOBEC-1 with characteristics of a cytoplasmic retention signal (CRS) or a nuclear export signal (NES). These data suggest, therefore, that the nuclear import of APOBEC-1 may not be mediated by a positive NLS; rather, it may be achieved by overcoming the effect of a CRS/NES. We also demonstrated that the nuclear distribution of APOBEC-1 occurred only in cell lines that were capable of editing apoB RNA. We propose that the cellular distribution of APOBEC-1 is determined by multiple domains within this protein, and a nuclear localization of the enzyme may be regulated by cell type-specific factors that render these cells uniquely editing competent.
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PMID:Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apolipoprotein B mRNA editing. 937 2

Apolipoprotein B (apoB) RNA editing involves a cytidine to uridine transition at nucleotide 6666 (C6666) 5' of an essential cis -acting 11 nucleotide motif known as the mooring sequence. APOBEC-1 (apoB editing catalytic sub-unit 1) serves as the site-specific cytidine deaminase in the context of a multiprotein assembly, the editosome. Experimental over-expression of APOBEC-1 resulted in an increased proportion of apoB mRNAs edited at C6666, as well as editing of sites that would otherwise not be recognized (promiscuous editing). In the rat hepatoma McArdle cell line, these sites occurred predominantly 5' of the mooring sequence on either rat or human apoB mRNA expressed from transfected cDNA. In comparison, over-expression of APOBEC-1 in HepG2 (HepG2-APOBEC) human hepatoma cells, induced promiscuous editing primarily 5' of the mooring sequence, but sites 3' of the C6666 were also used more efficiently. The capacity for promiscuous editing was common to rat, rabbit and human sources of APOBEC-1. The data suggested that differences in the distribution of promiscuous editing sites and in the efficiency of their utilization may reflect cell-type-specific differences in auxiliary proteins. Deletion of the mooring sequence abolished editing at the wild type site and markedly reduced, but did not eliminate, promiscuous editing. In contrast, deletion of a pair of tandem UGAU motifs 3' of the mooring sequence in human apoB mRNA selectively reduced promiscuous editing, leaving the efficiency of editing at the wild type site essentially unaffected. ApoB RNA constructs and naturally occurring mRNAs such as NAT-1 (novel APOBEC-1 target-1) that lack this downstream element were not promiscuously edited in McArdle or HepG2 cells. These findings underscore the importance of RNA sequences and the cellular context of auxiliary factors in regulating editing site utilization.
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PMID:Apolipoprotein B RNA sequence 3' of the mooring sequence and cellular sources of auxiliary factors determine the location and extent of promiscuous editing. 951 34


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