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)

To elucidate the role of the microsomal triglyceride transfer protein (MTP) in lipoprotein assembly, MTP and apolipoprotein B-53 (apoB 53; the N-terminal 53% of apoB) were expressed in HeLa cells. The results showed that apoB-53 could be expressed in HeLa cells with or without expression of MTP. In contrast, efficient secretion of apoB-53 required expression of MTP. Ultracentrifugal density flotation analysis showed that apoB-53 was secreted predominantly as a particle with the density of high density lipoprotein. An essentially identical apoB-53 particle density distribution was obtained after transient expression of apoB-53 in McArdle RH-7777 rat hepatoma cells. The mass of apoB-53 secreted was greater, and the flotation density was lower, from cells fed lipid, suggesting that apoB secretion in HeLa cells was regulated by lipid availability, similar to what has been described for lipoprotein-producing cell lines. These results indicate that MTP is necessary and sufficient to direct the regulated secretion of apoB-53 in HeLa cells.
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PMID:Secretion of apolipoprotein B-containing lipoproteins from HeLa cells is dependent on expression of the microsomal triglyceride transfer protein and is regulated by lipid availability. 805 32

We studied the role of microsomal triglyceride transfer protein (MTP) in the synthesis, secretion, and cotranslational degradation of apolipoprotein (apo) B using nonhepatic COS-7 cells that expressed C-terminally truncated forms of apoB (from apoB15 to apoB94) with or without the large subunit of human MTP. With the exception of apoB15 and apoB18, secretion of all of the apoB forms was stimulated by expression of MTP, even though a small amount of short apoB forms (</=apoB48) could be secreted by cells transfected with apoB alone. The majority of the apoB protein, including apoB72 and apoB94, was secreted as high density lipoprotein (1.08-1.17 g/ml). Pulse-chase experiments revealed that the secretion efficiency of apoB94 and apoB72 was low (ranging from 2 to 12%). The failure to secrete buoyant lipoproteins and the low secretion efficiency were associated with insufficient lipid synthesis by the cells. The incorporation of [3H]oleate into cellular triglyceride and phosphatidylcholine by COS cells over a 2-h period was 28 and 38%, respectively, of that by rat hepatoma (McA-RH7777) cells. In addition to the desired full-length apoB, cells transfected with large constructs (>/=apoB60) also produced smaller species with a size of approximately220 kDa (designated B48-like protein). Coexpression with MTP decreased formation of the B48-like proteins by 40-60%. The reduction in B48-like protein formation was specific to MTP expression; coexpression with other proteins (e.g. apoA-I or apoB15) did not alter B48-like protein production. Kinetic analysis suggested that B48-like proteins were produced concurrently (cotranslational) with the full-length apoB94 and apoB72 and were not products of post-translational degradation. Although some of the B48-like proteins might be derived from truncated species (approximately 7 kb in size) of apoB mRNA that were found in cells transfected with large apoB constructs, MTP coexpression did not affect the relative levels of the aberrant 7-kb RNA with respect to the full-length mRNA. However, coexpression of MTP decreased the accessibility of apoB to exogenous trypsin by 2-fold for apoB72 and by 10-fold for apoB94 in isolated microsomes. Thus, the reduced B48-like protein formation by MTP may be a consequence of attenuated cotranslational degradation during apoB translocation across the ER membrane. Formation of B48-like proteins was insensitive to N-acetyl-leucyl-leucyl-norleucinal, a cysteine protease inhibitor known to block post-translational degradation of apoB. These results indicate that MTP facilitates the assembly and secretion of lipoproteins containing apoB and also attenuates the formation of B48-like proteins, probably by assisting apoB translocation across the ER membrane.
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PMID:The microsomal triglyceride transfer protein facilitates assembly and secretion of apolipoprotein B-containing lipoproteins and decreases cotranslational degradation of apolipoprotein B in transfected COS-7 cells. 866 86

Apolipoprotein B (apoB), the major protein component of triglyceride-rich lipoproteins, is assembled into a lipoprotein particle via a complex, multistep process. Recent studies indicate that triglyceride-rich lipoprotein assembly requires the activity of the heterodimeric protein, microsomal triglyceride transfer protein (MTP). We identified a novel inhibitor of apolipoprotein B secretion using the human hepatoma cell line, HepG2. CP-10447, a derivative of the hypnotic drug methaqualone (Quaalude), inhibited apoB secretion from HepG2 cells with an IC50 of approximately 5 microM. CP-10447 also inhibited apoB secretion from Caco-2 cells, a model of intestinal lipoprotein production. In experiments using [3H]glycerol as a precursor for triglyceride synthesis, CP-10447 (20 microM) inhibited radiolabeled triglyceride secretion by approximately 83% (P < 0.0001) in HepG2 cells and 76% (P < 0.05) in Caco-2 cells with no effect on radiolabel incorporation into cellular triglyceride, indicating that CP-10447 inhibited triglyceride secretion without affecting triglyceride synthesis. RNA solution hybridization assay indicated that CP-10447 did not affect apoB or apoA-I mRNA levels. Pulse-chase experiments in HepG2 cells confirmed that CP-10447 inhibited the secretion of apoB (not its synthesis) without affecting secretion of total proteins or albumin and suggested that CP-10447 stimulates the early intracellular degradation of apoB in the endoplasmic reticulum (ER). Further studies demonstrated that CP-10447 is a potent inhibitor of human liver microsomal triglyceride transfer activity (IC50 approximately 1.7 microM) in an in vitro assay containing artificial liposomes and partially purified human MTP. These data suggest that CP-10447 may inhibit apoB and triglyceride secretion by inhibiting MTP activity and stimulating the early ER degradation of apoB. CP-10447 should provide a useful tool for further study of the mechanisms of apoB secretion and triglyceride-rich lipoprotein assembly.
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PMID:Inhibition of apolipoprotein B and triglyceride secretion in human hepatoma cells (HepG2). 882 19

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

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

1. Apolipoprotein B (apoB) is necessary for the assembly and secretion of both chylomicrons from the small intestine and very low-density lipoproteins (VLDL) from the liver. ApoB is also the major protein in low-density lipoproteins (LDL) and is the ligand for the LDL receptor. Studies in humans suggest that increased production of apoB-containing lipoproteins, particularly VLDL, is a common abnormality in dyslipidaemias. 2. Studies in primary and long-term cultures of hepatocytes and hepatoma cells indicate that a significant proportion of newly synthesized apoB is rapidly degraded and that this is the major mechanism for regulation of apoB secretion. The availability of newly synthesized lipids, particularly triglyceride and cholesteryl ester, appears to be a critical factor in targeting apoB for secretion rather than degradation. 3. ApoB is an atypical secretory protein in that cotranslational translocation across the endoplasmic reticulum membrane, a feature of all secretory proteins, seems to slow or stop in the absence of adequate lipid availability (or in the absence of microsomal triglyceride transfer protein), allowing for rapid degradation of apoB. 4. The degradation of apoB seems to be facilitated by the association of nascent apoB with the major cytosolic chaperone protein, heat shock protein 70. Additionally, degradation of nascent apoB appears to occur, to a large degree, via the proteasomal pathway for degradation of cytosolic proteins.
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PMID:Role of lipid synthesis, chaperone proteins and proteasomes in the assembly and secretion of apoprotein B-containing lipoproteins from cultured liver cells. 914 94

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

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(a) (apo(a)) is synthesized and secreted from liver cells and represents one of the two major protein components of the atherogenic lipoprotein, Lp(a). Little is known, however, of the factors that regulate the secretion of this protein. We have undertaken an analysis of the response to oleate supplementation in stable clones of HepG2 and McA-RH7777 cells expressing either a 6 K-IV or 17 K-IV isoform of apo(a). These cell lines were examined by pulse-chase analysis and each demonstrated an increase (range 2-6-fold) in apo(a) secretion following supplementation with 0.8 mM oleate. Microsomal membranes, prepared from HepG2 cells expressing a 6 K-IV apo(a) isoform, demonstrated that oleate supplementation increased the apparent protection of apo(a) from protease digestion, suggesting that alterations in the translocation efficiency of apo(a) may accompany the addition of oleate. Cells incubated with brefeldin A demonstrated increased recovery of the precursor form of apo(a) with oleate supplementation, suggesting that alterations in post-translational degradation may also contribute to the observed increase in apo(a) secretion following oleate addition. To further characterize the oleate-dependent increase in apo(a) secretion, cells were incubated with an inhibitor of the microsomal triglyceride transfer protein. These experiments demonstrated a dose-dependent decrease in apo(a) secretion from both cell lines. Furthermore, addition of either the microsomal triglyceride transfer protein inhibitor or triacsin C, an inhibitor of acyl-CoA synthase, completely abrogated the oleate-dependent increase in apo(a) secretion. Taken together, these data provide evidence that apo(a) secretion from hepatoma cells may be linked to elements of cellular triglyceride assembly and secretion.
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PMID:Apolipoprotein(a) synthesis and secretion from hepatoma cells is coupled to triglyceride synthesis and secretion. 965 81

The microsomal triglyceride transfer protein (MTP) is a heterodimeric lipid transfer protein that is required for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins. A key unresolved question is whether the MTP-mediated step is rate limiting. To address this, a unique experimental strategy was used that allowed the in situ modulation and measurement of MTP triglyceride transfer activity. In order to accomplish this, an irreversible photoaffinity inhibitor, BMS-192951, was designed and synthesized. When incubated with purified MTP and irradiated with UV light at 360 nm, BMS-192951 inhibits triglyceride transfer by covalently binding to the protein. HepG2 cells were treated with either increasing concentrations of BMS-192951 (0-15 microM) with 5 min of ultraviolet irradiation, or 3.0 microM BMS-192951 with various lengths (0-15 min) of ultraviolet irradiation. Microsomal extracts were prepared exhaustively dialyzed to remove unbound inhibitor, and assayed for MTP-mediated triglyceride transfer activity. BMS-192951 was shown to reduce MTP activity in both a dose- and UV exposure time-dependent fashion. Measurement of apoB concentration in the media showed that apoB secretion was reduced in proportion to the in situ inhibition of MTP activity, while no change was observed in apoA-I secretion. Experiments performed in McArdle RH-7777 rat hepatoma cells and primary rat hepatocytes gave nearly identical results; the decrease in apoB secretion was proportional to the decrease in MTP activity. These results indicate that MTP-mediated lipid transfer is limiting in the assembly and secretion of apoB-containing lipoproteins in hepatic cells under the conditions tested.
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PMID:Evidence that microsomal triglyceride transfer protein is limiting in the production of apolipoprotein B-containing lipoproteins in hepatic cells. 968 48

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