The distal enhancer implicated in the developmental regulation of the tyrosine aminotransferase gene is bound by liver-specific and ubiquitous factors

1993 ◽  
Vol 13 (8) ◽  
pp. 4494-4504
Author(s):  
D Nitsch ◽  
G Schütz
Keyword(s):  
Thymidine Kinase ◽  
Specific Binding ◽  
Regulatory Element ◽  
Hepatoma Cell ◽  
Tyrosine Aminotransferase ◽  
Hepatoma Cells ◽  
Hepatoma Cell Line ◽  
Distal Enhancer ◽  
Binding Studies ◽  
Enhancer Sequences

Tyrosine aminotransferase gene expression is confined to parenchymal cells of the liver, is inducible by glucocorticoids and glucagon, and is repressed by insulin. Three enhancers control this tissue-specific and hormone-dependent activity, one of which, located at -11 kb, is implicated in establishing an active expression domain. We have studied in detail this important regulatory element and have identified a 221-bp fragment containing critical enhancer sequences which stimulated the heterologous thymidine kinase promoter more than 100-fold in hepatoma cells. Within this region, we have characterized two essential liver-specific enhancer domains, one of which was bound by proteins of the hepatocyte nuclear factor 3 (HNF3) family. Analyses with the dedifferentiated hepatoma cell line HTC suggested that HNF3 alpha and/or -gamma, but not HNF3 beta, are involved in activating the tyrosine aminotransferase gene via the -11-kb enhancer. Genomic footprinting and in vitro protein-DNA binding studies documented cell-type-specific binding of ubiquitous factors to the second essential enhancer domain, which by itself stimulated the thymidine kinase promoter preferentially in hepatoma cells. These results will allow further characterization of the role of these enhancer sequences in developmental activation of the tyrosine aminotransferase gene.

scholarly journals The distal enhancer implicated in the developmental regulation of the tyrosine aminotransferase gene is bound by liver-specific and ubiquitous factors.

1993 ◽  
Vol 13 (8) ◽  
pp. 4494-4504 ◽  
Author(s):  
D Nitsch ◽  
G Schütz
Keyword(s):  
Thymidine Kinase ◽  
Specific Binding ◽  
Regulatory Element ◽  
Hepatoma Cell ◽  
Tyrosine Aminotransferase ◽  
Hepatoma Cells ◽  
Hepatoma Cell Line ◽  
Distal Enhancer ◽  
Binding Studies ◽  
Enhancer Sequences

Tyrosine aminotransferase gene expression is confined to parenchymal cells of the liver, is inducible by glucocorticoids and glucagon, and is repressed by insulin. Three enhancers control this tissue-specific and hormone-dependent activity, one of which, located at -11 kb, is implicated in establishing an active expression domain. We have studied in detail this important regulatory element and have identified a 221-bp fragment containing critical enhancer sequences which stimulated the heterologous thymidine kinase promoter more than 100-fold in hepatoma cells. Within this region, we have characterized two essential liver-specific enhancer domains, one of which was bound by proteins of the hepatocyte nuclear factor 3 (HNF3) family. Analyses with the dedifferentiated hepatoma cell line HTC suggested that HNF3 alpha and/or -gamma, but not HNF3 beta, are involved in activating the tyrosine aminotransferase gene via the -11-kb enhancer. Genomic footprinting and in vitro protein-DNA binding studies documented cell-type-specific binding of ubiquitous factors to the second essential enhancer domain, which by itself stimulated the thymidine kinase promoter preferentially in hepatoma cells. These results will allow further characterization of the role of these enhancer sequences in developmental activation of the tyrosine aminotransferase gene.

Activation of the silent endogenous cholesterol-7-alpha-hydroxylase gene in rat hepatoma cells: a new complementation group having resistance to 25-hydroxycholesterol

1991 ◽  
Vol 11 (4) ◽  
pp. 2049-2056
Author(s):  
J K Leighton ◽  
S Dueland ◽  
M S Straka ◽  
J Trawick ◽  
R A Davis
Keyword(s):  
Cell Line ◽  
Cholesterol Biosynthesis ◽  
Hepatoma Cell ◽  
Regulatory Elements ◽  
Hepatoma Cells ◽  
Complementation Group ◽  
Hepatoma Cell Line ◽  
Cytotoxic Action ◽  
Hydroxylase Activity ◽  
Rat Hepatoma

The oxysterol 25-hydroxycholesterol acts both as a regulatory sterol determining the expression of genes governed by sterol regulatory elements and as a substrate for 7-alpha-hydroxylase, the first and rate-limiting enzyme in the bile acid synthetic pathway. Most wild-type nonhepatic cells are killed by the cytotoxic action of 25-hydroxycholesterol. In contrast, liver cells, which express 7-alpha-hydroxylase activity, are resistant to killing by 25-hydroxycholesterol. We examined the possibility that selection for resistance to 25-hydroxycholesterol might lead to the derivation of a cell line expressing 7-alpha-hydroxylase. A rat hepatoma cell line (7-alpha-hydroxylase minus) was transfected with human DNA and screened for resistance to 25-hydroxycholesterol. Although parental hepatoma cells were all killed within a week, a 25-hydroxycholesterol-resistant cell line (L35 cells) which showed stable expression of 7-alpha-hydroxylase activity and mRNA was obtained. These cells exhibited normal inhibition of cholesterol biosynthesis by 25-hydroxycholesterol. Blocking 7-alpha-hydroxylase activity with ketoconazole also blocked the resistance of L35 cells to 25-hydroxycholesterol. Isolation of microsomes from these cells showed levels of 7-alpha-hydroxylase activity (22.9 pmol/min/mg of protein) that were comparable to the activity (33.2 pmol/min/mg) of microsomes isolated from the livers of rats killed during the high point of the diurnal cycle. Parental cells had no detectable activity. These data show a new complementation group for 25-hydroxycholesterol resistance: expression of 7-alpha-hydroxylase. Dexamethasone increased both the activity and the cellular content of mRNA coding for 7-alpha-hydroxylase. Since dactinomycin blocked the ability of dexamethasone to induce mRNA, active transcription is required. Southern analysis of genomic DNA showed that L35 cells contain the rat (endogenous) gene but not the human gene. Furthermore, the RNA expressed by L35 cells is similar in size to rat RNA and is distinct from the human form of 7-alpha-hydroxylase. The combined data indicate that L35 cells are resistant to 25-hydroxycholesterol because they express 7-alpha-hydroxylase. The mechanism responsible involves activation of the endogenous (silent) gene of the parental rat hepatoma cell.

Inhibitory Effects of Lycopene on the Adhesion, Invasion, and Migration of SK-Hep1 Human Hepatoma Cells

2006 ◽  
Vol 231 (3) ◽  
pp. 322-327 ◽  
Author(s):  
Eun-Sun Hwang ◽  
Hyong Joo Lee
Keyword(s):  
Cell Growth ◽  
Hepatoma Cell ◽  
Hepatoma Cells ◽  
Hepatoma Cell Line ◽  
Dependent Manner ◽  
Human Hepatoma ◽  
Human Hepatoma Cells ◽  
Human Hepatoma Cell ◽  
Invasion And Migration ◽  
And Migration

Lycopene, which is the predominant carotenoid in tomatoes and tomato-based foods, may protect humans against various cancers. Effects of lycopene on the adhesion, invasion, migration, and growth of the SK-Hep1 human hepatoma cell line were investigated. Lycopene inhibited cell growth in dose-dependent manners, with growth inhibition rates of 5% and 40% at 0.1 μM and 50 μM lycopene, respectively, after 24 hrs of incubation. Similarly, after 48 hrs of incubation, lycopene at 5 μM and 10 μM decreased the cell numbers by 30% and 40%, respectively. Lycopene decreased the gelatinolytic activities of both matrix metalloproteinase (MMP)-2 and MMP-9, which were secreted from the SK-Hep1 cells. Incubation of SK-Hep1 cells with 110 μM of lycopene for 60 mins significantly inhibited cell adhesion to the Matrigel-coated substrate in a concentration-dependent manner. To study invasion, SK-Hep1 cells were grown either on Matrigel-coated Transwell membranes or in 24-well plates. The cells were treated sequentially for 24 hrs with lycopene before the start of the invasion assays. Cell growth and death were assessed under the same conditions. The invasion of SK-Hep1 cells treated with lycopene was significantly reduced to 28.3% and 61.9% of the control levels at 5 μM and 10 μM lycopene, respectively (P < 0.05). In the migration assay, lycopene-treated cells showed lower levels of migration than untreated cells. These results demonstrate the antimetastatic properties of lycopene in inhibiting the adhesion, invasion, and migration of SK-Hep1 human hepatoma cells.

Simultaneous expression of salivary and pancreatic amylase genes in cultured mouse hepatoma cells

1986 ◽  
Vol 6 (4) ◽  
pp. 969-975
Author(s):  
G J Darlington ◽  
C C Tsai ◽  
L C Samuelson ◽  
D L Gumucio ◽  
M H Meisler
Keyword(s):  
Hepatoma Cell ◽  
Experimental System ◽  
Hepatoma Cells ◽  
Hepatoma Cell Line ◽  
Cell Type ◽  
Specific Expression ◽  
Consensus Sequences ◽  
Mouse Hepatoma ◽  
Amylase Genes

The tissue-specific expression of two types of mouse amylase genes does not overlap in vivo; the Amy-1 locus is transcribed in the parotid gland and the liver, while expression of Amy-2 is limited to the pancreas. We identified a mouse hepatoma cell line, Hepa 1-6, in which both amylase genes can be simultaneously expressed. Amy-1 is constitutively active in these cells and is inducible by dexamethasone at the level of mRNA. We demonstrated that the liver-specific promoter of Amy-1 is utilized by the dexamethasone-treated hepatoma cells, and that glucocorticoid consensus sequences are present upstream of this promoter. Amy-2 is not detectable constitutively, but can be activated if the cells are cultured in serum-free medium containing dexamethasone. Expression of Amy-2 in a nonpancreatic cell type has not previously been observed. We speculate that induction of Amy-1 and activation of Amy-2 may involve different regulatory mechanisms. Hepa 1-6 cells provide an experimental system for molecular analysis of these events.

scholarly journals Activation of the silent endogenous cholesterol-7-alpha-hydroxylase gene in rat hepatoma cells: a new complementation group having resistance to 25-hydroxycholesterol.

1991 ◽  
Vol 11 (4) ◽  
pp. 2049-2056 ◽  
Author(s):  
J K Leighton ◽  
S Dueland ◽  
M S Straka ◽  
J Trawick ◽  
R A Davis
Keyword(s):  
Cell Line ◽  
Cholesterol Biosynthesis ◽  
Hepatoma Cell ◽  
Regulatory Elements ◽  
Hepatoma Cells ◽  
Complementation Group ◽  
Hepatoma Cell Line ◽  
Cytotoxic Action ◽  
Hydroxylase Activity ◽  
Rat Hepatoma

The oxysterol 25-hydroxycholesterol acts both as a regulatory sterol determining the expression of genes governed by sterol regulatory elements and as a substrate for 7-alpha-hydroxylase, the first and rate-limiting enzyme in the bile acid synthetic pathway. Most wild-type nonhepatic cells are killed by the cytotoxic action of 25-hydroxycholesterol. In contrast, liver cells, which express 7-alpha-hydroxylase activity, are resistant to killing by 25-hydroxycholesterol. We examined the possibility that selection for resistance to 25-hydroxycholesterol might lead to the derivation of a cell line expressing 7-alpha-hydroxylase. A rat hepatoma cell line (7-alpha-hydroxylase minus) was transfected with human DNA and screened for resistance to 25-hydroxycholesterol. Although parental hepatoma cells were all killed within a week, a 25-hydroxycholesterol-resistant cell line (L35 cells) which showed stable expression of 7-alpha-hydroxylase activity and mRNA was obtained. These cells exhibited normal inhibition of cholesterol biosynthesis by 25-hydroxycholesterol. Blocking 7-alpha-hydroxylase activity with ketoconazole also blocked the resistance of L35 cells to 25-hydroxycholesterol. Isolation of microsomes from these cells showed levels of 7-alpha-hydroxylase activity (22.9 pmol/min/mg of protein) that were comparable to the activity (33.2 pmol/min/mg) of microsomes isolated from the livers of rats killed during the high point of the diurnal cycle. Parental cells had no detectable activity. These data show a new complementation group for 25-hydroxycholesterol resistance: expression of 7-alpha-hydroxylase. Dexamethasone increased both the activity and the cellular content of mRNA coding for 7-alpha-hydroxylase. Since dactinomycin blocked the ability of dexamethasone to induce mRNA, active transcription is required. Southern analysis of genomic DNA showed that L35 cells contain the rat (endogenous) gene but not the human gene. Furthermore, the RNA expressed by L35 cells is similar in size to rat RNA and is distinct from the human form of 7-alpha-hydroxylase. The combined data indicate that L35 cells are resistant to 25-hydroxycholesterol because they express 7-alpha-hydroxylase. The mechanism responsible involves activation of the endogenous (silent) gene of the parental rat hepatoma cell.

scholarly journals NPC1L1 (Niemann–Pick C1-like 1) mediates sterol-specific unidirectional transport of non-esterified cholesterol in McArdle-RH7777 hepatoma cells

2007 ◽  
Vol 406 (2) ◽  
pp. 273-283 ◽  
Author(s):  
J. Mark Brown ◽  
Lawrence L. Rudel ◽  
Liqing Yu
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Free Cholesterol ◽  
Hepatoma Cell ◽  
Hepatoma Cells ◽  
Cholesterol Depletion ◽  
Hepatoma Cell Line ◽  
Cholesterol Uptake ◽  
Sterol Transport ◽  
Niemann Pick

Recent evidence suggests that NPC1L1 (Niemann–Pick C1-like 1) is critical for intestinal sterol absorption in mice, yet mechanisms by which NPC1L1 regulates cellular sterol transport are lacking. In the study we used a McArdle-RH7777 rat hepatoma cell line stably expressing NPC1L1 to examine the sterol-specificity and directionality of NPC1L1-mediated sterol transport. As previously described, cholesterol-depletion-driven recycling of NPC1L1 to the cell surface facilitates cellular uptake of non-esterified (free) cholesterol. However, it has no impact on the uptake of esterified cholesterol, indicating free sterol specificity. Interestingly, the endocytic recycling of NPC1L1 was also without effect on β-sitosterol uptake, indicating that NPC1L1 can differentiate between free sterols of animal and plant origin in hepatoma cells. Furthermore, NPC1L1-driven free cholesterol transport was unidirectional, since cellular cholesterol efflux to apolipoprotein A-I, high-density lipoprotein or serum was unaffected by NPC1L1 expression or localization. Additionally, NPC1L1 facilitates mass non-esterified-cholesterol uptake only when it is located on the cell surface and not when it resides intracellularly. Finally, NPC1L1-dependent cholesterol uptake required adequate intracellular K+, yet did not rely on intracellular Ca2+, the cytoskeleton or signalling downstream of protein kinase A, protein kinase C or pertussis-toxin-sensitive G-protein-coupled receptors. Collectively, these findings support the notion that NPC1L1 can selectively recognize non-esterified cholesterol and promote its unidirectional transport into hepatoma cells.

Vitronetcin promotes cell growth and inhibits apoptotic stimuli in a human hepatoma cell line via the activation of caspases

2014 ◽  
Vol 92 (5) ◽  
pp. 363-368 ◽  
Author(s):  
Wei Zhu ◽  
Yingzhi Liu ◽  
Konghe Hu ◽  
Wenxue Li ◽  
Jianling Chen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Tumor Cell ◽  
Hepatoma Cell ◽  
Hepatoma Cells ◽  
Signaling Molecules ◽  
Hepatoma Cell Line ◽  
Dependent Manner ◽  
Tumor Cell Growth ◽  
Induced Apoptosis

This study sought to understand the effects of vitronectin (VTN) on the growth of SMMC-7721 hepatoma cells. In addition, this study examined how VTN inhibits the induction of apoptosis in SMMC-7721 cells by 3,3′-diindolylmethane (DIM), a metabolite of natural phytochemicals, and preliminarily investigated the signaling molecules involved in this process. A cell proliferation reagent was used to observe the effects of VTN on cell proliferation rates. Laser scanning confocal microscopy was performed to observe the effects of VTN on the morphology of tubulin, a component of the cytoskeleton. Flow cytometry and Western blotting assays were used to observe the inhibitory effects of VTN on DIM-induced apoptosis in SMMC-7721 cells and changes in the expression levels of the signaling molecules involved in this process. VTN promoted tumor cell growth in a concentration-dependent manner and inhibited apoptosis caused by the effects of apoptosis-inducing agents. Under in vitro experimental conditions, VTN contributed to the growth of SMMC-7721 hepatoma cells and protected them from the effects of an apoptosis-inducing agent. These findings suggest that during hepatocellular carcinogenesis, VTN may promote tumor cell growth and inhibit chemically induced apoptosis.

scholarly journals Structure and properties of an under-sulfated heparan sulfate proteoglycan synthesized by a rat hepatoma cell line.

1984 ◽  
Vol 98 (3) ◽  
pp. 946-953 ◽  
Author(s):  
J Robinson ◽  
M Viti ◽  
M Höök
Keyword(s):  
Heparan Sulfate ◽  
Rat Liver ◽  
Culture Medium ◽  
Hepatoma Cell ◽  
Hepatoma Cells ◽  
Heparan Sulfate Proteoglycan ◽  
Heparan Sulfate Proteoglycans ◽  
Hepatoma Cell Line ◽  
Sulfate Proteoglycan ◽  
Rat Hepatoma Cell Line

A rat hepatoma cell line was shown to synthesize heparan sulfate and chondroitin sulfate proteoglycans. Unlike cultured hepatocytes, the hepatoma cells did not deposit these proteoglycans into an extracellular matrix, and most of the newly synthesized heparan sulfate proteoglycans were secreted into the culture medium. Heparan sulfate proteoglycans were also found associated with the cell surface. These proteoglycans could be solubilized by mild trypsin or detergent treatment of the cells but could not be displaced from the cells by incubation with heparin. The detergent-solubilized heparan sulfate proteoglycan had a hydrophobic segment that enabled it to bind to octyl-Sepharose. This segment could conceivably anchor the molecule in the lipid interior of the plasma membrane. The size of the hepatoma heparan sulfate proteoglycans was similar to that of proteoglycans isolated from rat liver microsomes or from primary cultures of rat hepatocytes. Ion-exchange chromatography on DEAE-Sephacel indicated that the hepatoma heparan sulfate proteoglycans had a lower average charge density than the rat liver heparan sulfate proteoglycans. The lower charge density of the hepatoma heparan sulfate can be largely attributed to a reduced number of N-sulfated glucosamine units in the polysaccharide chain compared with that of rat liver heparan sulfate. Hepatoma heparan sulfate proteoglycans purified from the culture medium had a considerably lower affinity for fibronectin-Sepharose compared with that of rat liver heparan sulfate proteoglycans. Furthermore, the hepatoma proteoglycan did not bind to the neoplastic cells, whereas heparan sulfate from normal rat liver bound to the hepatoma cells in a time-dependent reaction. The possible consequences of the reduced sulfation of the heparan sulfate proteoglycan produced by the hepatoma cells are discussed in terms of the postulated roles of heparan sulfate in the regulation of cell growth and extracellular matrix formation.

scholarly journals Multiple regulatory elements in the intergenic region between the alpha-fetoprotein and albumin genes.

1986 ◽  
Vol 6 (2) ◽  
pp. 477-487 ◽  
Author(s):  
R Godbout ◽  
R Ingram ◽  
S M Tilghman
Keyword(s):  
Thymidine Kinase ◽  
Hela Cells ◽  
Hepatoma Cell ◽  
Regulatory Elements ◽  
Alpha Fetoprotein ◽  
Hepatoma Cell Line ◽  
Thymidine Kinase Gene ◽  
Hep G2 ◽  
Transient Expression Assay ◽  
Kinase Gene

Three enhancer elements spanning a distance of 7 kilobases have been found at the 5' end of the alpha-fetoprotein (AFP) gene. These elements were identified by transient expression assay after the introduction of a modified mouse AFP gene with variable amounts of 5' flanking sequence into a human hepatoma cell line, Hep G2. These regulatory elements function in a position-independent and orientation-independent manner that is typical of enhancers. All three elements will stimulate transcription from the promoter of the herpes simplex virus thymidine kinase gene. In Hep G2 cells, transcriptional activation from the heterologous promoter was approximately 25- to 50-fold higher than the basal levels obtained in the absence of AFP enhancer elements. In HeLa cells, the increase in thymidine kinase gene transcription varied from 6- to 14-fold, indicating that the enhancer elements exhibit some cell type specificity. Deletion analysis of the region proximal to the AFP transcription initiation site identified an essential region between 85 and 52 bases upstream of the site of initiation of transcription whose removal resulted in almost complete extinction of transcriptional activity. This region, which has been shown to be dispensable for transcription in HeLa cells, defines a second tissue-specific regulatory region in the gene.

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