Negative regulation of catalase gene expression in hepatoma cells

1992 ◽  
Vol 12 (6) ◽  
pp. 2525-2533
Author(s):  
K Sato ◽  
K Ito ◽  
H Kohara ◽  
Y Yamaguchi ◽  
K Adachi ◽  
...  
Keyword(s):  
Rat Liver ◽  
Catalase Activity ◽  
Hepatoma Cell ◽  
Nuclear Extract ◽  
Hepatoma Cells ◽  
Negative Regulation ◽  
Regulation Of Transcription ◽  
Catalase Gene ◽  
Core Sequence ◽  
Silencer Element

For an understanding of the molecular basis of the marked decrease in catalase activity of various tumor cells, expression of the catalase gene was studied in rat and human hepatoma cell lines and in rat liver, which was used as a control with high activity. RNA blot hybridization profiles and run-on assays indicated that the decrease in catalase activity was due to depression of catalase gene transcription. Chloramphenicol acetyltransferase (CAT) assays for the fragments with various lengths of the 5'-flanking region (up to -4.5 kb from the ATG codon) of the catalase gene revealed the presence of several cis-acting elements involved in the negative regulation of transcription. The most-upstream element with the strongest activity (-3504 to -3364 bp), when linked to the catalase promoter region (-126 bp) of the CAT construct and subjected to an in vitro transcription assay, did not yield transcripts in experiments with the hepatoma nuclear extract, whereas the unlinked template did yield transcripts. A gel shift competition assay using hepatoma nuclear extract showed the core sequence of the silencer element to be 5'-TGGGGGGAG-3'. A homology search found that the same core sequence was also present in 5'-flanking regions of the albumin gene and of some other liver enzyme genes, the expression of which has been reported to be down regulated in some hepatoma cells. Southwestern (DNA-protein) analysis demonstrated that an approximately 35-kDa nuclear protein bound to the silencer element was present in hepatoma cells but not in rat liver cells.

scholarly journals Negative regulation of catalase gene expression in hepatoma cells.

1992 ◽  
Vol 12 (6) ◽  
pp. 2525-2533 ◽  
Author(s):  
K Sato ◽  
K Ito ◽  
H Kohara ◽  
Y Yamaguchi ◽  
K Adachi ◽  
...  
Keyword(s):  
Rat Liver ◽  
Catalase Activity ◽  
Hepatoma Cell ◽  
Nuclear Extract ◽  
Hepatoma Cells ◽  
Negative Regulation ◽  
Regulation Of Transcription ◽  
Catalase Gene ◽  
Core Sequence ◽  
Silencer Element

For an understanding of the molecular basis of the marked decrease in catalase activity of various tumor cells, expression of the catalase gene was studied in rat and human hepatoma cell lines and in rat liver, which was used as a control with high activity. RNA blot hybridization profiles and run-on assays indicated that the decrease in catalase activity was due to depression of catalase gene transcription. Chloramphenicol acetyltransferase (CAT) assays for the fragments with various lengths of the 5'-flanking region (up to -4.5 kb from the ATG codon) of the catalase gene revealed the presence of several cis-acting elements involved in the negative regulation of transcription. The most-upstream element with the strongest activity (-3504 to -3364 bp), when linked to the catalase promoter region (-126 bp) of the CAT construct and subjected to an in vitro transcription assay, did not yield transcripts in experiments with the hepatoma nuclear extract, whereas the unlinked template did yield transcripts. A gel shift competition assay using hepatoma nuclear extract showed the core sequence of the silencer element to be 5'-TGGGGGGAG-3'. A homology search found that the same core sequence was also present in 5'-flanking regions of the albumin gene and of some other liver enzyme genes, the expression of which has been reported to be down regulated in some hepatoma cells. Southwestern (DNA-protein) analysis demonstrated that an approximately 35-kDa nuclear protein bound to the silencer element was present in hepatoma cells but not in rat liver cells.

Immunofluorescent localization of type II insulin-like growth factor receptor in rat liver and hepatoma cells

1989 ◽  
Vol 121 (2) ◽  
pp. 221-NP ◽  
Author(s):  
M. A. Hartshorn ◽  
C. D. Scott ◽  
R. C. Baxter
Keyword(s):  
Rat Liver ◽  
Hepatoma Cell ◽  
Rat Hepatocytes ◽  
Hepatoma Cells ◽  
Type Ii ◽  
Adult Rat ◽  
Hepatoma Cell Lines ◽  
Igf Receptors ◽  
Immunofluorescent Technique ◽  
Igf Receptor

ABSTRACT We have used an immunofluorescent technique to localize type II insulin-like growth factor (IGF) receptors in rat liver, rat hepatocytes and three rat hepatoma cell lines (HTC, H-35 and 5123) using a polyclonal antibody (C-1) raised to purified rat liver type II IGF receptor. Specificity of the antiserum was confirmed by Western blotting of microsomal membranes prepared from hepatocytes and hepatoma cells which showed a single class of receptor in all cells, of Mr approximately 210 000 for hepatocytes, HTC and H-35 cells and approximately 220 000 for 5123 cells, on non-reduced, 4–15% polyacrylamide gradient gels. The specificity of the immunofluorescent technique was also verified by abolition of labelling after preincubation of antiserum with purified type II IGF receptor. Rat liver cryosections contained areas of juxtanuclear labelling in hepatocytes, consistent with the presence of type II IGF receptor in the Golgi region. Brightest immunofluorescence was seen in sections from fetal and neonatal rats with adult rat hepatocytes staining brightly only around central veins. Areas of labelling were also seen in connective tissue surrounding larger veins. Cultured adult rat hepatocytes and rat hepatoma cell lines also showed bright areas of juxtanuclear immunofluorescence, with HTC and H-35 cells staining more than 5123 and adult hepatocytes. Fetal rat hepatocytes in culture also labelled very brightly both in a juxtanuclear location and in small clusters over the cell, possibly on the cell surface. These observations indicate that type II IGF receptors are located predominantly on intracellular membranes and are most abundant in rapidly growing cells and tissues (such as fetal liver and hepatoma cells). Journal of Endocrinology (1989) 121, 221–227

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 Expression of laminin γ 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter

1996 ◽  
Vol 313 (3) ◽  
pp. 745-752 ◽  
Author(s):  
Françoise LEVAVASSEUR ◽  
Jocelyne LIÉTARD ◽  
Kohei OGAWA ◽  
Nathalie THÉRET ◽  
Peter D. BURBELO ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Hepatoma Cell ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
Hepatoma Cells ◽  
Basement Membranes ◽  
Major Protein ◽  
Cultured Hepatocytes

Laminin γ1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin γ1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin γ1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5´ flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin γ1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that a Mr 60000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures.

scholarly journals Cyclic AMP-dependent protein kinase regulates transcription of the phosphoenolpyruvate carboxykinase gene but not binding of nuclear factors to the cyclic AMP regulatory element.

1990 ◽  
Vol 10 (7) ◽  
pp. 3357-3364 ◽  
Author(s):  
P G Quinn ◽  
D K Granner
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Catalytic Subunit ◽  
Nuclear Factors ◽  
Dependent Protein

We have examined the binding of factors in rat liver nuclear extracts to the phosphoenolpyruvate carboxykinase (PEPCK) gene cyclic AMP (cAMP) response element (CRE) and other CREs and have isolated a rat liver CRE-binding protein (CREBP) cDNA. In addition, we have examined the influence of altering the phosphorylation state of nuclear factors on both CRE binding and in vitro transcription. Specific binding to the PEPCK CRE was measured in a mobility shift assay. CRE sequences of the PEPCK, somatostatin, and glycoprotein hormone alpha subunit genes competed equally for binding of rat liver nuclear factors to the PEPCK CRE, whereas mutant PEPCK CRE sequences did not compete for binding. Oligonucleotides complementary to rat pheochromocytoma CREBP (Gonzalez et al., Nature [London] 337:749-752, 1989) were used to prime rat liver and brain cDNA in the polymerase chain reaction. The predominant CREBP molecule obtained was identical to the rat pheochromocytoma CREBP except for a 14-amino-acid deletion in the N-terminal half that was also present in a human placental cDNA (Hoeffler et al., Science 242:1430-1433, 1988). The regulation of transcription by cAMP was examined by coincubation of rat liver nuclear extract with the purified catalytic subunit of cAMP-dependent protein kinase (protein kinase A). Although binding to the CRE was unaffected, in vitro transcription directed by the PEPCK promoter was stimulated by catalytic subunit, and this effect was blocked by protein kinase inhibitor peptide. In contrast, when nuclear extract was coincubated with phosphatase, there was substantial inhibition of in vitro transcription directed by the PEPCK promoter, but there was no effect on binding to the CRE. The major effects of catalytic subunit were exerted through the CRE, but residual stimulation was evident in promoter fragments containing only the TATA element. These data suggest that factors are bound to the CRE at constitutively high levels and that their capacity for transcriptional activation is regulated by phosphorylation.

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.

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.

Sign in / Sign up

Export Citation Format

Share Document