Reuber H35 Cells Research Articles

Metabolism of palmitic and docosahexaenoic acids in Reuber H35 hepatoma cells.

In this work, we have modified the fatty acid composition of Reuber H35 hepatoma cells by supplementation of the culture medium with a saturated (palmitic) or a polyunsaturated (docosahexaenoic) acid. These fatty acids were incorporated into total lipids and phospholipids of hepatoma cells. Palmitic acid readily increased the percentage of its monounsaturated derivative (16:1 n-7). When both fatty acids were supplemented at the same concentration, the percentage of docosahexaenoic acid in the total lipids and phospholipids of Reuber H35 cells increased more than that of palmitic acid. Although the levels of 16:0 increased, the addition of docosahexaenoic acid to the culture medium decreased the percentages of monoenoic acids. From our results, it can be concluded that palmitic and docosahexaenoic acids modify the fatty acid composition of Reuber H35 hepatoma cells. The profound changes induced by docosahexaenoic acid, especially those in the phospholipid fraction, may be of great interest given the main role of these components in the regulation of chemical and physical properties of biological membranes and/or membrane systems.

Novel delayed-early and highly insulin-induced growth response genes. Identification of HRS, a potential regulator of alternative pre-mRNA splicing

We have identified 41 novel and many previously known growth response genes induced in regenerating liver and insulin-treated Reuber H35 cells, a rat hepatoma cell line that grows in response to physiologic concentrations of insulin and retains some properties of regenerating liver. Although many genes are expressed similarly in the two systems, there are important differences in the kinetics of induction of some genes. These differences allowed us to identify and characterize novel genes that are highly insulin-induced and expressed as delayed-early genes in regenerating liver. Sequence analysis of CL-6, the most abundant insulin-induced gene, resulted in the identification of a highly hydrophobic hepatic protein. Sequence analysis of HRS, a highly insulin-induced delayed-early gene, demonstrated that it is a member of the family of regulators of alternative pre-mRNA splicing. Different forms of HRS mRNA are temporally regulated during the growth response, suggesting that HRS could autoregulate processing of its pre-mRNA. Given the dramatic increase in RNA production during late G1, proteins induced by mitogens like insulin that control RNA processing are likely to have important roles in cell cycle regulation.

Differential expression of tyrosine aminotransferase by glucocorticoids and insulin

The differential response of the tyrosine aminotransferase (TAT) gene to glucocorticoids and insulin in HTC cells and cell clones derived from Reuber H35 cells (FaO and Fu5.5) have been analyzed by nuclear run-on assay. It has been previously shown that clones of cells from HTC and Reuber H35 cell lines, exhibit different sensitivities for the induction of TAT mRNA and enzyme activity. The purpose of the present study was to determine whether this difference in TAT expression between hepatocytes and hepatoma cell lines occurs at the level of TAT gene transcription or mRNA stability. A study of the TAT mRNA accumulation in all cell types showed that TAT mRNA in the Reuber H35 cell clones and hepatocytes was synthesized at a higher rate than in HTC cells. However, dexamethasone induction of α 1 AGP mRNA and glutamine synthetase was comparable to glucocorticoid bound receptors. In addition, cycloheximide decreased the rate at which induced levels of TAT mRNA were degraded. We also show that a heterologous fusion gene constructed from 3.0 kilobases (kb) 5′ to the transcription initiation site of the rat TAT gene and the bacterial chloramphenicol acetyltransferase gene (CAT) responds similarly to dexamethasone in Fu5.5 and HTC cells as determined by transient transfection assay; and insulin inhibits dexamethasone mediated transcription in Reuber H35 cells and primary adult hepatocytes. These data indicate that DNA sequences involved in the differential response of the TAT gene to hormone treatments between HTC and Reuber H35 cell lines are not located in the first 3.0 kb fragment.

Regulation of Ki-ras expression in Reuber H35 cells

Glucagon at a low concentration has a stimulatory effect on Ki-ras expression, whereas, at high concentrations the hormone suppresses the level of the Ki-ras transcripts. Incubation of the hepatoma cells with 10 microM dibutyryl cyclic AMP results in suppression of Ki-ras expression but the phorbol ester, 21-O-tetradecanoylphorbol 13-acetate (TPA) causes an increase. Down regulation of protein kinase C by prolonged exposure of hepatoma cells to TPA causes a dramatic decrease in the glucagon-stimulated effect on Ki-ras expression. The presence of diacylglycerol for 2 h in the culture medium results in a significant increase in Ki-ras expression, while treatment of the cells with 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine, a potent inhibitor of protein kinase C, leads to a dramatic reduction. The calcium ionophore, A23187 is able to stimulate Ki-ras expression, whereas, addition of verapamil or EGTA results in its suppression. The present findings suggest that the inductive effect of glucagon on Ki-ras expression at low concentrations is via the activation of protein kinase C which causes phosphorylation of some regulatory proteins that may eventually affect the level of Ki-ras mRNA. The suppressive effect of glucagon at higher concentrations is via an increase in cAMP through activation of adenylate cyclase.

Transcriptional regulation of the liver beta-galactoside alpha 2,6-sialyltransferase by glucocorticoids.

Hepatic expression of the beta-galactoside alpha 2,6-sialyltransferase is at least in part specified by circulatory glucocorticoids. In this report we use the glucocorticoid agonist, RU362, and the antagonist, RU486, to demonstrate the participation of the glucocorticoid receptor pathway in beta-galactoside alpha 2,6-sialyltransferase regulation. The existing pool of sialyltransferase mRNA is turned over with an approximate half-life of 13 h, and presence of dexamethasone does not alter this rate of degradation. By means of nuclear run-off assays and measurement of nuclear unprocessed transcripts we demonstrate that dexamethasone induction of beta-galactoside alpha 2,6-sialyltransferase mRNA in rat Reuber H35 cells is mediated by a transcriptional enhancement mechanism. The same initiation site is utilized for sialyltransferase transcription in both basal- and hormone-stimulated synthesis. Sialyltransferase sequences residing upstream of this transcriptional initiation point are used to control chloramphenicol acetyltransferase expression in fusion constructs following transient transfection into H35 cells to demonstrate the presence of a functional promoter. Although no element with similarity to the known GRE consensus sequence resides within this promoter region, chloramphenicol acetyltransferase expression under the control of the sialyltransferase promoter is subject to a low (1.6-fold) but reproducible induction in response to dexamethasone. Implications of this observation to glucocorticoid regulation are discussed.

An Inducible 50-Kilodalton NFκB-Like Protein and a Constitutive Protein Both Bind the Acute-Phase Response Element of the Angiotensinogen Gene

The rat angiotensinogen gene is induced in the course of the hepatic acute-phase response. We demonstrate that monocyte conditioned medium can stimulate transcription of a stably introduced reporter construct driven by 615 base pairs of the angiotensinogen 5'-flanking sequence, as well as the endogenous gene, in Reuber H35 cells. Point mutations of a cis-acting element, located 545 base pairs from the transcription start site and sharing sequence identity with known nuclear factor kappa B (NF kappa B)-binding sites, led to loss of cytokine inducibility. When cloned upstream of a minimal promoter, this cis-acting element imparted transcriptional inducibility by monocyte conditioned medium, interleukin-1, and tumor necrosis factor on a luciferase reporter gene in HepG2 cells. Two distinct proteins bound this element in vitro: a heat-stable, constitutively present, hepatic nuclear protein that gave rise to a DNase I-protected footprint covering the functionally defined element; and a binding protein of different mobility, induced by monocyte conditioned medium, which also recognized the NF kappa B-binding site of the murine kappa light-chain enhancer. UV cross-linking showed this inducible protein to have an apparent molecular mass of 50 kilodaltons, similar to that described for NF kappa B and distinct from the constitutively present protein that was shown by Southwestern (DNA-protein) blot to have a molecular mass of 32 kilodaltons. Methylation interference analysis showed that the induced species made contact points with guanine residues in the NF kappa B consensus sequence typical of NF kappa B. Induction of this binding activity did not require new protein synthesis, and 12-O-tetradecanoylphorbol-13-acetate could mimic the induction by cytokines. We thus provide direct evidence for involvement of NF kappa B or a similar factor in the hepatic acute-phase response and discuss the potential role of the presence of a constitutive nuclear factor binding the same cis element.

An inducible 50-kilodalton NF kappa B-like protein and a constitutive protein both bind the acute-phase response element of the angiotensinogen gene.

The rat angiotensinogen gene is induced in the course of the hepatic acute-phase response. We demonstrate that monocyte conditioned medium can stimulate transcription of a stably introduced reporter construct driven by 615 base pairs of the angiotensinogen 5'-flanking sequence, as well as the endogenous gene, in Reuber H35 cells. Point mutations of a cis-acting element, located 545 base pairs from the transcription start site and sharing sequence identity with known nuclear factor kappa B (NF kappa B)-binding sites, led to loss of cytokine inducibility. When cloned upstream of a minimal promoter, this cis-acting element imparted transcriptional inducibility by monocyte conditioned medium, interleukin-1, and tumor necrosis factor on a luciferase reporter gene in HepG2 cells. Two distinct proteins bound this element in vitro: a heat-stable, constitutively present, hepatic nuclear protein that gave rise to a DNase I-protected footprint covering the functionally defined element; and a binding protein of different mobility, induced by monocyte conditioned medium, which also recognized the NF kappa B-binding site of the murine kappa light-chain enhancer. UV cross-linking showed this inducible protein to have an apparent molecular mass of 50 kilodaltons, similar to that described for NF kappa B and distinct from the constitutively present protein that was shown by Southwestern (DNA-protein) blot to have a molecular mass of 32 kilodaltons. Methylation interference analysis showed that the induced species made contact points with guanine residues in the NF kappa B consensus sequence typical of NF kappa B. Induction of this binding activity did not require new protein synthesis, and 12-O-tetradecanoylphorbol-13-acetate could mimic the induction by cytokines. We thus provide direct evidence for involvement of NF kappa B or a similar factor in the hepatic acute-phase response and discuss the potential role of the presence of a constitutive nuclear factor binding the same cis element.

Increase of thermoresistance after growth stimulation of resting Reuber H35 hepatoma cells: Alteration of nuclear characteristics, non-histone chromosomal protein phosphorylation and basal heat shock protein synthesis

In this paper we demonstrate an increase in thermoresistance of resting Reuber H35 cells upon growth stimulation by serum-containing medium: late G1/early S-phase cells were thermoresistant as compared with G0 phase cells. Increase of thermoresistance during early cell cycle runs parallel with increased tolerance of structural and molecular properties of the cell nucleus. Nuclear shape and chromatin structuring became thermotolerant as determined by geometric and densitometric analysis of Feulgen-stained nuclei. Moreover, increased tolerance was demonstrated by means of the capability for endogenous phosphorylation of isolated non-histone chromosomal proteins (NHCPs). We discuss the molecular basis for this increased thermoresistance after growth stimulation and make a comparison with induction of ‘acquired thermotolerance’ such as has been observed in studies on fractionated hyperthermia. Both after growth stimulation and after heat shock, an increase of endogenous phosphorylation capacity of isolated NHCPs was observed, while a main enhancement of phosphorylation was found for a NHCP of M r 95000. Moreover, the basal synthesis of proteins inducible by heat shock (heat shock proteins) and indicated as HSP65, HSP68 and HSP84 was enhanced in thermoresistant late G1/early S phase cells as compared with thermo-sensitive G0 phase cells. A role for chromatin structuring, NHCP phosphorylation and HSPs in the regulation of thermosensitivity and cell cycling is discussed.

Hepatocyte-hepatoma cell hybrids. Characterization and demonstration of bile acid synthesis.

Hybrids were created by fusion of primary rat hepatocytes with well-differentiated Reuber H35 rat hepatoma cells. Seventeen hybrids were screened for bile acid synthesis using [26-14C]cholesterol. As [26-14C]cholesterol was converted to bile acid, 14CO2 was released. Using this assay, four hybrids (8B, 12C, 13C, and 13D) were identified which synthesized bile acid. These four hybrids also incorporated [14C]taurine into bile acid. Bile acids were identified by capillary gas chromatography/mass spectrometry, and their rates of synthesis were quantitated by isotope dilution. Reuber H35 cells synthesized little or no bile acid. However, hybrids 8B, 12C, 13C, and 13D synthesized chenodeoxycholic acid, alpha-muricholic acid, and cholic acid and secreted them into the media. The rates of synthesis of individual bile acids varied among these hybrids. For example, the relative percentage of cholic acid ranged from 11.1% (hybrid 8B) to 50.4% (hybrid 13C). The bile acids synthesized and secreted by the most active hybrid, 12C, were greater than 93% conjugated. In summary, hybrids were created that retain the capacity to synthesize, conjugate, and secrete three major rat bile acid species. Such hybrids are unique model systems that will allow the study of the biochemical and genetic regulation of bile acid synthesis.

Inhibition of steroid-inducible tyrosine aminotransferase gene expression by N-methyl-N'-nitro-N-nitrosoguanidine in a rat hepatoma cell line.

Treatment of rat hepatoma cells (the Fao clone of Reuber H35 cells) with a 500 ng/ml concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a marked inhibition of steroid-inducible tyrosine aminotransferase (TAT) gene expression. Following a 1-h treatment period with MNNG, addition of 1 microM dexamethasone (DEX) for 6 and 24 h resulted in, respectively, 78 and 51% less induction of TAT enzyme activity in MNNG-treated than in solvent-treated cells. MNNG had little effect on the basal level of TAT activity 6 h after treatment with the carcinogen, but the uninduced level of TAT activity declined to 36% of the control value 24 h following MNNG treatment. MNNG-treated cells concurrently exhibited 75% less total steroid-induced TAT RNA following addition of 1 microM of DEX for 4 h, but showed little or no change in the basal level of total TAT RNA or in the level of total alpha-tubulin RNA. In addition, MNNG treatment resulted in 45% less induction of TAT enzyme activity following a 4-h treatment period with 0.5 mM N6,O2'-dibutyryl-adenosine 3',5'-cyclic monophosphate. The concentration of MNNG used in these experiments caused only a 14% decline in cell number 24 h following carcinogen treatment and had no effect on cell viability as determined by trypan blue exclusion. The data indicate that the inhibitory effects of MNNG appear to be mediated at a pre-translational step. MNNG inhibits the accumulation of total DEX-inducible TAT RNA either by preventing the steroid-mediated increase of the rate of TAT RNA transcription or decreasing total TAT RNA stability, or both.

Effect of external K+ on protein and DNA synthesis during and after heat shock in rat hepatoma cells.

The effects of extracellular K+ concentrations on protein and DNA synthesis after non-lethal heat shock were studied in the hepatoma cell lines Reuber H35 and HTC. Elevation of the extracellular K+ concentration by equimolar replacement of Na+ by K+ in growth media of Reuber H35 and HTC cells caused an increase of the intracellular K+ content in both cell lines. This property was subsequently used to study the effect of elevated intracellular K+ concentrations on protein and DNA synthesis after hyperthermic treatment at 42 degrees C for 30 min. In normal K+ medium, protein and DNA synthesis were inhibited rapidly after the start of the hyperthermic treatment in both Reuber H35 and HTC cells. Increasing the external K+ concentration of the medium did not influence the inhibition and subsequent recovery of protein synthesis after heat shock in both cell lines. In contrast, in media with elevated K+ concentrations, DNA synthesis after heat-shock was inhibited less in Reuber H35 cells than in cells incubated in normal K+ medium and, furthermore, showed no inhibition in HTC cells. The protective effect of external K+ on DNA synthesis after heat shock was maximal between 50 and 70 mM in the temperature range 42-44 degrees C.

Further evidence for translational regulation of tyrosine aminotransferase synthesis by dibutyryl cyclic AMP in Reuber H35 hepatoma cells

Cyclic AMP derivatives increase the rate of synthesis of tyrosine aminotransferase in Reuber H35 hepatoma cells. Various studies lend support to the hypothesis that cyclic AMP increases the synthesis of tyrosine aminotransferase by acting at a posttranscriptional site. The presence of a limited non-translatable pool of tyrosine aminotransferase mRNA prior to the formation of the translatable tyrosine aminotransferase mRNA implicates a possible site of action of cyclic AMP. We compared the capacity of N 6,O 2′- dibutyryl cyclic AMP to induce tyrosine aminotransferase synthesis when untranslatable tyrosine aminotransferase mRNA sequences are present or absent. The transition of a condition in which non-translatable tyrosine aminotransferase mRNA sequences were present to a condition in which they were absent was established by preinduction of Reuber H35 cells with dexamethasone, followed by addition of actinomycin D. In the time period thereafter, the amount of non-translatable mRNA decreased and 1.5–2 h after addition of actinomycin D, only translatable tyrosine aminotransferase mRNA was present. It can be seen that the induction of tyrosine aminotransferase synthesis by dibutyryl cyclic AMP follows the normal decrease of tyrosine aminotransferase mRNA. We present evidence that dibutyryl cyclic AMP in Reuber H35 hepatoma cells regulates tyrosine aminotransferase synthesis at a posttranscriptional site independent of the pool of nontranslatable tyrosine aminotransferase mRNA sequences, but influencing the efficiency of translation of active tyrosine aminotransferase mRNA.

Effect of Methylxanthines on Binding of the Glucocorticoid Receptor to DNA‐Cellulose and Nuclei

The binding of [3H]dexamethasone-receptor complex from rat liver cytosol to isolated nuclei or DNA-cellulose can be greatly enhanced at low temperature by the presence of theophylline. Aminophylline and caffeine can mimic this effect; however, papaverine and 1-methyl-3-isobutylxanthine, at concentrations inhibitory to phosphodiesterase, are without effect on glucocorticoid receptor binding to DNA. Furthermore, theophylline can be added when adenosine 3':5'-monophosphate-(cAMP) hydrolysis is already complete and still enhance DNA binding. These results imply that this effect of theophylline is independent of its known effect on cAMP levels. Activation by methylxanthines does not alter the sedimentation of the glucocorticoid-receptor complex in sucrose gradients but does alter the pI and in this respect brings about changes resembling those which occur upon activation by heat. Recently we have shown that pyridoxal phosphate inhibits the binding of heat-activated receptor to DNA-cellulose. Similarly, we have shown here that pyridoxal phosphate also inhibits the DNA-cellulose binding of theophylline-treated receptor. The presence of theophylline also enhances the rate of binding of [3H]dexamethasone to the receptor and increases its apparent affininty for the steroid. The data suggest that the effect of theophylline is on some cytosolic component, perhaps the receptor itself. Enhanced DNA binding as a result of exposure to theophylline at low temperature can also be demonstrated using the glucocorticoid receptor of kidney, thymus and Reuber H35 cells.

Infulence of hormones and medium composition on the degradation of phosphoenolpyruvate carboxykinase (GTP) and total protein in Reuber H35 cells.

Reuber H35 cells were pulse-labeled with radioactive leucine and the influence of hormones, serum, and amino acids on protein degradation was investigated during a subsequent chase period. Radioactive, immunoprecipitable phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) had a half-life of 5 to 6 hours which was not influenced by either N6, O2-dibutyryl adenosine 3':5'-monophosphate, dexamethasone, or insulin. The rate of phosphoenolpyruvate carboxykinase degradation was the same under steady state conditions as during the approach to a new steady state following hormonal induction or deinduction of the enzyme. Therefore, hormonal regulation of enzyme activity in vivo is the result of changes in the rate of enzyme synthesis. The rate of proteolysis for total cell proteins was increased under nutritional step-down conditions produced by the removal of serum or amino acids, or both, from the medium. This effect was completely prevented by insulin. Cycloheximide and puromycin, but not actinomycin D or cordycepin, inhibited protein degradation under step-down conditions but did not further decrease the basal rate of proteolysis measured in the presence of either insulin or serum plus amino acids. There was a good correlation between changes in proteolysis produced by serum and amino acids and changes in the degradation rate of phosphoenolpyruvate carboxykinase. Also, inhibition of proteolysis with cycloheximide and puromycin was accompanied by a decrease in the degradation rate for enzyme antigen. It is suggested that nutritional step-down leads either to the synthesis or activation of a proteolytic system.