Cultured Mouse Hepatoma Cells Research Articles

N-hydroxylation of 4-aminobiphenyl by CYP2E1 produces oxidative stress in a mouse model of chemically induced liver cancer.

4-Aminobiphenyl (ABP) is a trace component of cigarette smoke and hair dyes, a suspected human carcinogen and a potent rodent liver carcinogen. Postnatal exposure of mice to ABP results in a higher incidence of liver tumors in males than in females, paralleling the sex difference in human liver cancer incidence. A traditional model of ABP tumorigenesis involves initial CYP1A2-mediated N-hydroxylation, which eventually leads to production of mutagenic ABP-DNA adducts that initiate tumor growth. However, several studies have found no correlation between sex or CYP1A2 function and the DNA-damaging, mutagenic, or tumorigenic effects of ABP. Oxidative stress may be an important etiological factor for liver cancer, and it has also been linked to ABP exposure. The goals of this study were to identify novel enzyme(s) that contribute to ABP N-oxidation, and to investigate a potential role for oxidative stress in ABP liver tumorigenicity. Isozyme-selective inhibition experiments using liver microsomes from wild-type and genetically modified mice identified CYP2E1 as a major ABP N-hydroxylating enzyme. The N-hydroxylation of ABP by transiently expressed CYP2E1 produced oxidative stress in cultured mouse hepatoma cells. In vivo postnatal exposure of mice to a tumorigenic dose of ABP also produced oxidative stress in male wild-type mice, but not in male Cyp2e1(-/-) mice or in female mice. However, a stronger NRF2-associated antioxidant response was observed in females. Our results identify CYP2E1 as a novel ABP-N-oxidizing enzyme, and suggest that sex differences in CYP2E1-dependent oxidative stress and antioxidant responses to ABP may contribute to the observed sex difference in tumor incidence.

Dual-specificity phosphatases are targets of the Wnt/β-catenin pathway and candidate mediators of β-catenin/Ras signaling interactions

The Wnt/β-catenin and the Ras/mitogen-activated protein kinase (MAPK) pathways play important roles in cancer development. Both pathways have been studied discretely, but the mechanisms of possible crosstalk are still not fully understood. We have previously shown that β-catenin and MAPK signaling interfere with each other in murine liver in vivo and in vitro. Here, we show that dual specificity phosphatases (Dusps) 6 and 14, known to play an essential role in regulating MAPK pathway activity via feedback mechanisms, are up-regulated by activation of β-catenin in murine liver cells, whereas the epidermal growth factor receptor, an upstream effector in the Ras/MAPK cascade, is down-regulated by β-catenin. In addition, we identified a β-catenin-binding site within the Dusp6 promoter, which is responsible for the activation of the promoter by β-catenin signaling, and demonstrated reduced inducibility of MAPK signaling in cultured mouse hepatoma cells following β-catenin activation. Thus, β-catenin is able to inhibit activation of the Egfr/Ras/MAPK signaling cascade, both at the receptor level and by interfering with MAPK activity via Dusps.

Comparison of gene expression in cell culture to that in the intact animal: relevance to drugs and environmental toxicants. Focus on “Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes”

to study the effects of drugs and other environmental toxicants on the liver, many believe that the use of hepatocyte-derived cultures offers advantages over studies in the liver of intact animals. Cell cultures can be manipulated more easily, and they are far less expensive to maintain than a

Oral administration of trans-resveratrol to guinea pigs increases cardiac DT-diaphorase and catalase activities, and protects isolated atria from menadione toxicity

Resveratrol (3,4′,5-trihydroxy- trans-stilbene) is a natural phytoalexin found in grapes and wine. It has antioxidant and antiproliferative activities, and has been shown to induce NAD(P)H:quinone oxidoreductase, also known as DT-diaphorase, in cultured mouse hepatoma cells. DT-diaphorase is a detoxifying enzyme for quinone-containing substances, due to its ability to prevent their one-electron reduction and the consequent generation of reactive oxygen species (ROS). The aim of the present study was to investigate whether oral administration of trans-resveratrol to guinea pigs (60 mg/l in tap water for 16 days, ad libitum) increases cardiac DT-diaphorase and, consequently, reduces the response of isolated atria to 2-methyl-1,4-naphthoquinone (menadione), the positive inotropic effect of which is related to the amount of ROS generated by its cardiac metabolism. In the cardiac tissue of resveratrol-treated animals, DT-diaphorase activity was significantly higher than that measured in control animals, the V max of the enzyme reaction being 75.47 ± 3.87 and 50.73 ± 0.63 nmoles/mg protein/min, respectively (p < 0.05). Resveratrol administration also significantly increased the activity of cardiac catalase (32.20 ± 2.39 vs. 25.14 ± 3.85 units/mg protein in treated and control animals, respectively; p < 0.001). As a consequence, menadione metabolism by the cardiac homogenate obtained from resveratrol-treated animals generated a smaller amount of ROS and, in electrically driven left atria, menadione produced a significantly lower increase in the force of contraction than in atria isolated from control animals. These results indicate that oral administration of resveratrol exerts cardioprotection against ROS-mediated menadione toxicity.

Enzyme induction by L-buthionine (S,R)-sulfoximine in cultured mouse hepatoma cells.

Induction of Phase II enzymes of the [Ah] gene battery by L-buthionine (S,R)-sulfoximine (BSO) and other agents was examined in mouse hepatoma Hepa-1c1c7 cells. BSO, a nonelectrophilic inhibitor of gamma-glutamylcysteine synthetase (GCS), is routinely used to examine the toxicological implications of GSH depletion. Exposure to BSO for 24 h produced a 75-85% depletion of GSH levels, proportional to the inhibition of GCS activity, as well as small increases in the UDP-glucuronosyltransferase (UGT, 60%) and glutathione transferase (GST, 30%) enzyme activities in Hepa-1 wild-type (wt) cells. However, for the NAD(P)H:menadione oxidoreductase (NMO1) and cytosolic aldehyde dehydrogenase class 3 (AHD4) enzyme activities, BSO produced larger increases (110% and 170%, respectively). The mechanisms of NMO1 and AHD4 induction were examined further. In Hepa-1 wt cells, NMO1 and AHD4 activities were increased by the aromatic hydrocarbon inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and by the electrophile tert-butylhydroquinone (tBHQ), known inducing agents for these enzymes. However, NMO1 and AHD4 were induced in Ah receptor nuclear translocation-defective mutant (c4) cells by BSO and tBHQ, but not by TCDD, suggesting that the induction by BSO and tBHQ is not Ah receptor-mediated. In wt cells, N-acetylcysteine produced a concentration-dependent increase in intracellular cysteine levels, but not GSH levels, in the absence or presence of BSO. Furthermore, N-acetylcysteine had no effect on NMO1 activity under any conditions examined, suggesting that GSH levels per se, rather than change in overall thiol status, might be mediating increased NMO1 activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal regulation of expression of the angiotensinogen gene in cultured mouse hepatoma cells.

To investigate the hormonal regulation of expression of the angiotensinogen (ANG) gene in the liver, we constructed fusion genes with various lengths of the 5'-flanking region of the rat ANG gene linked to a bacterial chloramphenicol acetyl transferase (CAT) gene as reporter and introduced them into mouse hepatoma cells (Hepa 1-6). As a negative control, we introduced them into a nonhepatic cell line, a mouse testicular Sertoli (TM4) cell line. The level of expression of ANG-CAT fusion genes, pOCAT (ANG N-1498/+18), pOCAT (ANG N-688/+18), pOCAT (ANG N-110/+18), pOCAT (ANG N-53/+18) and (ANG-35/+18) were 3.7, 4, 1.1, 4, and 3-fold higher than promoterless pOCAT in Hepa 1-6 cells. No significant expression of any of these ANG-CAT fusion genes over the promoterless pOCAT was observed in Sertoli TM4 cells. The addition of dexamethasone (10(-10) to 10(-4) mol/L) stimulated the expression of the pOCAT (ANG N-1498/+18) fusion gene in Hepa 1-6 cells in a dose-dependent manner with a maximum stimulation at 10(-4) mol/L and a half-maximal stimulation at 10(-8) mol/L. A combination of dexamethasone (10(-6) mol/L) and 8-bromo-cyclic AMP (cAMP) (10(-3) mol/L) further enhanced the effect of the dexamethasone alone although cAMP alone had no effect. Testosterone (10(-6) mol/L), estradiol (10(-6) mol/L), progesterone (10(-6) mol/L), and thyroid hormone (L-T3, 10(-6) mol/L) did not have this effect in either the presence or absence of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the haemopexin-transport system in cultured mouse hepatoma cells. Links between haemopexin and iron metabolism

Minimal deviation hepatoma (Hepa) cells, from the mouse hepatoma B7756, synthesize and secrete haemopexin and express both the haemopexin receptor and the membrane haem-binding protein (MHBP) associated with the receptor, making this cell line the first available for detailed study of both haemopexin metabolism and hepatic transport. The 17.5 kDa MHBP was detected in Triton X-100 extracts of Hepa cells by immunoblotting with goat anti-rabbit MHBP. Scatchard-type analysis of haem-125I-haemopexin binding at 4 degrees C revealed 35,000 receptors per cell of high affinity (Kd 17 nM). Haemopexin-mediated haem transport at 37 degrees C is saturable, having an apparent Km of 160 nM and a Vmax. of 7.5 pmol of haem/10(6) cells per h during exponential growth. Haem-transport capacity is highest in the period just before the cells enter their exponential phase of growth and slowest in stationary phase. Interestingly, haem-haemopexin serves as effectively as iron-transferrin as the sole source of iron for cell growth by Hepa cells. Furthermore, depriving Hepa cells of iron by treatment with desferrioxamine (DF) increases the number of cell-surface haemopexin receptors to 65,000 per cell and consequently increases haemopexin-mediated haem transport. The effects of DF do not appear to require protein synthesis since they are not prevented by cycloheximide. Treatment of Hepa cells with hydroxyurea, an inhibitor of the iron-requiring enzyme ribonucleotide reductase that is obligatory for DNA synthesis, enhanced haemopexin-mediated haem transport. Thus, these studies provide the first evidence for regulation of haem transport by the iron status of cells and suggest a linkage between haemopexin, iron homeostasis and cell growth.

Simultaneous Expression of Salivary and Pancreatic Amylase Genes in Cultured Mouse Hepatoma Cells

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.

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

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.

Translation kinetics in cultured mouse hepatoma cells. Regulation of albumin synthesis by amino acids.

The synthesis of albumin in the liver has been shown to correlate with the availability of essential amino acids in the diet. We have investigated this phenomenon in the highly differentiated mouse hepatoma cell line, Hepa. Cells were grown for three days in complete medium with daily changes. The cells were then incubated for 22 h in media containing varying concentrations of individual essential amino acids. The deficient media were then changed; 1.5 h later the cells were labeled for 0.5 h with [3H]leucine. Albumin was immunoprecipitated and total protein was acid-precipitated from postribosomal supernatants of detergents-solubilized cells. With the exception of isoleucine, the relative rates of albumin synthesis decreased as a function of amino acid concentration from 4.3% in complete medium to 2.5% in totally deficient media. This specific reduction in albumin synthesis was confirmed by analysis of labeled Hepa proteins displayed on sodium dodecyl sulfate/polyacrylamide gels. Essential amino acid limitation reduced total protein synthesis by 50%. This is the result of a decrease in the translation efficiency of total mRNA from 5 to 3 polypeptides/message min-1 and is consistent with a reduction in the initiation rate. In contrast, the 70% decrease in albumin synthesis was a result of a reduced number of functional albumin messages/cell. The translation efficiency of these albumin messages remained unchanged at 1.

Formation of transcribing mononucleosome-eukaryotic RNA polymerase II complexes in vitro as a simple model of active chromatin.

Mononucleosomes obtained from cultured mouse hepatoma cells were incubated with RNA polymerase II from wheat germ. No free DNA was liberated as available templates under the experimental condition employed. Size analysis of the transcripts showed that the polymerase initiated transcription from either terminus and read through the DNA template of mononucleosomes. Sucrose density gradient centrifugation of the reaction mixture resolved mononucleosome-polymerase complexes from free materials. The complexes were characterized by the enrichment of DNA fragments containing the nucleosome linker region, the presence of H1 histone, and the increased susceptibility to DNase I. Both the complexes formed in the presence and absence of precursor nucleotides were susceptible. These suggest that RNA polymerase II prefers to bind to the linker region, and the polymerase-bound nucleosomes are structurally altered. The data were discussed in context with possible mechanisms of transcription of the nucleosome structure.

Effects of cytochrome P1-450 inducers on the cell-surface receptors for epidermal growth factor, phorbol 12,13-dibutyrate, or insulin of cultured mouse hepatoma cells.

Hepa-1c1c7, a mouse hepatoma cell line, was used to study the effect of cytochrome P1-450 inducers on the binding of 125I-epidermal growth factor (EGF), 125I-insulin, or [20-3H]phorbol 12,13-dibutyrate each to its specific cell-surface receptor. After a 24-h exposure to the cultured cells, several polycyclic hydrocarbon P1-450 inducers decrease the binding of EGF to EGF receptors much more than phenobarbital does. There appears to be a selectivity in the inhibitory effects: whereas EGF binding to EGF receptors is blocked, the binding of either phorbol ester or insulin each to its specific cell-surface receptors remains unaffected. The rank order of binding affinities of these chemicals to the cytosolic Ah receptor (2,3,7,8-tetrachlorodibenzo-p-dioxin much greater than benzo[a]pyrene greater than benzo[a]anthracene greater than 6-aminochrysene much greater than phenobarbital) is not correlated with their effects on EGF binding capacity. The effect of polycyclic hydrocarbons on EGF binding takes 24 h at 37 degrees C to be maximal, whereas phorbol 12-myristate 13-acetate, a potent tumor-promoting compound, inhibits EGF binding in less than 30 min. Removal of benzo[a]anthracene from the growth medium after 24 h results in a gradual recovery in EGF binding, indicating that the effect is reversible. Benzo[a]pyrene and benzo[a]anthracene are relatively ineffective at decreasing EGF binding to the EGF receptors in Hepa-1 mutant clones c2 and c4, which lack a normally functioning Ah receptor and inducible aryl hydrocarbon hydroxylase activity (P1-450). The very toxic metabolite (+)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, when added directly to the growth medium of c4 cells, however, is effective at decreasing EGF binding. These data suggest that electrophilic metabolites of polycyclic aromatic compounds, formed by P1-450 induced during the exposure of Hepa-1 cells to these chemicals, are important in decreasing EGF binding to the EGF cell-surface receptor. Occupancy of the Ah receptor per se does not affect EGF binding.

Ethylated fluoresceins: Assay of cytochrome P-450 activity and application to measurements in single cells by flow cytometry

Four ethylated derivatives of fluorescein were synthesized and their metabolism by mouse liver homogenates and cultured mouse hepatoma cells was investigated. Two of the ethylfluoresceins are specifically metabolized by the polycyclic aromatic hydrocarbon inducible cytochrome P-450 to yield fluorescein, which is about 15 times more fluorescent than the ethylated compounds. One of these fluorogenic derivatives, ethoxyfluorescein ethyl ester, is useful for flow cytometric analysis and sorting of intact, viable cells on the basis of cytochrome P-450 activity. The structure-activity relationship of the ethylfluoresceins is discussed.

Response of mouse hepatoma cell methylenetetrahydrofolate polyglutamates to folate deprivation

The behavior of methylenetetrahydrofolate polyglutamate conjugates in cultured mouse hepatoma cells which were starved of folate has been investigated. Folate deprivation caused methylenetetrahydrofolate levels to decrease an order of magnitude. This diminished pool consisted essentially completely of the octaglutamate form. Replenishment of the media with folate caused a slow recovery to normal levels of methylenetetrahydrofolate with undetectable quantities of shorter chain length polyglutamates observable during recovery. Leucovorin, on the other hand, caused a much more rapid recovery to normal levels and gave rise to the early appearance of short chain length polyglutamate intermediates.

Cell cycle patterns of thymidylate synthetase and 5,10-methylenetetrahydrofolate polyglutamates in cultured mouse hepatoma cells.

The regulation of thymidylate synthetase activity was investigated throughout the first cell cycle after release from an isoleucine block in synchronous cultures of mouse hepatoma (Hepa) cells. Activity in cell extracts increased with the onset of S phase and the increased activity was attributed to a parallel increase in enzyme concentration as determined by titration with tritiated fluorodeoxyuridylate. The polyglutamate chain length of reduced folate cofactors, which could also influence thymidylate synthetase activity, was unchanged.

Immunoprecipitation Assay of Alpha-fetoprotein Synthesis by Cultured Mouse Hepatoma Cells Treated with Estrogens and Glucocorticords

This investigation was to study the biosynthesis of 3H-labeled alpha-fetoprotein (AFP) by cultured mouse hepatoma (HEPA-2) cells. Both the function and regulation of this oncodevelopmental gene are unknown. However, evidence indicates that mechanisms controlling the expression of AFP involve aspects of both normal embryonic development and neoplastic transformation. the secretion of AFP was analyzed during different phases of the growth cycle to provide information on AFP production using standard culture conditions. The highest rate of secretion occurred during the stationary phase, followed by the late logarithmic and early logarithmic phases of growth, respectively. The production of AFP was then determined following the addition of glucocorticoids and estrogens in an attempt to understand hormonal factors that may be involved. Studies utilizing estradiol-17 beta indicated that the secretion of AFP did not appear to be sensitive to this steroid even though sucrose density gradient analysis of HEPA-2 cytosol, for estrogenic receptors, revealed competitive binding moieties on the 8S and 4S regions of the gradient. In contrast, the secretion of the total complement of proteins, including AFP, was significantly stimulated by the glucocorticoids, dexamethasone and corticosterone. Analysis of HEPA-2 cytosol for glucocorticoid receptors revealed binding components in the 7S and 3-4S regions of the gradient. The 3H-dexamethasone binding appeared to be stereospecific since nonlabeled dexamethasone, but not nonlabeled estradiol-17 beta, effectively displaced the bound radioactivity. The glucocorticoid-binding component in HEPA-2 therefore displayed characteristics reported for glucocorticoid receptors in normal liver and other hepatomas.

Effects of cyclic AMP on the kinetics of serum protein synthesis in cultured mouse hepatoma cells

The mouse hepatoma cell line, Hepa, was cultured in the presence of either 1 mM N 6, O 2′ -dibutyryl cyclic AMP (Bt 2cAMP), 0.5 mM 3-isobutyl-1-methylxanthine, or 1 μg/ml cholera toxin. The synthesis and secretion of albumin, α-fetoprotein, and transferrin were elevated above controls by 24 h reaching two- to fourfold stimulations within 72 h. These effects were reversible and were specific for the serum proteins. The stimulation of serum protein synthesis was accompanied by a decrease in the rate of cell proliferation. Protein synthetic parameters were analyzed in Hepa cells 72 h after exposure to N 6,O 2′ -dibutyryl cyclic AMP. The cellular rate of albumin synthesis was increased fourfold and the relative rate of albumin synthesis was increased approximately threefold. N 6,O 2′ -Dibutyryl cyclic AMP did not affect the size distribution of either total Hepa polyribosomes or of albumin-synthesizing polyribosomes. The elongation rate on total mRNA and on albumin mRNA was decreased by approximately 40%. These results indicate that the rate of initiation of total Hepa mRNA and of albumin mRNA also decreased by 40%. The nonspecific nature of the N 6,O 2′ -dibutyryl cyclic AMP effect on Hepa protein synthetic parameters must be due to an alteration in the level of a common substrate, perhaps ATP. The specific threefold increase in the relative rate of albumin synthesis with no alteration in polyribosome sizes requires a threefold increase in the relative amount of functional albumin mRNA in Hepa cells. This prediction was confirmed by cell-free translation of Hepa polyribosomes.

Increased thymidylate synthetase in 5-fluorodeoxyuridine resistant cultured hepatoma cells

A FdUrd resistant line of cultured mouse hepatoma cells has been obtained. The resistant cell line had 6- to 10-fold higher levels of thymidylate synthetase, but dihydrofolate reductase and thymidine kinase were unchanged. No impairment of FdUrd incorporation by the resistant cell line could be detected. The increased thymidylate synthetase in resistant cells had the same turnover number and I 50 for FdUMP as the enzyme found in sensitive cells, making it unlikely that a new gene product had been obtained. Sensitive cells could be completely rescued by the addition of thymidine, suggesting that the primary mode of drug action is to diminish thymidine metabolites. Resistant cells, removed from FdUrd for several generations, did not proliferate immediately upon reintroduction of the drug; however, loss of sensitivity was much more rapid than upon initial exposure. These results are interpreted in terms of a mechanism for resistance.

Analysis of translational parameters in cultured cells

A method is described for the measurement of protein synthesis parameters in cultured cells. Ribosome transit times, polyribosome size distribution and relative synthetic rates are measured on individual cultures of cells. This method is applied to an analysis of cycloheximide (1 μM) inhibition of protein synthesis in cultured mouse hepatoma cells.

Synthesis and Secretion of Transferrin by Cultured Mouse Hepatoma Cells

The mouse hepatoma cell (Hepa-1) in tissue culture has been shown to synthesize and secrete three electrophoretically distinct transferrins. Each of these forms of transferrin has a molecular weight of 77,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentration of each form is indicated by its staining intensity, which is highest in the form with the fastest mobility and lowest in the form with the slowest mobility. The relative rate of transferrin synthesis has been determined in log-phase and stationary-phase cells; the data indicate that the relative rate of synthesis increases twofold in stationary-phase cells. When the incorporation of [3H]leucine into transferrin reaches steady state, the rate of secretion is equal to the rate of synthesis; the rate of secretion also increases twofold in stationary-phase cells. Our studies also show that transferrin synthesis accounts for 0.98% of the total protein synthesis in log-phase cells and for 1.8% in stationary-phase cells. This is the level of synthesis that has been determined by in vivo studies. We conclude that after continuous culture for several years these hepatoma cells have maintained one of the characteristics of the differentiated liver cell, namely, the ability to synthesize and secrete transferrin.