HTC Cell Line Research Articles

Phosphorylation of Telomeric Repeat Binding Factor 1 (TRF1) by Akt Causes Telomere Shortening

Telomeric repeat binding factor 1 (TRF1) belongs to the shelterin complex, which modulates the telomere structures. Akt/protein kinase B activation caused genomic instability and contributes to tumorigenesis, although the molecular mechanism remained little known. Here, we show the direct interaction between Akt and TRF1. In vitro kinase assays showed the phosphorylation of a putative Akt phosphorylation site (Threonine 273) in wild type TRF1, but not the mutant TRF1 (T273A), by Akt. Overexpression of Akt decreased telomere length in a HTC cell line. These results indicate that Akt plays an important role in telomere length regulation, contributing to genomic instability and tumorigenesis.

Initial results in the development of a reporter cell line for toxicology studies at gene expression level: Activation of the electrophile-responsive element by copper and methyl parathion

Induction of many genes encoding detoxifying enzymes and antioxidant proteins is mediated through a common mechanism, which is controlled by electrophile-responsive elements (EpRE) within the regulatory region of those genes. Copper and methyl parathion are environmental pollutants known to induce the expression of EpRE-mediated genes. In order to evaluate the molecular response triggered by these pollutants, a stable cell line was produced, which carries a transgene comprised of the green fluorescent protein (GFP) reporter gene under transcriptional control of the mouse glutathione- S-transferase ( gst1) electrophile-responsive element fused to the mouse metallothionein ( mt1) minimal promoter. The rat HTC hepatoma cells were transfected with the EpREmt–GFP construct and successfully selected with G418 antibiotic. EpREmt–GFP HTC cells were treated with 0.002 mg L −1, 0.02 mg L −1, 0.2 mg L −1 and 2 mg L −1 copper sulfate and 0.001 mg L −1, 0.01 mg L −1, 0.1 mg L −1 and 1 mg L −1 methyl parathion for 48 h. GFP expression was directly quantified in living cells using a microplate fluorimeter. GFP expression was significantly increased in higher concentrations of both pollutants, showing a 1.80- and 2.58-fold induction of GFP at 2 mg copper L −1 and 1 mg methyl parathion L −1, respectively ( p < 0.01). The results obtained in the present study demonstrate that the EpREmt–GFP HTC cell line can be an interesting model for further development for the study of the cellular response to aquatic pollutants as well as a new tool for environmental monitoring at the molecular level.

Differential Modulation of TNf&amp;#945; -Induced Cell Death By 3-Methyladenine, an Autophagy Inhibitor

Cell death by apoptosis is a fundamental mechanism for the maintenance of tissue homeostasis, activated to get rid of excess, damaged, or infected cells. It may be induced by several stimuli such as growth factor withdrawal, oxidative stress, drugs, or humoral mediators such as cytokines. The present study is focused on the cytotoxic activity of TNFα, a pleiotropic cytokine that promotes a variety of biological effects. TNFα cytotoxicity depends on the cell type and state. Although apoptosis (or type I cell death) has long been considered as the paradigm, various observations have stressed the possibility that TNFα-induced cell death may also develop with other features. Particularly interesting, in this regard, is the possibility that TNFα effects include stimulation of autophagy and/or induction of autophagic (type II) cell death. Aim of the present study has been to investigate if TNFα-induced death in cell types of different origin can bemodulated by interference with the autophagic process. The work has been performed on U937 (lymphoid), HT-29 (colon carcinoma), L929 (fibrosarcoma) and HTC (hepatoma) cell lines. All of them undergo apoptosis orapoptosis-like death in response to TNFα alone (U937, L929), or combined with transcription or translationinhibitors (HT-29, HTC). However, when TNFα is coupled with 3-methyladenine, a well known inhibitor of theautophagic process, the various cell lines show different behaviors: HTC cells are partially protected fromTNFα-induced death, whereas HT-29, L929 and U937 cells are sensitized, albeit to varying degrees, since thepercentage of apoptotic cells is higher than in cultures exposed to the cytokine alone. These results show that 3-methyladenine can modulate TNFα-induced death both positively and negatively,depending on the cell type. Moreover, they are consistent with the view that modulation of autophagy can berelevant to the design of therapeutic strategies aimed to reduce excess apoptosis, such as that occurring inacute hepatitis, or to enhance cell death in pathologies characterized by defective apoptosis, such as cancer.

Evaluation of antimutagenic activity and mechanisms of action of β-glucan from barley, in CHO-k1 and HTC cell lines using the micronucleus test

Due to the need to identify new antimutagenic agents and to determine their mechanism of action, the present study examined the mechanism of action of the β-glucan with regard to antimutagenicity using the micronucleus assay in CHO-k1 and HTC cell lines. The mutagenicity experiments were performed with three different concentrations of β-glucan (5, 10, and 20 μg/mL), in wich only the highest dose showed mutagenic activity. In the antimutagenicity experiments, the same concentrations of β-glucan were combined with a mutagenic agent, methylmethane sulfonate, or 2-aminoanthracene, using four different treatment protocols: pre-treatment, simultaneous treatment (simple and with pre-incubation), and post-treatment. The results indicate that the CHO-k1 cell line treated with MMS presented a chemopreventive activity for all the doses of β-glucan in the different treatment protocols, except for the lowest dose in post-treatment. When HTC cell line treated with MMS is analysed, a chemopreventive activity can be verified for the highest dose in both pre- and post-treatment. For the simple simultaneous treatment, the three doses demonstrated efficacy, while for the simultaneous treatment with pre-incubation only the intermediate concentration was effective. In HTC treated with 2AA both the lowest dose in the pre-treatment protocol and the post-treatment protocol did not show efficacy in preventing DNA damage. The evaluation of the different protocols and the damage decrease percentages observed suggest that β-glucan has both desmutagenic and bioantimutagenic activity. It is necessary, however, to note that efficacy and mechanism of action are subject to variation when compared the two cell lines, since in HTC, representing a drug-metabolizing system, this substance can show a diminished chemopreventive capacity.

Development of a flow cytometric assay to study glucocorticoid receptor-mediated gene activation in living cells

A flow cytometry-based reporter gene assay was developed and utilized to measure glucocorticoid receptor (GR)-mediated gene activation at the single cell level in living cells. A reporter gene was generated that contains two copies of the glucocorticoid response element and an E1b TATA box upstream of a destabilized enhanced green fluorescent protein. Glucocorticoid activation of the reporter gene in Cos-1 and HTC cell lines was measured in vivo by flow cytometry and was shown to be dose dependent, leading to an increase in total fluorescence of the cell population. Flow cytometric analysis indicated this increase in total fluorescence per sample resulted from an increase in the number of cells expressing the activated green fluorescent protein (GFP) reporter as well as an overall increase in the mean GFP fluorescence within cells. Activation of reporter gene activity was time dependent occurring as early as 1–2 h after dexamethasone addition. Activation of the reporter gene was specific as it exhibited different sensitivities to a range of glucocorticoids and activation could be blocked with glucocorticoid receptor antagonists. Coexpression of the coactivator SRC-1a or P65 subunit of NF-kappa B with GR led to enhancement or repression, respectively. Taken together, these data suggest the reporter-based flow cytometry assay is an effective method for analyzing glucocorticoid receptor-mediated gene expression at the single cell level in living cells.

Comparative Evaluation of Cytotoxicity and Metabolism of Four Aldehydes in Two Hepatoma Cell Lines

ABSTRACTThe metabolism of acetaldehyde (ACA), benzaldehyde (BA), propionaldehyde (PA) and valeraldehyde (VA) has been studied in two hepatoma cell lines, the rat HTC and mouse Hepa 1c1c7 cells. The cytotoxicity of the four aldehydes to these two cell lines has been compared. The end-points for evaluating cytotoxicity were 1) total macromolecular content (TMC) of confluent cultures, and 2) colony forming ability of dividing cells. These two assay systems had different sensitivities for the toxicity of aldehydes, probably due to different numbers of target cells.The activities of aldehyde dehydrogenases (NAD- and NADP-dependent, ALDH), alcohol dehydrogenase and aldehyde reductase were markedly greater in the HTC cell line compared to the Hepa 1c1c7 cell line, especially with BA as substrate.The cytotoxicities of aldehydes were generally stronger in the HTC cell line than in the Hepa 1c1c7 cell line, with the CF test. Particularly, BA was highly toxic to the HTC cells, which possessed the highest ALDH levels. Moreover, the treatment with (diethylamino)benzaldehyde, an ALDH inhibitor, completely abolished the toxicity of BA.Taken together, all these findings suggest that several cell lines expressing different aldehyde metabolizing activities could be used especially in the prescreening phase to distinguish the metabolism-dependent cytotoxic effects from the metabolism independent effects.

Enhanced secretion of glycocholic acid in a specially adapted cell line is associated with overexpression of apparently novel ATP-binding cassette proteins.

Secretion of anionic endo- and xenobiotics is essential for the survival of animal and plant cells; however, the underlying molecular mechanisms remain uncertain. To better understand one such model system--i.e., secretion of bile acids by the liver--we utilized a strategy analogous to that employed to identify the multidrug resistance (mdr) genes. We synthesized the methyl ester of glycocholic acid (GCE), which readily enters cells, where it is hydrolyzed to yield glycocholic acid, a naturally occurring bile acid. The rat hepatoma-derived HTC cell line gradually acquired resistance to GCE concentrations 20-fold higher than those which inhibited growth of naive cells, yet intracellular accumulation of radiolabel in resistant cells exposed to [14C]GCE averaged approximately 25% of that in nonresistant cells. As compared with nonresistant cells, resistant cells also exhibited (i) cross-resistance to colchicine, a known mdr substrate, but not to other noxious substances transported by hepatocytes; (ii) increased abundance on Northern blot of mRNA species up to 7-10 kb recognized by a probe for highly conserved nucleotide-binding domain (NBD) sequences of ATP-binding cassette (ABC) proteins; (iii) increased abundance, as measured by RNase protection assay, of mRNA fragments homologous to a NBD cRNA probe; and (iv) dramatic overexpression, as measured by Western blotting and immunofluorescence, of a group of 150- to 200-kDa plasma membrane proteins recognized by a monoclonal antibody against a region flanking the highly conserved NBD of mdr/P-glycoproteins. Finally, Xenopus laevis oocytes injected with mRNA from resistant cells and incubated with [14C]GCE secreted radiolabel more rapidly than did control oocytes. Enhanced secretion of glycocholic acid in this cell line is associated with overexpression of ABC/mdr-related proteins, some of which are apparently novel and are likely to include a bile acid transport protein.

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.

Evaluation of the Cytotoxicity of the First 20 MEIC Chemicals in Two Hepatoma Cell Lines with Different Xenobiotic Metabolism Capacities

The cytotoxicity of the first 20 chemicals on the MEIC list was evaluated using two hepatoma cell lines having different xenobiotic metabolic capacities, one derived from the rat (HTC), and one of human origin (Hep G2). Two endpoints were measured to evaluate cytotoxicity: colony-forming ability (CF), and cell viability of the attached monolayer (CV), evaluated as total macromolecular content. The CF test provided the most sensitive endpoint, due to the lower number of exposed cells in comparison with the CV test. Using the CF assay, the Hep G2 cell line showed higher sensitivity than the HTC cell line to some chemicals known to be metabolised in vivo. The IC50s obtained under the different experimental conditions varied as a consequence of phenotypic differences between the cell lines, and of the nature of the endpoints. The most toxic chemicals were malathion, diazepam and amitriptyline (IC50: 0.001-0.1mM). The solvents tested produced the lowest toxic effects (IC50: &gt; 10mM). These findings suggest that hepatoma cell lines possessing various specific enzyme activities could be usefully employed in a battery of tests designed to reproduce in vitro the wide range of biochemical properties expressed by the cells in the whole organism.

Specific growth stimulation by linoleic acid in hepatoma cell lines transfected with the target protein of a liver carcinogen.

The hepatic carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) was shown previously to interact specifically with its target protein, liver fatty acid binding protein (L-FABP), early during hepatocarcinogenesis in rats. In search of the significance of the interaction, rat L-FABP cDNA in the sense and antisense orientations was transfected into a subline of the rat hepatoma HTC cell line that did not express L-FABP. After the transfections, the basal doubling times of the cells were not significantly different. However, at 10(-5)-10(-7) M, linoleic acid, which is an essential fatty acid, a ligand of L-FABP, and the precursor of many eicosanoids and related lipids, stimulated the incorporation of [3H]thymidine in three randomly isolated and stably transfected cell clones that expressed L-FABP, but virtually did not stimulate the incorporation of [3H]thymidine in three L-FABP-nonexpressing clones transfected with the antisense DNA. Linoleic acid at 10(-6) M increased cell number almost 3-fold (38% vs. 14%; P less than 0.0001) and thymidine incorporation nearly 5-fold (23.2% vs. 4.9%; P less than 0.001) in the L-FABP-expressing cells compared to that in the transfected nonexpressing cells. L-FABP acted specifically and cooperatively with linoleic acid, inasmuch as all the proteins other than L-FABP in the transfected L-FABP nonexpressing cells and four other fatty acids (gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, and palmitoleic acid) were unable to effect a significant elevation or difference in the level of DNA synthesis that was attributable to the transfection. Metabolism of the linoleic acid to oxygenated derivatives was apparently necessary, since the cyclooxygenase inhibitor indomethacin partly inhibited and the antioxidant lipoxygenase inhibitors nordihydroguariaretic acid and alpha-tocopherol completely abolished the growth stimulation. The evidence supports the idea that L-FABP, the target protein of the liver carcinogen, acts specifically in concert with oxygenated metabolites of linoleic acid to modulate the growth of hepatocytes.

Use of Cultured Hepatoma Cell Lines in the Assessment of Aldehyde Metabolism

Aldehyde dehydrogenases (ALDH) represent a major pathway for the enzymatic removal of many potentially toxic aldehydes. The purpose of this study was to examine the basal levels of ALDH in five hepatoma cell lines chosen as representatives of three different species (man, rat, mouse) and their inducibility by some xenobiotics. Human HepG2, rat MH1C1, HTC, H4IIEC3 and mouse Hepa 1c1c7 cell lines were grown as monolayers. The ALDH activities were determined in cell homogenates from both unexposed control cultures and cells exposed to phenobarbital (PB), 3-methylcholanthrene (MC) and β-naphthoflavone (BNF). The ALDH activity was detected using benzaldehyde (BA) and propionaldehyde (PA) as substrates and both NAD and NADP as co-enzymes.Great variability in basal ALDH levels was found in the five cell lines: BA/NAD and BA/NADP enzyme activities were very high in the HTC cell line, intermediate in MH1C1 cells (near to normal rat hepatocytes) and very low in the remaining three cell lines. In HTC cells only, the PA/NAD activity was slightly induced by PB, but it remained unchanged under all the other experimental conditions. MH1C1 cells showed highly significant increases of all the activities with MC and BNF (up to 10-fold). The low basal activity of the H4IIEC3 cell line was significantly increased by MC and BNF, but only with BA/NADP. The Hepa 1c1c7 cell line responded only to BNF, as inducing compound, whereas the low basal enzyme levels of the human-derived HepG2 cell line were not significantly increased.These results suggest various applications of hepatoma cell cultures in in vitro toxicology.

Stable ornithine decarboxylase in a rat hepatoma cell line selected for resistance to α-difluoromethylornithine

Ornithine decarboxylase (ODC) is extremely unstable in mammalian cells. This unusual characteristic facilitates rapid fluctuations in the activity of this enzyme in response to variations in its biosynthesis. Unfortunately, very little is known about the mechanism or regulation of this ODC-specific proteolytic pathway. This study describes the production and characterization of a variant of the rat hepatoma HTC cell line that is strikingly deficient in this pathway. This cell variant was induced by selection for growth in stepwise increasing concentrations (up to 10 m m) of the irreversible ODC inhibitor, α-difluoromethylornithine (DFMO). Resistance to this inhibitor appears to result from a combination of elevated (10×) ODC biosynthesis and inhibited degradation, producing greater than a 2000-fold increase in the level of ODC protein. In these variant cells (DH23b) inhibition of protein synthesis by cycloheximide did not result in rapid loss of enzyme activity or ODC protein determined by radioimmunoassay. Pulse-chase studies with [ 35S]methionine confirmed that this enzyme was not preferentially degraded, even when spermidine was added to the media. ODC purified from the variant cells was found to be identical to the control cell enzyme in size, isoelectric point, substrate binding kinetics, and sensitivity to the inhibitor DFMO. Also, as in the control cells, a major fraction of the ODC molecules extracted from DH23b cells was shown to be phosphorylated on a serine residue. The inability to detect physical or kinetic differences between the parent and the variant cell ODC suggests that the unusual stability of ODC in this cell is associated with a defect in a cellular mechanism for ODC-specific degradation.

Evaluation of different enzyme activities involved in the sensitivity to xenobiotics in HTC and HEPG2 hepatoma cell lines

Evaluation of different enzyme activities involved in the sensitivity to xenobiotics in HTC and HEPG2 hepatoma cell lines

Role of estrogen receptors and antiestrogen binding sites in an early effect of antiestrogens, the inhibition of cholesterol biosynthesis

The effects of estradiol (E 2), 4-hydroxy-tamoxifen (OH-Tam), and LY117018 on cholesterogenesis were investigated in two human breast cancer cell lines (MCF-7 and BT20), and in rat hepatoma (HTC) and fibroblastic (NRK-49F) cell lines. It was found that 10 −10 M E 2 stimulated and 10 −8 M OH-Tam inhibited cholesterol synthesis in the estrogen-sensitive MCF-7 cell line. The OH-Tam effect occurred in less than 15 min whereas E 2 only stimulated after 8 h. The inhibition of cholesterol synthesis was not reversed by E 2 · E 2 was without effect in the HTC and estrogenresistant BT20 cell lines whereas OH-Tam was as effective as in the MCF-7 cells. LY117018 had nearly as much effect on cholesterol synthesis as OH-Tam, in both MCF-7 and BT20 cells. Neither E 2 nor OH-Tam had any effect on the NRK-49F cell line, even at micromolar concentrations. The three lines (MCF-7, BT20, HTC), whose cholesterol synthesis has been shown to be OH-Tam sensitive, appeared to contain high-affinity antiestrogen binding sites (AEBS); since the OH-Tamresistant line (NRK) only contained low-affinity AEBS, there appears to be some relationship between OH-Tam sensitivity and high-affinity AEBS content. This suggests that the cholesterogenesis inhibition induced by antiestrogens is ER-independent and may involve AEBS. The cholesterogenesis stimulation induced by E 2 occurred via a different pathway that appears to be related to the presence of ER in the cells.

Inhibition of hepatoma cell growth by a steroid anti-hormone

The antiproliferative effect of the potent antiglucocorticoid and antiprogestin RU38486, an 11β-substituted nor-steroid, was investigated on the HTC cell line. Its action clearly visualized in absence of serum, is reversible. Dexamethasone does whereas progesterone does not protect the cells against the cytostatic effect. Thus, the latter rather depends on the antiglucocorticoid-and not on the antiprogestin-function of RU38486. Taking into account the antiproliferative action on HTC cells together with the antiglucocorticoid behaviour in hepatocytes and hepatomas, our results suggest that RU38486 might represent a potential drug for liver tumour therapy.

Dual regulation of protein maturation in viral infected rat HTC hepatoma cells by glucocorticoids and progesterone.

Dexamethasone, a synthetic glucocorticoid, is required for full posttranslational maturation of mouse mammary tumor virus (MMTV) phosphoproteins and glycoproteins in M1.54 cells, a viral infected rat hepatoma (HTC) cell line. Pulse-chase radiolabeling with [35S]methionine revealed that steroids with known glucocorticoid activity (such as dexamethasone and hydrocortisone) regulated the maturation of both MMTV polyproteins in a manner proportional to their occupancy for glucocorticoid receptors and their biological potency. In contrast, progesterone selectively induced the proteolytic processing of MMTV phosphoproteins but simultaneously antagonized the dexamethasone-regulated maturation of MMTV glycoproteins and all other tested glucocorticoid responses. Exposure to suboptimal concentrations of both progesterone and dexamethasone fully stimulated the processing of MMTV phosphoproteins, suggesting that steroid receptors occupied with combinations of either steroid functionally interact at the putative maturation gene. Moreover, treatment with either actinomycin D, a potent inhibitor of de novo RNA synthesis, or RU38486, a synthetic antagonist of glucocorticoid and progesterone action, prevented both the dexamethasone and progesterone-regulated induction of MMTV phosphoprotein maturation. Sedimentation velocity and saturation binding analysis revealed that the sizes and concentrations of hepatoma cell progesterone and dexamethasone binding activities are similar while specific binding of the active progestin R5020 was not detected in either M1.54 cells or the glucocorticoid receptor deficient HTC cell line MSN6.10.2. Taken together, our results demonstrate that two distinct classes of steroid hormones can uniquely alter the posttranslational maturation of a specific subset of phosphoprotein substrates by a common glucocorticoid receptor-dependent process.

In vitro stimulation of cell growth by hepatic stimulator substance.

An extract of normal weanling or adult regenerating rat liver called hepatic stimulator substance (HSS) stimulates incorporation of tritiated thymidine ([3H]dThd) into liver DNA. In vivo, it is organ specific, species nonspecific, displays diurnal rhythm, and has a molecular weight of approximately 10,000-20,000. It has now been further characterized in vitro. HSS retained organ specificity, stimulating normal adult hepatocytes in primary culture, the HTC, PLC, and MH1C1 hepatoma cell lines, and the PRC rat liver cell line. It did not stimulate human or mouse lymphocytes, 3T3 mouse fibroblasts, Y1BS1 mouse adrenal cells, Chinese hamster ovary cells, rat hypernephroma cells, C6 rat glial cells, or L1210 mouse leukemic cells. More detailed studies of the response of HTC cells to HSS revealed a dose-dependent increase in [3H]dThd incorporation (up to 30-fold) with a response to as little as 140-ng purified extract/ml culture medium. Autoradiography confirmed that the thymidine was taken up by nuclear DNA. This represented a true stimulation of growth as shown by an almost twofold increase in cell number after 6 days of exposure to low doses of HSS.

Two distinct mechanisms for ornithine decarboxylase regulation by polyamines in rat hepatoma cells

Exogenous diamines and polyamines added to rat hepatoma (HTC) cells in culture rapidly decrease ornithine decarboxylase (ODC) activity. Previous evidence has suggested that these amines set either at the level of blocking new enzyme synthesis or by the induction of a non-competitive protein inhibitor, termed antizyme, which complexes with ODC to form an inactive complex. Wth the use of HMOA cells, a recently cloned rat hepatoma cell line that has a greatly stabilized ODC, it has been possible to demonstrate that 10(-5) M of exogenous putrescine blocks the increase in ODC activity, but unlike in the parent HTC cell line, without induction of the antizyme or formation of any inactive ODC-antizyme complex. However, complete blockade of ODC at 10(-2) M putrescine is effected by induction of antizyme and formation of the ODC-antizyme complex, as now evidenced by the isolation of the active enzyme and antizyme components after Sephadex column chromatography in the presence of 250 mM NaCl. These findings indicate clearly that two polyamine-regulatory mechanisms for ODC exist and are separable in this cell line.

Interaction of glucocorticoid hormones and cyclic nucleotides in induction of tyrosine aminotransferase in cultured hepatoma cells.

Reproducible induction of the enzyme tyrosine aminotransferase by dibutyryl cAMP (Bt2cAMP) in a line of HTC hepatoma cells in suspension culture requires that the cells be preinduced with dexamethasone, a synthetic glucocorticoid which itself induces tyrosine aminotransferase. Concentrations of dexamethasone that do not induce tyrosine aminotransferase fail to support Bt2cAMP induction, removal of the steroid from the medium leads to a loss of the Bt2cAMP effect, and an HTC cell line whose aminotransferase is not steroid-inducible does not respond to the cyclic nucleotide. We show that the further induction of tyrosine aminotransferase by Bt2cAMP in dexamethasone-treated cells is due to an increased rate of enzyme synthesis. The cyclic nucleotide has no effect on aminotransferase synthesis in cells grown in the absence of steroid. Several lines of evidence suggest that dexamethasone acts at a step beyond the activation of protein kinase by cAMP: (a) basal levels of cAMP are not altered by growth of HTC cells in dexamethasone; (b) accumulation of cAMP from the medium is not enhanced; (c) the glucocorticoid does not induce cAMP-dependent protein kinase in HTC cells; and (d) there is no augmentation of cAMP binding to the regulatory protein, nor is there any change in cAMP activation of protein kinase caused by growth in dexamethasone. These results help define a system that should be useful in studying the interaction of cyclic nucleotides and steroid hormones.

Regulation of Tyrosine α-Ketoglutarate Transaminase in Rat Liver: IX. STUDIES OF THE MECHANISMS OF HORMONAL INDUCTIONS IN CULTURED HEPATOMA CELLS

Abstract Synthesis of the messenger RNA that codes for tyrosine α-ketoglutarate transaminase (l-tyrosine:2-oxo-glutarate aminotransferase, EC 2.6.1.5) was blocked by actinomycin and its stability was determined in cultured hepatoma cells by measuring rates of transaminase synthesis. By this criterion the transaminase messenger RNA is degraded with a half-life of 2 to 3 hours in both H-35 and HTC cell lines; this rate is not changed by hydrocortisone. Transaminase synthesis is decelerated by high and low concentrations of actinomycin; high concentrations of this antibiotic block transaminase degradation as well. At low concentrations of actinomycin, transaminase induction by hydrocortisone is blocked but induction by insulin is unaffected for a period of time consistent with messenger stability. Preliminary treatment with hydrocortisone primes the cells to respond to insulin with a rapid increase in enzyme, but preliminary treatment with insulin does not alter the response to hydrocortisone. With the use of experimentally derived rates of degradation of both the transaminase and its messenger RNA, a mathematical model was developed which satisfactorily predicts kinetic behavior of the enzyme level upon addition and withdrawal of inducing hormones. The data are consistent with the conclusion that hydrocortisone acts transcriptionally while insulin acts on some posttranscriptional or translational step in enzyme synthesis. We find no indication of posttranscriptional mechanisms in the steroid-mediated induction. A discussion of the mathematical model used is appended.