Ketoconazole inhibition and glucocorticoid action in the human lymphoblastic leukemia cell line CEM-C7

The antifungal drug, ketoconazole, was reported to antagonize the induction of the enzyme tyrosine aminotransferase (TAT) by glucocorticoids in hepatoma tissue culture (HTC) cells, and to compete with glucocorticoids for binding to the glucocorticoid receptor. Since glucocorticoids inhibit the growth of the human leukemia cell line CEM-C7, ketoconazole might be expected to reverse this inhibition. Unexpectedly, ketoconazole inhibited CEM-C7 cell growth without utilizing glucocorticoid receptors. This was confirmed by ketoconazole inhibition of the growth of a receptor-less subline of CEM-C7 cells which are insensitive to glucocorticoids. Ketoconazole competed with triamcinolone acetonide (TA) for binding to the glucocorticoid receptor in cell-free supernatant prepared from CEM-C7 cells, but this was greatly reduced if ketoconazole and TA were incubated with intact cells prior to preparation of the cell-free supernatant. Ketoconazole inhibited induction by TA of the enzyme glutamine synthetase only at concentrations of 45-90 microM. We conclude that ketoconazole antagonism of glucocorticoid activity in CEM-C7 cells is probably not of pharmacologic significance due to the large concentrations required, and its reduced interaction with receptors in intact cells. The growth inhibitory activity of ketoconazole may be of interest in cancer chemotherapy.     

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.     

RU 38486: a potent antiglucocorticoid in vitro and in vivo

The antiglucocorticoid activity of RU 38486, was studied both in vitro and in vivo. In vitro studies, RU 38486 was characterized by a high affinity (3 times higher than that of dexamethasone) for the cytosolic glucocorticoid receptor in rat hepatoma tissue culture (HTC) cells. This high affinity was due to a very low dissociation rate of the complexes formed with the receptor. In whole cells it was a potent full antagonist of dexamethasone-induced tyrosine aminotransferase (TAT) activity: the IC50 was 6-7 times lower than the concentration of the dexamethasone used. It was devoid of any glucocorticoid activity up to a concentration of 10 microM. In in vivo studies using adrenalectomized rats, RU 38486 totally inhibited dexamethasone-induced hepatic tryptophan oxygenase (TO) activity. It is also the first pure antagonist of dexamethasone-induced hepatic TAT. However, doses as high as 5 mg/kg of body weight were required for a 50% inhibition of the effect of dexamethasone at 0.01 mg/kg. RU 38486 did not display any glucocorticoid effect on these two responses up to 50 mg/kg.     

Induction of tyrosine aminotransferase under the influence of D-galactosamine

The induction of the tyrosine aminotransferase by tyrosine and by tryptophan + methionine is completely inhibited by 375 mg/kg D-galactosamine-HCl. The hydrocortisone induction is reduced in dependence on the amount of D-galactosamine. Tryptophan protects to some extent the influence of low doses of D-galactosamine on the hydrocortisone induction of tyrosine aminotransferase.     

Pretranslational control of tyrosine aminotransferase synthesis by 8-bromo-cyclic AMP in H-4 rat hepatoma cells

The H-4 rat hepatoma cell line grown in tissue culture was used as a model system to investigate the action of cAMP in tyrosine aminotransferase induction. An immunoprecipitation technique was used to quantitate the amount and the rate of synthesis of tyrosine aminotransferase; the level of mRNA coding for tyrosine aminotransferase was determined by in vitro translation of poly(A)+ RNA isolated from hepatoma cells. Our results demonstrated that 8-bromo-cAMP gave time-dependent and proportionate increases in the tyrosine aminotransferase activity, the amount of immunoprecipitable tyrosine aminotransferase, the rate of synthesis of tyrosine aminotransferase, and the level of mRNATAT in H-4 hepatoma cells. The time course of increase in mRNATAT preceded the increase in synthesis of tyrosine aminotransferase and was dependent on the continuous production of poly(A)+ RNA. Pretreatment of the cells with cordycepin completely abolished the 8-bromo-cAMP-evoked increase in mRNATAT activity. These results provided evidence that the primary action of cAMP in tyrosine aminotransferase induction is the increase of functional mRNATAT and that this increase can completely account for the increase in tyrosine aminotransferase activity.     

Insulin-mediated inhibition of the induction of tyrosine aminotransferase by dexamethasone.

Insulin-mediated regulation of glucocorticoid-induced expression of the liver-specific gene tyrosine aminotransferase was studied in a clone of the Reuber rat hepatoma cells. Insulin inhibited dexamethasone-induced chloramphenicol acetyltransferase expression from approximately 4 kb of TAT 5' flanking sequence. The degree of this inhibition was comparable to the response of the endogenous gene. A construct of approximately 3 kbp of 5' flanking sequence exhibited no significant basal expression but retained sensitivity to glucocorticoids and to insulin inhibition of the glucocorticoid response. Results of further analysis of the insulin response in deletion constructs and constructs containing glucocorticoid responsive elements ligated to a heterologous promoter suggest that in addition to the glucocorticoid response elements a region close to the start site in the TAT promoter is necessary for insulin to inhibit glucocorticoid-mediated induction of expression.     

Inhibition by ultraviolet light of pole cell formation in Smittia sp (Chironomidae,Diptera): Action spectrum and photoreversibility

The formation of pole cells (primordial germ cells) in Smittia sp can be inhibited by ultraviolet (uv) irradiation without causing significant mortality. Until 70 min after egg deposition, pole cells are suppressed by low uv doses applied to the posterior pole region. Microbeam irradiation of a target area including the oosome inhibits pole cell formation; this is not observed after irradiation of other target areas. The action spectrum for uv inhibition of pole cells shows a distinct peak at 260 nm; its shape suggests that a nucleic acid or nucleic acid-protein complex acts as an effective target. Independent evidence for the involvement of a nucleic acid moiety is derived from the fact that uv inhibition of pole cell formation is photoreversible. The results are discussed in the context of pole cell determination by localized cytoplasmic components.     

Glucocorticoid regulation of hepatic cytosolic glucocorticoid receptors in vivo and its relationship to induction of tyrosine aminotransferase

Regulation of rat hepatic cytosolic glucocorticoid receptors was studied using our newly developed exchange assay. Injecting 1 mg of dexamethasone or corticosterone into 150-250 g adrenalectomized rats caused a rapid decline in glucocorticoid receptor binding. Glucocorticoid receptor levels were depressed 80-90% in less than 15 min after hormone treatment, and remained low for about 24-48 h after glucocorticoid administration. 80-90% of glucocorticoid receptor binding was regenerated by 48 h, and complete binding was recovered by 72 h. Regenerated glucocorticoid receptor binding (48-72 h after first hormone injection) could be re-depressed by a second injection of the hormone. Similar results were obtained using normal (intact) rats. Optimum induction of tyrosine aminotransferase activity was obtained within 2 h following the first hormonal injection. Induction of tyrosine aminotransferase activity (measured 2 h after a second injection of the glucocorticoid) correlated with glucocorticoid receptor levels. Thus, 1 mg of dexamethasone or corticosterone greatly enhanced the liver tyrosine aminotransferase activity in the adrenalectomized rats (not previously hormone treated) and in adrenalectomized rats previously injected (48-72 h) with 1 mg of the glucocorticoid hormone. Enhancement of tyrosine aminotransferase activity was lowest 16-24 h after the first hormone injection (when receptor levels were extremely low). These results indicate that the induction of liver tyrosine aminotransferase activity by glucocorticoid hormones is correlated with cytosolic glucocorticoid receptor levels.     

Evidence of far ultraviolet light-mediated changes in plasma membrane structure and function

Irradiation of plasma membrane preparations with 254 nm light increases its apparent microviscosity as measured with fluorescent polarimetry. Doses of $\text{3 · 10}{}^4$ J/m² increase the fluorescent polarization of a diphenylhexatriene probe by 10%. A similar increase is seen when whole cells are irradiated. The fate of membrane protein following irradiation was examined using SDS-polyacrylamide gel electrophoresis. Increasing the 254 nm doses reduces the amount of material in distinct bands on the gel and increases the amount of very low mobility material. No new bands of Coomassie blue staining material were observed. Irradiation of whole cells inhibited their attachment to concanavalin A-coated surfaces, an indication of a change in membrane function.     

Inhibition of semiconservative DNA synthesis in ICR 2A frog cells by pyrimidine dimers and nondimer photoproducts induced by ultraviolet radiation

DNA synthesis was examined in ultraviolet (uv)-irradiated ICR 2A frog cells in which either pyrimidine dimers or nondimer photoproducts represented the major class of DNA lesions. Dimers were induced by exposure of cells to 254 nm uv, while nondimer photoproducts were induced by irradiation of cells with uv produced by a fluorescent sunlamp (FSL) that was filtered through 48A Mylar (removes wavelengths less than 310 nm). The FSL-irradiated cultures were also treated with photoreactivating light (PRL) which removed most of the small number of dimers induced by the irradiation, leaving a relatively pure population of nondimer photoproducts. In addition, cells were exposed to 60Co gamma rays. The cultures were pulse-labeled and the size distribution of the DNA synthesized was estimated using both sucrose gradient sedimentation and alkaline step elution. Using either of these techniques, it was found that the presence of dimers resulted in a reduction principally in the synthesis of high molecular weight (MW) DNA. In contrast, nondimer photoproducts caused a strong inhibition in the synthesis of low MW DNA, as was also observed in gamma-irradiated cells. Hence the induction of pyrimidine dimers in DNA mainly affected the elongation of replicons, whereas nondimer lesions primarily caused an inhibition of replicon initiation.     

Inhibition by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, of enzyme induction by glucocorticoid and of nuclear translocation of glucocorticoid-receptor complexes

Induction of tyrosine aminotransferase by glucocorticoid in rat hepatocytes was inhibited concentration-dependently by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase C, but not by N- [2-(methyl-amino)-ethyl]-5-isoquinolinesulfonamide dihydrochloride, an inhibitor of cyclic nucleotide dependent protein kinases. H-7 also inhibited the accumulation of glucocorticoid-receptor complexes in the nuclear fraction with associated accumulation of these complexes in the cytoplasmic fraction, but did not affect incorporation of glucocorticoid into hepatocytes. These results indicate that protein kinase C may be essential in translocation of glucocorticoid-receptor complexes to the nuclei.     

Lysosomal involvement in apoptosis

Abstract

The involvement of reactive oxygen species (ROS) in the induction of DNA damage to Escherichia coli cells caused by UVC (254 nm) irradiation was studied. We verified the expression of the soxS gene induced by UVC (254 nm) and its inhibition by sodium azide, a singlet oxygen (¹O₂) scavenger. Additional results showed that a water-soluble carotenoid (norbixin) protects against the lethal effects of UVC. These results suggest that UVC radiation can also cause ROS-mediated lethality.     

The role of messenger RNA in tyrosine aminotransferase superinduction: effects of camptothecin on hepatoma cells in culture

Camptothecin inhibited the hydrocortisone but not the insulin induction of tyrosine aminotransferase activity in hepatoma cells in culture. However, camptothecin did not cause “superinduction” of tyrosine aminotransferase activity even though it reportedly inhibits messenger RNA synthesis. In hydrocortisone pre-induced cultures, camptothecin treatment caused a rapid decline in tyrosine aminotransferase activity suggesting it did not block degradation of the enzyme. A comparison of actinomycin D with camptothecin indicated that some of the effects of actinomycin D on tyrosine aminotransferase activity may not be mediated through inhibition of messenger RNA synthesis.     

Biphasic effects of translational inhibitors on liver tyrosine aminotransferase.

Low doses of cycloheximide or emetine cause rat liver tyrosine aminotransferase activity to rise up to twice the control levels in 2 h. By contrast, in the same interval no changes, or only a slight decrease, are produced by either drug at high dosage. Adrenalectomised animals display the same pattern of response. High doses of either antibiotic virtually afford a complete inhibition of 14C-labelled amino acid incorporation into liver and plasma proteins, whereas no more than a 30% decrease is observed with low doses. When administered in the course of the induction by cortisol, high doses of inhibitor prevent any further change in tyrosine aminotransferase activity, stabilising it at the level already attained; low doses, while slightly affecting the synthetic phase evoked by cortisol, drastically interfere with the deinduction. Six hours after various doses of either inhibitor the tyrosine aminotransferase activity is markedly increased, this late effect being largely dependent on the presence of adrenals. The amino acid incorporating actitivy of the liver may exceed that of controls, as observed particularly after small doses of emetine.     

The influence of culture conditions on the induction of tyrosine aminotransferase by cyclic nucleotides in rat hepatoma cells.

The increase in tyrosine aminotransferase activity which occurs in rat hepatoma tissue culture (HTC) cells in response to cyclic AMP analogs has been shown to be an enzyme induction, similar to the larger response observed in certain other hepatoma cells and in liver. A specific antibody to tyrosine aminotransferase has been used to show that the number of enzyme molecules and the rate of enzyme synthesis are increased by N6,O2'-dibutyryl cyclic AMP in HTC cells. The effect on tyrosine aminotransferase is also produced by various 8-substituted derivatives of cyclic AMP and occurs whether or not the enzyme has been preinduced with a glucocorticoid. The response of the enzyme is greater when HTC cells are maintained in monolayer than in suspension cultures. Neither cell growth nor serum is required for the response.     

Effects of UV-C radiation on extension growth, H+-extrusion and transmembrane electric potential in maize coleoptile segments

The effect of 253.7 nm ultraviolet radiation on elongation growth, medium acidification and changes in electric potential difference between vacuole and external medium in cells of maize ( Zea mays L.) coleoptile segments was investigated. It was found that irradiation with 390, 1170, 3900 and 5 850 J m⁻² UV-C (ultraviolet radiation 253.7 nm) inhibited elongation growth, whereas at 195 J m⁻² stimulation of growth was observed. The administration of IAA (10⁻⁵ M ) to the incubation medium of coleoptile segments partially abolished the inhibitory effect of UV-C. The pH of the incubation medium, measured simultaneously with growth, showed that the exposure of the segments to UV-C caused inhibition of H⁺-extrusion (or stimulation of H⁺ uptake). The presence of IAA (10⁻⁵ M ) in the incubation medium promoted (except after 5850 J m⁻² irradiation) H⁺-extrusion to a level comparable with that produced by IAA in non-irradiated segments. In UV-C irradiated segments the potential difference underwent significant alterations. Irradiation of coleoptile segments with 390 J m⁻² caused a transient depolarization, which was fully reversible within 30 min, while at higher doses depolarization was irreversible. The hyperpolarization of the membrane potential (MP) in cells of maize coleoptile induced by IAA was completely nullified by subsequent irradiation with UV-C. It is suggested that UV-C inhibited IAA-induced growth by a mechanism independent of cell wall acidification.     

Control of ketogenesis from amino acids: III. In vitro and in vivo studies on ketone body formation, lipogenesis and oxidation of tyrosine by rats

Ketone body formation from tyrosine was studied in rat liver in vitro with special references to the activities of tyrosine aminotransferase (EC 2.6.1.5) and p-hydroxyphenylpyruvate hydroxylase (EC 1.14.2.2). Liver was obtained from rats which had been given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase. The enzyme activities of the preparations were plotted against the amounts of ketone body formed from tyrosine. It was found that over a low range of tyrosine aminotransferase activities, activity was proportional to the amount of ketone body formed. However, above this range, ketone body formation ceased to increase and p-hydroxyphenylpyruvate started to accumulate. This inhibition of ketone body formation and accumulation of the p-hydroxyphenylpyruvate could be prevented by addition of ascorbate. These results suggest that the primary factor regulating metabolism of tyrosine in vitro is tyrosine aminotransferase and when the activity of this is high so that it is no longer rate limiting, p-hydroxyphenylpyruvate hydroxylase becomes the rate limiting step because its activity is inhibited by the accumulation of p-hydroxyphenylpyruvate.For in vivo studies rats were given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase and then injected with a tracer dose of [U- or 1-¹⁴ C]tyrosine. Then their respiratory ¹⁴CO₂ and the incorporation of ¹⁴C into total lipids of liver were measured. The amounts of radioactivity in CO₂ and lipids were found to be proportional to the tyrosine aminotransferase activity and were not affected by the free tyrosine concentration in the liver. After injection of [U-¹⁴C] acetate the radioactivities in CO₂ and lipids were not proportional to the tyrosine aminotransferase activity. These results indicate that the enzyme activity also regulates tyrosine metabolism in vivo. In vivo studies gave no evidence of the participation of p-hydroxyphenylpyruvate hydroxylase in regulation of tyrosine metabolism.     

Effects of UV-C and UV-B on cytomorphology and water permeability of inner epidermal cells of Allium cepa

Changes of cytomorphology and water permeability of inner epidermal cells of Allium cepa L. cvs Spartan and Keep Well were investigated with the light microscope after irradiation with UV-C (254 nm) and UV-B (280 and 310 nm). The sequence of the investigated changes of viscosity, protoplasmic streaming, organelle shape and water permeability was the same with 254 as with 280 nm. although a higher dose of 280 nm was needed to produce the same biologically equivalent effect. However, when calculated as a percentage of the lethal dose, the initial doses that produced the effects were the same for 254 and 280 nm. No changes could he observed at the cellular level after the cells were irradiated with 310 nm. The lethal dose depended upon the time between irradiation and observation. At 254 nm it was 1.1 kJ m⁻² up to 1 h after irradiation and dropped to 860 J m⁻² when measured 24 and 48 h later. At 280 nm a dose of 2.8 kJ m⁻² killed the cells within I h while the dose needed after 24 and 48S h was 1.99 kJ m⁻². The minimum doses which caused the different cytomorphological effects did not depend upon the observation time. Normal cell structure and functions that were altered immediately alter irradiation did not recover. Doses that were not immediately effective alter irradiation caused no later damage. Doses which increased water permeability were much higher than doses which influenced cytomorphological parameters of the cell.