Hormonal induction of tyrosine aminotransferase and RNA synthesis in the cells of Morris hepatoma strain 7777

The transfer of Morris hepatoma cells induced by the hormone within 10-60 min in to a hormone-free medium is associated with the augmentation of tyrosine aminotransferase synthesis. The kinetics of this process does not differ from that of the hormone-induced enzyme. The return of tyrosine aminotransferase synthesis to the basal level occurs 15-20 hours after the hormone withdrawal from the medium, although the concentration of the intranuclear hormone sharply decreases already after 3 hours. It was demonstrated that the presence in the hepatoma cell nuclei of 20-25% of the initially bound hormone for at least 20 hours after the cell transfer to the hormone-free medium is not sufficient for maintaining a high level of tyrosine aminotransferase gene expression. Using two-dimensional electrophoresis of 3H-labeled hepatoma cell proteins, it was demonstrated that the observed high activity of tyrosine aminotransferase is due to the de novo synthesis of enzyme molecules rather than to the existence of preformed long-living tyrosine aminotransferase molecules inside the cell. Study of [14C]uridine incorporation into non-ribosomal nuclear RNA of hepatoma cells showed a long-term presence of the label in the RNA throughout the chase experiment. It was assumed that the high activity of the enzyme for 10-15 hours after the hormone release from the hepatoma cell nuclei is due to the accumulation in the nuclei of long-living pre-mRNA molecules synthesized after the hormone addition to the cells and during the first hours after the cell transfer to the hormone-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase in hepatic tyrosine aminotransferase mRNA during enzyme induction by N6,O2'-dibutyryl cyclic AMP.

Tyrosine aminotransferase mRNA was quantitated by translation in a cell-free system derived from wheat germ followed by specific immunoprecipitation of the newly synthesized enzyme subunit. Hepatic poly(A)-containg RNA prepared from rats treated for 4 h with N6, O2'-dibutyryl cyclic AMP and theophylline was approximately 5.6 times more active in directing the synthesis of the tyrosine aminotransferase subunit relative to untreated controls. The overall template activity of the RNA prepared from control and cyclic AMP-treated animals was virtually identical, demonstrating that the cyclic nucleotide effect was specific for the tyrosine aminotransferase mRNA. At all times, after a single injection of dibutyryl cyclic AMP and theophylline, the increase in hepatic enzyme activity was accompanied by corresponding induction in the level of functional tyrosine aminotransferase mRNA. Other inducers of tyrosine aminotransferase, such as glucagon and hydrocortisone, also increased the level of tyrosine aminotransferase mRNA in proportion to their effect on enzyme activity. The RNA polymerase II inhibitor, alpha-amanitin, completely blocked the dibutyryl cyclic AMP-mediated increase in tyrosine aminotransferase mRNA activity. These studies demonstrate that, in intact animals, the induction of tyrosine aminotransferase activity by dibutyryl cyclic AMP can be completely accounted for by a corresponding increase in the level of functional mRNA coding for the enzyme.     

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.     

Evidence for a dual effect of dibutyryl cyclic AMP on the synthesis of tyrosine aminotransferase in rat liver

A single injection of dibutyryl cyclic AMP (Bt2cAMP) into adrenalectomized rats results in rapid and proportionate increases in hepatic tyrosine aminotransferase catalytic activity and in the amount of functional mRNA coding for this enzyme. This effect is transient in that mRNATAT peaks at 0.065% of total poly(A)+RNA activity at 1 h and is back to the basal level of 0.012% in 2.5 h. Enzyme activity peaks at 2.5 h and is back to the basal level by 5 h. If Bt2cAMP is repeatedly injected (0, 1, 2.5, and 4 h), enzyme activity remains at maximal levels for 4 to 5 h, whereas changes in mRNATAT activity are identical with those observed in the single injected rats. The rate of tyrosine aminotransferase synthesis at 5.5 h in the multiply injected rats, a time when mRNATAT has already returned to the basal level, is 3 to 4 times greater than that in either control or singly injected rats at the same time (0.3% of total protein versus 0.07%) and is equivalent to the maximal rate seen 1 h after the initial injection of the cyclic nucleotide. Since the rate of synthesis is increased in proportion to the increase in enzyme catalytic activity, stabilization of the enzyme against degradation is excluded as an induction mechanism at this late time point. These responses are not due to differences in the metabolism of Bt2cAMP, and the effect depends on the presence of metabolically active derivatives of this nucleotide. It thus appears that Bt2cAMP induces the synthesis of tyrosine aminotransferase in rat liver in two distinct ways. One is pretranslational and involves a transient and rapid increase in mRNATAT activity. The second appears to involve a delayed but sustained increase in translation of a basal level of mRNATAT.     

Effect of cortisol on the content of mRNA coding inducible tyrosine aminotransferase isoenzyme in rat liver]

In vitro estimation of synthesis of inducible tyrosine aminotransferase isoenzyme, directed by poly-A-containing RNA from liver of intact and corticol treated rats, is carried out. Total poly-A-containing RNA from liver polyribosomes of intact and induced rats was translated in cell-free system from wheat germs. Two antibodies immunoprecipitation was used to identify the translocation product (tyrosine aminotransferase). It was found that a synthesis of a specific protein product, precipitated by antibodies to tyrosine aminotransferase, takes place in cell-free system under translation of polysomic poly-A-containing liver RNA. The amount of immunoprecipitated product indicates, that the content of individual poly-A-containing mRNA for inducible tyrosine aminotransferase isoenzyme in liver of cortisol-induced rats is considerably higher than in intact animals.     

Dibutyryl cyclic AMP increases the amount of functional messenger RNA coding for tyrosine aminotransferase in rat liver.

The administration of N6,O2-dibutyryl cyclic AMP and theophylline to adrenalectomized rats results in an increase in the amount of functional mRNA coding for tyrosine aminotransferase that can be isolated from liver. The induction of this specific mRNA, as quantitated in a mRNA-dependent reticulocyte lysate system, and using poly(A)+ mRNA extracted from total tissue and polysomes, is very rapid. Within an hour after the intraperitoneal injection of the cyclic AMP derivative there is a 5- to 7-fold elevation of functional mRNA coding for tyrosine aminotransferase (mRNATAT), and by 3 h this has returned to basal levels. In contrast, the 4- to 5-fold induction of tyrosine aminotransferase catalytic activity is maximal at 2 h and is still significantly greater than the basal level at 5 h. In the basal state, tyrosine aminotransferase mRNA codes for 0.019 +/- 0.003% of the protein synthesized in the in vitro system, whereas after cyclic nucleotide treatment this value 0.115 +/- 0.015%, hence the increase in mRNATAT activity is relatively specific. Cordycepin, at a concentration which prevents the accumulation in cytoplasm of poly(A)+ mRNA, completely blocks the increase in both the catalytic and mRNA activity of this enzyme. The marked increase in functional mRNA, the requirement for continued synthesis of poly(A)+ RNA, and the rapid induction and deinduction suggest that the cyclic nucleotide is enhancing specific mRNA synthesis and/or, processing, however an effect on mRNA degradation cannot be excluded.     

The effect of sodium butyrate on tyrosine aminotransferase induction in primary cultures of normal adult rat hepatocytes

Treatment of primary cultures of adult rat hepatocytes with 5 mM butyrate inhibited the spontaneous decrease in basal activity and mRNA levels of tyrosine aminotransferase (TAT) that occurred during culture (Staecker et al., submitted). We report here that butyrate treatment of primary cultures of rat hepatocytes initially inhibited the induction of TAT. This inhibition was followed by a period of accelerated TAT induction. TAT induction in butyrate-treated primary cultures of adult rat hepatocytes occurred only after metabolism of butyrate by the cultured hepatocytes. The accelerated induction of TAT in hepatocyte cultures treated with sodium butyrate was reflected by increased TAT activity and mRNA levels. Cultured hepatocytes rapidly metabolized butyrate, but the addition of more butyrate into cultures after its initial metabolism resulted in a rapid reduction in TAT activity. These findings indicate that butyrate treatment can affect the expression of TAT in primary hepatocyte cultures in both a positive (increased basal TAT expression) and a negative (inhibition of the induced expression of TAT) manner.     

The effects of theophylline and certain other purine derivatives on tyrosine aminotransferase activity in hepatoma cells in culture

The induction of tyrosine aminotransferase in HTC cells by derivatives of adenosine 3′,5′-monophosphate is not potentiated by theopylline, a commonly used inhibitor of cyclic nucleotide phosphodiesterase. In fact, the addition of theophylline to HTC cell cultures produces a rapid decrease in the level of tyrosine aminotransferase activity. The magnitude of this decrease is dependent upon the added concentration of theopylline in both the presence and absence of enzyme inducers. Among several other purines and pyrimidines tested, caffeine and adenine most strongly resemble theophylline in affecting tyrosine aminotransferase activity. Theophylline inhibits growth and both protein and RNA synthesis in HTC cells, but the inhibition of protein synthesis cannot account completely for the effect on tyrosine aminotransferase. Theophylline also seems to increse the rate of degradation of the enzyme without affecting the degradation rate for general cellular protein. The mechanism of this apparently specific increase in degradation rate differs from both the normal degradation process for the enzyme and the enhanced degradation produced by nutritional depletion of the medium.     

Cycloheximide causes increased accumulation of translatable mRNA for tyrosine aminotransferase and tryptophan oxygenase in livers of cortisol-treated rats.

Messenger RNA activities for two cortisol-inducible enzymes, tyrosine aminotransferase and tryptophan oxygenase, have been determined by translation in a wheat germ system. The effects of cycloheximide on the two mRNA activities have been evaluated. Cortisol leads to an increase of the translatable mRNAs for tyrosine aminotransferase and tryptophan oxygenase with a maximum at approximately 6 h. Cycloheximide was administered 4 h after treatment with cortisol; 2 h later, the activities of tyrosine aminotransferase and tryptophan oxygenase mRNA had increased five-fold and two-fold, respectively, compared to the activities reached with cortisol alone. Thereafter the amount of the two translatable mRNAs declined, though 14 h after cortisol administration the mRNA activities were still several fold higher than in control animals. Application of alpha-amanitin together with cycloheximide did not prevent an increased accumulation of specific translatable mRNAs. The increase in tyrosine aminotransferase and tryptophan oxygenase activity by cortisol was immediately blocked by cycloheximide. Whereas tryptophan oxygenase activity rapidly declined after cycloheximide application, tyrosine aminotransferase activity remained at the same level. Approximately 4 h thereafter, both enzyme activities increased again.     

Translation of poly-A RNA from rat liver in vitro. Evidence for a high molecular weight subunit of tyrosine aminotransferase

Poly-A RNA extracted from the rat liver was translated in a cell-free wheat germ system and a rabbit reticulocyte lysate. The subunit of tryptophan pyrrolase precipitated by specific antiserum after synthesis in vitro has the same molecular weight as the corresponding subunit derived from the rat liver. With specific antiserum prepared against tyrosine aminotransferase, however, a radioactive protein from both the in vitro assays was precipitated with an about 5% higher molecular weight than the tyrosine aminotransferase subunit precipitated from rat liver. The immunological evidence and the comparison of the specific peptide patterns prepared by cyanogen bromide treatment showed that the in vitro product corresponds to tyrosine aminotransferase. Various concentrations of potassium or spermidine used in the wheat germ translation system did not alter the size of the enzyme subunit synthesized. The run of the tyrosine aminotransferase purified form the rat liver in the SDS-polyacrylamide gel electrophoresis was not influenced by treatment with Escherichia coli alkaline phosphatase. The possibility is discussed that the larger enzyme synthesized in vitro represents a precursor molecule which is cleaved proteolytically in vivo.     

Quantification of the importance of individual steps in the control of aromatic amino acid metabolism.

The quantitative importance of the individual steps of aromatic amino acid metabolism in rat liver was determined by calculation of the respective Control Coefficients (Strengths). The Control Coefficient of tryptophan 2,3-dioxygenase for tryptophan degradation was determined in a variety of physiological conditions and with a range of activities of tryptophan 2,3-dioxygenase. The Control Coefficient varied from 0.75 with basal enzyme activity to 0.25 after maximal induction of the enzyme by dexamethasone. The remainder of the control for tryptophan degradation was associated with the transport of the amino acid across the plasma membrane, with only very small contributions from kynureninase and kynurenine hydroxylase. The Control Coefficients of tyrosine aminotransferase for tyrosine degradation were approx. 0.70 and 0.20 with basal and dexamethasone-induced tyrosine aminotransferase activities respectively; the Control Coefficients of the transport of the amino acid into the cell were 0.22 and 0.58 respectively. Phenylalanine hydroxylase was found to have a Control Coefficient for the degradation of phenylalanine of approx. 0.50 under conditions of basal enzyme activity; after maximal activation by glucagon, the Control Coefficient decreased to 0.12. The transport of phenylalanine was responsible for the remaining control in the pathway. These results have important implications, directly for the regulation of aromatic amino acid metabolism in the liver, and indirectly for the regulation of neuroamine synthesis in the brain.     

Translation of mRNA from rat-liver polysomes into tyrosine aminotransferase and tryptophan oxygenase in a protein-synthesizing system from wheat germ. Effects of cortisol on the translatable levels of mRNA for these two enzymes.

Messenger RNA was isolated from rat liver polysomes by phenol/chloroform extraction and subsequent oligo(dT)-cellulose chromatography. The mRNA was translated in a protein-synthesizing system in vitro derived from wheat germ. The system was optimized in respect to Mg2+ and K+. The presence of spermidine or spermine is necessary for the synthesis of polypeptides having molecular weights of over 20 000. In the absence of the bases only small molecular weight products are formed. The amount of protein synthesized is linearly dependent on the amount of mRNA added up to concentrations of 80 mug mRNA/ml. The synthesis of tyrosine aminotransferase and tryptophan oxygenase in the system in vitro has been demonstrated by specific immunoprecipitation and sodium-dodecylsulfate polyacrylamide gel electrophoresis of the precipitate with enzyme proteins as marker. The amount of specific product formed is linearly dependent on the amount of mRNA present. The amount of translatable tyrosine aminotransferase mRNA and tryptophan oxygenase mRNA increases after administration of hydrocortisone to adrenalectomized rats. At low doses of hormone (2 mg/100 g body weight) maximal values are observed at 4 h, control levels being reached at 6-8 h after hormone application. With higher doses of hydrocortisone (20 mg/100 g body weight) maximal values are attained at 6 h, tending to control levels 14 h after treatment. The enzyme activity curves are parallel to the mRNA curves, the peak of enzyme activity occurring 2 h after the peak of mRNA activity.     

Immunological study of carbon tetrachloride-mediated induction of tyrosine aminotransferase in rat liver

Administration of CCl₄ (1.0 ml/kg) to rats resulted in a rise of liver tyrosine aminotransferase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) activity to a maximum of about 3.6 times the normal level 6 hr later. An immunological titration study proved that the phenomenon was due to increased enzyme content. Using an isotopic-immunochemical procedure the half-life of liver tyrosine aminotransferase at 3.5 hr after CCl₄ administration was shown to be 11.9 hr in contrast to 2.1 hr in the normal liver. Immunochemical analysis revealed that enzyme synthesis was decreased by CCl₄. Thus, in the early stage of CCl₄ poisoning, enzyme synthesis proceeded at a moderate rate while degradation was markedly impaired, resulting in the rise of tyrosine aminotransferase in the liver tissue.Several hours after administration of hydrocortisone to adrenalectomized rats, induced tyrosine aminotransferase reached its peak activity and then subsided to the basal level. At any time following hydrocortisone administration, administration of CCl₄ consistently caused an elevation of the enzyme activity above the level in controls not treated with CCl₄. Actinomycin D (5 mg/kg) also increased the enzyme at an early period of induction cycle but failed to do so at a later period.The CCl₄-mediated “superinduction” of hormonally preinduced tyrosine aminotransferase, like the induction of this enzyme by CCl₄ at a basal level, was found to be caused by the differential inhibitory effect of CCl₄ on the synthesis and degradation of this enzyme.     

Variations in some molecular events during the early phases of the Reuber H35 cell cycle. IV-regulation of tyrosine aminotransferase

Tyrosine aminotransferase activity increased during conversion of serum depleted quiescent Reuber H35 rat hepatoma cells into the proliferative state. Increased activity coincides with the actual increase of cells into S phase. The rate of tyrosine aminotransferase synthesis along the cell cycle was studied. The rate of enzyme synthesis fluctuated through the cell cycle but could not explain the increase of specific activity. Apparently enzyme activity is predominantly regulated by a post-translational event. Intracellular levels of cyclic AMP and cyclic GMP were measured at various times of G1 and S phases. In the early part of the cell cycle tyrosine aminotransferase decreased while intracellular levels of cyclic AMP increased. At later stages cyclic AMP rises concurrently with increased rates of enzyme synthesis. Induction of tyrosine aminotransferase by N6,O2'-dibutyryladenosine 3', 5'-monophosphate (Bt2cAMP) was studied. Inducibility by Bt2cAMP fluctuated through the cell cycle. Alternation of positive and negative control of tyrosine aminotransferase synthesis was observed. In early serum induced cells, Bt2cAMP increased enzyme activity without any increased rate of enzyme synthesis, on the contrary, a decreased rate of synthesis was observed. The data support the view that alternation of positive and negative control of tyrosine aminotransferase synthesis and temporary post-translational control of enzyme activity determine the enzyme level during the transition of quiescent hepatoma cells into proliferation.     

Morphological and biochemical differentiation in HeLa cells. Effects of cycloheximide on butyrate-induced process formation and ganglioside metabolism.

When butyrate-treated HeLa cells are trypsinized and replated in the absence of butyrate, their neurite-like processes re-extend transiently. Process formation after replating is prevented when the cells are exposed to cycloheximide during butyrate treatment, whereas it is not prevented by prior exposure to the calcium ionophore A23187 plus butyrate. These results indicate that butyrate induces protein(s) required for process extension which can accumulate in the absence of processing and promote processing in the absence of inducer. Transient process re-extension is followed by spontaneous retraction of processes and reversion to normal morphology. Reversion is not prevented or delayed by puromycin. Surprisingly, however, cycloheximide completely prevents reversion even at low concentrations (< 0.5 μg/ml). Levels of the ganglioside sialolactosylceramide (G_M3), synthesis of which is induced by butyrate, return to basal levels after removal of the inducer. Cycloheximide at 0.5 μg/ml prevents the decline of G_M3 levels after removal of butyrate although the biosynthetic enzyme sialyltransferase decays at the same rate in the presence or absence of the drug and the activity of the sialidase is not affected. The results further support the hypothesis that the ganglioside G_M3 is necessary for the morphological differentiation induced in HeLa cells by butyrate.     

Morphological alterations and ganglioside sialyltransferase activity induced by small fatty acids in HeLa cells

Incubation of HeLa cells in the presence of millimolar concentrations of propionate, butyrate, or pentanoate increases the specific activity of CMP-sialic acid:lactosylceramide sialyltransferase 7-20-fold within 24 h. Longer-chain saturated fatty acids or acetate are much less effective, decanoate showing no induction. Unsaturated fatty acid analogs of butyrate and other compounds are ineffective. Only the three most effective compounds also produce characteristic smooth extended cell processes in HeLa cells. Butyrate (5 mM) induces the sialyltransferase after a 4-h lag, producing maximum specific activity by 24 h. The amount of sialyl-lactosylceramide, the glycolipid product of the enzyme, increases during that time 3.5 times more than in control cultures. No other glycosphingolipid enzyme is significantly altered by butyrate exposure. The cellular shape changes occur 2-3 h later than the increase of sialyltransferase activity, and both processes require the continuous presence of inducer and the synthesis of RNA and protein but not the synthesis of DNA or the presence of serum.     

Effects of insulin on messenger RNA activities in rat liver

Liver poly(A) RNA, isolated from adrenalectomized rats after insulin treatment, was translated in a nuclease-treated lysate of rabbit reticulocytes and quantitated for both total activity and the capacity to synthesize the insulin-inducible enzyme tyrosine aminotransferase. Analysis of the translated products from poly(A) RNA isolated 1 h after insulin treatment showed a 2.7-fold increase in activity of tyrosine aminotransferase mRNA. During the same interval, the capacity of poly(A) RNA to direct the synthesis of total protein in lysates also changed, showing a 30 to 40% increase in translational activity/unit of RNA. Increased translatability was apparent in all fractions of poly(A) RNA separated by centrifugation on sucrose gradients. Insulin thus appears to mediate a generalized change in mRNAs leading to increased capacity for translation; induction of tyrosine aminotransferase may reflect sensitivity to effect of the hormone.