Nucleus-dependent regulation of tyrosine aminotransferase degradation in hepatoma tissue culture cells.

Upon removal of the nucleus from rat hepatoma tissue culture cells, levels of the enzyme tyrosine aminotransferase no longer change in response to withdrawal of glucocorticoids. The rate of tyrosine aminotransferase degradation is drastically reduced in rat hepatoma tissue culture cytoplasts leading to stabilization of pre-existing levels of tyrosine aminotransferase. Moreover, the rate of synthesis of the enzyme in cytoplasts is very low near that observed in uninduced whole cells. These effects of enucleation occur very rapidly and appear to be specific for tyrosine aminotransferase and a small number of other unstable hepatoma proteins. A nuclear effect is thus directly involved in the control of tyrosine aminotransferase degradation and synthesis.     

The involvement of cyclic GMP in tyrosine aminotransferase degradation in rat hepatoma tissue culture cells

Theophylline, a cyclic nucleotide phosphodiesterase inhibitor, increases the rate of tyrosine aminotransferase (TAT) degradation in rat hepatoma tissue culture (HTC) cells. Theophylline (0.1-10 mM) causes a two- to five-fold increase in intracellular cAMP concentration but a 30-60% decrease in cGMP concentration. The decrease in cGMP occurs at doses of theophylline which increase the rate of TAT degradation. When cGMP levels are increased by incubating the cells with either Mn2+, an activator of guanylate cyclase, or 8-bromo-cGMP, an analog of cGMP, the effect of theophylline is reversed and the rate of TAT degradation is slowed. Thus, the rate of TAT degradation is inversely related to the concentration of cGMP in HTC cells. This raises the possibility that a cGMP-dependent event is involved in the control of specific protein degradation.     

Inverse correlation between cyclic GMP and tyrosine aminotransferase degradation in rat hepatoma tissue culture cells

[1,2-³H]Cholesterol was epoxidized to radioactive cholesterol α- and β-epoxides (5,6α-epoxy-5α- and 5,6β-epoxy-5β-cholestan-3β-ols) in the ratio 1:4 by hepatic microsomal lipid hydroperoxides (MsOOH, 1 mM as active oxygen) in the presence of ferrous ion. MsOOH could be replaced by methyl linoleate hydroperoxides (MOOH) under the same conditions although the latter was less effective than the former. None of cumene hydroperoxide, t-butyl hydroperoxide, and hydrogen peroxide was an effective oxidant even at 10 mM. Neither ADP nor EDTA had an effect on the epoxidation of cholesterol by MsOOH as well as by MOOH. Ferrous ion could not be replaced by ferric ion in the hydroperoxide-mediated epoxidation. Cyanide anion potentially inhibited the reaction.     

The stability of tyrosine aminotransferase and other proteins in enucleated rat hepatoma tissue culture cells.

The role of the nucleus in bringing about the induction of tyrosine aminotransferase (TAT) by glucocorticosteroid hormone and its deinduction upon steroid removal has been studied in enucleated rat hepatoma tissue culture cells (FU5-5). Both processes require the presence of the nucleus. However, cytoplasts from preinduced cells show an initial rapid decline in enzyme activity immediately after enucleation followed by maintenance of a constant level of activity. This initial decline in enzyme activity can be partially prevented by trypan blue, an inhibitor of lysosomal activity. This suggests that the early fall in enzyme activity could be due to an increase in the level of lysosomal activity immediately after enucleation. The subsequent constant level of activity seems due to maintenance rather than synthesis and degradation since it is not affected by cycloheximide. The absence of degradation applies to other kinds of proteins in enucleated FU5-5 cells and enucleated mouse fibroblast L cells. These experiments suggest that some kind of labile RNA or protein dependent on the presence of the nucleus is required for the degradation of all classes of proteins in different kinds of cells.     

Effect of concanavalin A on tyrosine aminotransferase in rat hepatoma tissue culture cells. Rapid reversible inactivation of soluble enzyme.

Concanavalin A added to intact cells at 37 degrees caused rapid and reversible inactivation of a soluble enzyme, tyrosine aminotransferase, in two lines of rat hepatoma tissue culture cells grown in monolayer culture. This temperature-dependent process was independent of de novo protein and RNA synthesis and independent of increased uptake of Ca2+ and Mg2+ or glucose. The inactivation could be reversed by adding alpha-methyl-D-mannopyranoside a competing sugar for concanavalin A binding. Other lectins known to bind to different sugars did not bring about the inactivation of tyrosine aminotransferase. Addition of concanavalin A did not result in the inactivation of another soluble enzyme, lactic dehydrogenase. The maintenance of tyrosine aminotransferase in an inactive form after the binding of concanavalin A to the cells required the continued presence of concanavalin A. This effect of concanavalin A could not be mimicked either by dibutyryl cyclic adenosine or guanosine monophosphoric acid. Incubation of cell extracts with concanavalin A did not result in inactivation nor did mixing of extracts from concanavalin A-treated cells with extracts from untreated cells. On the basis of these results we conclude that the following are the essential requirements for concanavalin A to bring about the inactivation of tyrosine aminotransferase: (a) the binding of native concanavalin A to the cells; (b) integrity of certain structural elements of the cells.     

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.     

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)     

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.     

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.     

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.