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Interaction of 5-azacytidine and dexamethasone in the control of hepatic tyrosine aminotransferase
INTERACTION OF5-AZACYTIDINE AND DEXAMETHASONE IN THE CONTROL OF HEPATIC TYROSINEAMINOTRANSFERASE A. SCHULZE, H.-J. BOHME, W. GOLTZSCH and E. HOFMANN Institute of Biochemistry, University of Leipzig School of Medicine, 7010 Leipzig, Germany
INTRODUCTION
Perinatal development of rat liver is associated with major changes in morphology, notably loss of the hematopoietic cells with a concomitant increase in size and number of parenehymal liver cells and in metabolic capacity. The hepatocytes attain much of their mature functional abilities during this life period. Characteristic enzymes expressed in adult liver have been shown to be absent throughout most of intrauterine life and to appear at definite times just before or after birth (1, 2). There is now ample evidence that the basic mechanisms underlying this developmental regulation of gene expression act primarily at the transcriptional level. Methylation and demethylation of specific cytosine residues in DNA is one of the mechanisms controlling the responsiveness of eukaryotic genes towards transcriptional activators. Transcriptionally active genes are often less methylated than silent genes (for a review see Ref. (3)). 5-Azacytidine has been found to be a useful tool in working out the critical role of DNA methylation in cellular differentiation. When 5-azacytidine is incorporated into DNA it inhibits its enzymic methylation at position 5 of cytosine. In recent years, the growing interest in the role of enzymic DNA methylation in gene regulation has prompted studies on the effects of 5-azacytidine on cell differentiation and cell functions. It has been demonstrated that 5-azacytidine incorporation into genomic DNA leads to DNA hypomethylation (4, 5). Experimental studies have demonstrated the differentiation of muscle cells, adipocytes and chondrocytes in C3H 10T1/2 mouse embryonic fibroblasts exposed to 5-azacytidine for a short period of time (6-8). This differentiation never occurs in culture unless it is exposed to 5-azacytidine. Moreover, it has been shown in several systems that 5-azacytidine-indueed hypomethylation of genomic DNA is associated with specific activation of previously silent genes such as: metallothionine-1 inducibility in mice (9), cellular thymidine kinase (EC 2.7.1.21) in Chinese hamsters (10), fetal hemoglobin in baboon 247
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and human cells (11), prolactin (12) and tyrosine aminotransferase (TAT, L-tyrosine : 2-oxoglutarate aminotransferase, EC 2.6.1.5) (13-15) in fetal rats. The present paper deals with the action of 5-azacytidine on the inducibility of tyrosine aminotransferase in the liver of suckling rats. Dexamethasone multiples the 5-azacytidine effect, while adrenalectomy prevents the induction by the drug. These results point to an involvement of glucocorticoids. On the other hand, the antiglucocorticoid RU 486, being able to suppress the dexamethasone-induced accumulation of tyrosine aminotransferase very effectively, does not abolish the action of 5-azacytidine. In order to clarify the role of glucocorticoids in this mechanism, the influence of 5-azacytidine on the concentration of the glucocorticoid receptor, its binding affinity as well as its chromatographic behavior on an anion exchanger, were investigated. MATERIALS AND METHODS Chemicals. Substrates and 5-azacytidine were obtained from Boehringer Mannheim GmbH (FRG). Dexamethasone was purchased from KRKA, Novo Mesto (Yugoslavia), estradiol and progesterone from VEB Jenapharm, Jena (FRG). The antiglucocorticoid RU 486 was a generous gift from the Central Institute of Microbiology and Experimental Therapy, Jena. All other chemicals were of the highest purity commercially available. Animals. Rats of a Wistar strain bred in this Institute were used. 5-Azacytidine was administered by i.p. injection in saline immediately after preparation of the solution because of its relative instability. The animals were sacrificed by exsanguination under light ether anesthesia. Determination of glucocorticoid receptor. Livers were homogenized in 6 vol of ice-cold 50 mM Tris/HC1 buffer pH 7.5 containing 50 mM KCI, 20 mM 2-mercaptoethanol, 10 mM sodium molybdate and 20% (v/v) glycerol (buffer A). For the determination of the cytosolic receptor the homogenate was then centrifuged at 90,000 x g for 60 min at 4°C. The resulting supernatant was aspirated and used as cytosol. Aliquots of the cytosol were incubated with 100 nM [1,2,4-(n)-3H]dexamethasone (sp. act., 45 Ci/mmol, Amersham, Buckinghamshire (UK)) in the absence or presence of a 1000-fold excess of unlabelled hormone at 0°C for 4 hr. Unbound dexamethasone was removed by incubation with 5 vol of 2% (w/v) dextran-coated charcoal as described in (16) and subsequent centrifugation for 5 min at 11,000 x g. Aliquots of the supernatant were assayed for radioactivity. For the determination of the nuclear glucocorticoid receptor a modi-
5-AZACYTIDINE AND CONTROL OF HEPATICTAT
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fication of the procedure described in (17) was used. The liver homogenate was centrifuged at 900 x g for 10 rain. The pellet was suspended in 20 mM Tris/HCl buffer pH 7.5, 2.2 M sucrose and 3 mM CaCI 2 (TSS-buffer) and centrifuged at 40,000 x g for 30 min. The nuclei pellet was washed with TSS-Buffer and resuspended in the same buffer. Aliquots were incubated with [3H]-labeUed-dexamethasone as described above in the absence or presence of a 1000-fold excess of unlabelled hormone for 30 min at room temperature. The nuclei were then sedimented by centrifugation at 11,000 x g for 1 rain and the supernatant was carefully removed. The pellet was washed once with 0.25% (w/v) Triton X-100 in TSS-buffer in order to remove the outer nuclear membrane, which binds dexamethasone unspecifically, and three times with TSS-buffer. The final pellet was taken up in concentrated formic acid and transferred to scintillation vials containing 4 ml Bray solution.
High-performance ion-exchange chromatography. Cytosol was incubated for 4 hr at 4°C with 100 nM [1,2,4-(n)-aH]dexamethasone. After removal of unbound steroid by chromatography on Sephadex G-25, the samples were applied to a Mono Q H R 5/5 column connected to a Pharmacia FPLC system. The column was washed with buffer A at a flow rate of 1 ml/min. Elution was carried out with a linear 0-0.4 M NaC1 gradient in the equilibration buffer. Other assays. Tyrosine aminotransferase activity was assayed as described in (18) and serine dehydratase (SDH, L-serine hydrolyase, EC 4.2.1.13) by a modification of the procedure described in (19). Protein was measured using the dye-binding assay by Bradford (20) with human serum albumin as standard and D N A concentrations were estimated according to (21). RESULTS AND DISCUSSION
Induction of Tyrosine Aminotransferase Activity by 5-Azacytidine The nucleoside analogue 5-azacytidine is able to regulate hepatic gene expression in a highly targeted gene-specific manner. Only a subset of liver-specific genes, including tyrosine aminotransferase (13-15), phosphoenolpyruvate carboxykinase (EC 4.1.1.32) (15) and a gene of unknown specificity (gene 33) (15), is induced by the action of this compound in the liver of fetal rats (13, 14). These target genes share additional characteristics of regulation. They are all inducible via the cAMP pathway and by glucocorticoids. On the other hand, a number of enzymes, such as phosphofructokinase-2 (EC 2.7.1.105) (14), glucose 6phosphatase (EC 3.1.3.9) and tryptophan 2,3-dioxygenase (EC 1.13.11.11)
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(15), regulated by the same hormonal stimuli, are insensitive against 5-azacytidine. Treatment of fetal rats with 5-azacytidine also changes hepatic morphology. The hepatocytes appear larger, more basophilic and better aligned in sinusoidal array, wherees the erythroid cell population is little affected by the drug (15). These results gave rise to the conclusion that in fetal rat liver 5-azacytidine administration causes a drug-induced advancement of processes normally programmed to occur later. Moreover, because the potent antiglucocorticoid 5-ot-pregnane-3,20-dione abolishes the "overshoot" in tyrosine aminotransferase expression occurring about 12 hr after birth, but does not influence either the transition from the fetal to the adult level of tyrosine aminotransferase expression (22) or the precocious induction of tyrosine aminotransferase by 5-azacytidine (15), it has been concluded that glucocorticoids are not involved either in the normal or in the 5-azacytidine-induced activation of tyrosine aminotransferase expression (15). In other words, 5-azacytidine is believed to cause an activation of selected genes similar to those occurring during normal development. This activation elicits glucocorticoid responsiveness, and as the levels of these steroids are high in the neonatal rat the maturation appears to be amplified by glucocorticoid induction causing the typical "overshoot" production of tyrosine aminotransferase.
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FIG. 1. Time dependence of the induction of tyrosine aminotransferase by 5-azacytidine an¢ dexamethasone. A n i m a l s were injected with 15 ~ g 5-~acytidine/g body weight and 10 ~ d e x a m e t h a s o n e / g body weight. T h e controls obtained the same vol saline. Results are giver as m e a n s of 5--8 animals per group + S.E.M. C) - - - O control. Q - - - • d e x a m e t h a s o n e ..~7=,-,,t,n,,,,~. V __ _
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However, this concept does not explain the induction of tyrosine aminotransferase activity in suckling rat liver by a single injection of 5-azacytidine (15/zg/g body weight) which is clearly detectable already after 5 hr and reaches maximum values approximately 12 hr after the application of the drug (Fig. 1). In comparison to the induction by dexamethasone (10/~g/g body weight), the accumulation of tyrosine aminotransferase activity caused by 5-azacytidine occurs with a lower rate but reaches higher maximum values. Dexamethasone stimulates tyrosine aminotransferase within 5 hr about 4- to 5-fold, while in 5-azacytidine-treated animals only a 2- to 3-fold induction of tyrosine aminotransferase is observable at this time. Twelve hr after the injection, however, an 8- to 10-fold induction of tyrosine aminotransferase by dexamethasone and a 10- to 20-fold induction of the enzyme by 5-azacytidine is found. 5-Azacytidine also acts synergistically with progesterone which is a weak inducer of tyrosine aminotransferase in suckling rat liver under the conditions applied: enzyme levels 12 hr after treatment were 110.1 _+ 32.0 units/g protein in progesterone-treated animals, and 474.0 _+ 40.4 units/g protein in those treated with 5-azacytidine as well. The induction by estradiol of tyrosine aminotransferase is also slightly increased when the hormone is injected together with the drug, however, at a statistically not significant level (Table 1).
T A B L E 1. INFLUENCE OF 5-AZACYTIDINE ON THE INDUCTION OF TYROSINE AMINOTRANSFERASE AND SERINE D E H Y D R A T A S E IN THE LIVER OF SUCKLING RATS Activity (units/g protein) Treatment Saline 5-Azacytidine Dexamethasone Dexamethasone + 5-azacytidine Estradiol Estradiol + 5-azacytidine Progesterone Progesterone + 5-azacytidine
TAT
SDH
34.1 + 2.8 431.2 + 38.7* 327.5 _+ 50.9*
15.1 + 6.2 14.1 + 4.3 41.3 _+ 4.0*
625.9 + 73.5",t 224.3 + 34.0*
12.2 + 3.1t 29.3 + 3.8*
329.3 + 42.6* 110.1 + 32.0*
11.9 + 1.2t 25.0 + 4.5*
474.0 + 46.4",t
13.9 + 1.7t
Results are given as means of 5--6 animals per group + S.E.M. The hormones were applied to 21-day-old rats using the following dosages: 20 ~tg progesterone/g body weight; 20/zg estradiol/g body weight; 5 ~zg dexamethasone/g body weight; 15/zg 5-azacytidine/g body weight. The animals were killed 12 hr later. *Means significantly different from the control at the 0.05 level. tMeans significantly different at the 0.05 level from the group of animals treated with the respective hormone only.
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A. SCHULZE, et al. TABLE 2. INFLUENCE OF 5-AZACYTIDINE ON TYROSINE AMINOTRANSFERASE ACTIVITY ON ADRENALECTOMIZED SUCKLING RATS Treatment
TAT activity (units/g protein)
Control 5-Azacytidine Dexamethasone
133.8 _+ 9.5 (4) 156.1 _+ 33.6 (4) 702.9 ± 296.2 (4)
Bilateral adrenalectomy was performed via the dorsolateral route 3 days before the experiment at the 22rid day of life. The animals were supplemented by daily injection of 0.05 ~g deoxycorticosterone/g body weight. The animals obtained intraperitoneally 10/zg/g body weight dexamethasone or 10/z/g body weight 5-azacytidine and were killed 20 hr later. Control animals obtained the same vol saline. Results are given as mean -+ S.E.M. with the number of animals in parenthesis.
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FIG. 2. Influence of the antiglucocorticoid RU 486 on the induction of tyrosine aminotransferas¢ (A) and serine dehydratase (B) by dexamethasone. Dexamethasone (2/~g/g body weight) was injected i.p. 1 hr after RU 486. The animals were sacrificed 15 hr later. Results are given as means of 6-9 animals per group ± S.E.M.
5-AZACYTIDINE AND CONTROL OF HEPATIC TAT
253
Serine dehydratase which behaves very similarly to tyrosine aminotransferase in its developmental activation is not stimulated by 5-azacytidine under these conditions. On the contrary, the drug abolishes the induction of the enzyme by all three steroid hormones tested (Table 1). Bilateral adrenalectomy renders the liver unresponsive towards the stimulating effect of 5-azacytidine (Table 2). These results point to an involvement of glucocorticoids in the mechanism by which 5-azacytidine activates the tyrosine aminotransferase expression in suckling rat liver. This implication was explored further by studying the influence of the antiglucocorticoid RU 486 on the induction of tyrosine aminotransferase by 5-azacytidine. Administration of this antagonist at a dosage (20/~g/g body weight) which inhibits the induction of tyrosine aminotransferase and serine dehydratase by dexamethasone very effectively (Fig. 2) has no influence on the stimulation of tyrosine aminotransferase activity by 5-azacytidine (Fig. 3).
Influence of 5-Azacytidine on the Glucocorticoid Receptor The binding affinities of the glucocorticoid receptor for radioactive labelled dexamethasone were found to be similar in 5-azacytidine-
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FIG. 3. Ineffectiveness of RU 486 to inhibit induction of tyrosine aminotransferase by
5-azacytidine.Results are givenas means _+S.E.M. of 6-7 animalsper group.
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and saline-treated animals (Ka = 3.5 x 10-9 versus 4.5 x 10-9, Fig. 4). However, the number of cytosolic glucocorticoid receptors is decreased after the administration of 5-azacytidine (Table 3). The decline of cytosolic glucocorticoid receptor occurs very rapidly after treatment with 5-azacytidine. Apparently, this is not an experimental artifact caused by a competition of the drug with the labelled dexamethasone in the binding assay because it was found that 5-azacytidine (up to 0.25 mM)
TABLE 3. INFLUENCE OF 5-AZACYTIDINE ON THE CYTOSOLIC GLUCOCORTICOID RECEPTOR Time after injection (hr) 0.5 1 5 12 24 Control (without injection)
Glucocorticoid receptor (pmol/mg protein) Control 5-Azacytidine Dexamethasone 0.056 0.098 0.069 0.060 0.083
+ + + + +
0.012 0.004 0.012 0.008 0.012
0.046 0.041 0.036 0.029 0.039
+ + + + +
0.006 0.006 0.008 0.005 0.006
0.005 + 0.002 0.001 + 0.001 n.d. 0.002 + 0.001 0.018 + 0.004
0.079 + 0.008
n.d. - - not detectable. The animals (3 weeks old) were injected with 10/~g dexamethasone/g body weight or 15 ~g 5-azacytidine/g body weight. The control group received the same vol of the vehicle fluid only. The results are given as means + S.E.M. of 5-9 animals per group.
5-AZACYTIDINE AND CONTROL OF HEPATIC TAT
255
does not interfere with the receptor determination. The slight decrease of the cytosolic glucocorticoid receptor in the control animals detectable 30 rain after injection very likely reflects a stress effect due to the handling of the animals. In the 5-azacytidine-treated group, the concentration of cytosolic glucocorticoid receptor was found to be approximately 50% of the control even 24 hr after the application of the drug. The decrease of the glucocorticoid receptor concentration is comparable with the down-regulation of the glucocorticoid receptor by dexamethasone but smaller in magnitude and longer lasting. In the dexamethasone group, partial regeneration of the cytosolic glucocorticoid receptor can be seen about 24 hr after the injection (Table 3). As shown in Table 4, the fast decrease of cytosolic glucocorticoid receptor is obviously accompanied by an increase of the glucocorticoid receptor concentration in the nuclei. In all three groups of animals the increase of the glucocorticoid receptor in the nuclei is significantly different from the control group (untreated animals) with p < 0.05. In the saline-treated controls and in the dexamethasone group the level of nuclear glucocorticoid receptor returns within 12 hr to the normal value. In the 5-azacytidine group the concentration of nuclear glucocorticoid receptor remains elevated for this period of time; however, because of the high variation observed, only the 5 hr-value is statistically different from the respective saline-treated control. The antiglucocorticoid RU 486, which is unable to inhibit the induction by 5-azacytidine of tyrosine aminotransferase, is also not capable of preventing the decline of cytosolic glucocorticoid receptor in 5-azacytidine-treated animals. On the contrary, the glucocorticoid receptor concentration was found to be lower in animals which obtained the antiglucocorticoid along with 5-azacytidine (results not shown). However, it cannot be excluded
T A B L E 4. INFLUENCE OF D E X A M E T H A S O N E AND 5-AZACYTIDINE ON THE CONCENTRATION OF THE N U C L E A R GLUCOCORTICOID RECEPTOR Time (hr) 0.5 5.0 12.0 Control (without injection)
Glucocorticoid receptor (pmol/mg DNA) Saline Dexamethasone 5-Azacytidine 0.783 _+ 0.221 0.407 _+ 0.222 0.100 _+ 0.024
1.120 + 0.326 0.987 + 0.402 0.083 _+ 0.026
0.973 _+ 0.159 1.063 + 0.063* 0.417 + 0.125
0.040 _+ 0.012
The 21-day-old rats were injected with I0/~g dexamethasone/g body weight or with 15/tg 5-azacytidine/g body weight. The control group obtained the same vol saline. Results a r e given as means + S.E.M. of 4--8 animals per group. *Statistically different from the respective saline-injected animals at the 0.05 level.
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that this result is artificial, because RU 486 possesses a high affinity to the glucocorticoid receptor (25) and interferes with the receptor assay. The results point to an influence of 5-azacytidine on the activation of glucocorticoid receptor and cannot be explained in terms of alterations in the degree of DNA methylation since this process depends on de n o v o synthesis of DNA which takes place with a much slower rate than the observed induction of tyrosine aminotransferase and translocation of the glucocorticoid receptor into the nuclei. In the absence of steroid hormone a very large fraction of the glucocorticoid receptor is found in the cytosol and sediments as heterooligomer (designated as 8S-R) with an apparent mol. wt. of 300 k (24). In addition to the hormone-binding receptor species the fraction contains the heat-shock protein hsp 90 which is believed to be a "cap" for the receptor interacting with its DNA-binding site. Upon hormone binding, the 8S-receptor is physiologically transformed (activated). This change of structure is responsible for the release of hsp 90 from the hetero-oligomeric form of the receptor, forming the 4S-receptor species. Its putative DNA binding site may then interact with glucocorticoid responsive elements (GRE) of DNA. RU 486, a 19-norsteroid, stabilizes the 8S form of glucocorticoid receptor as shown by a number of studies in different mammalian tissues including rat liver (25). Therefore, the 5-azacytidine effect on the glucocorticoid receptor represents very likely not an influence of the drug on the transformation of the 8S- into the 4S-receptor species but may be a consequence of acting on a later step of glucocorticoid receptor activation such as translocation of the 4S-species into the nuclei.
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Anion Exchange Chromatographyof GlucocorticoidReceptor Chromatography of suckling rat liver cytosol on a Mono Q column in the presence of molybdate revealed independent of the treatment of the animals only the presence of the "classic" glucocorticoid receptor (designated as binder II) (Fig. 5). In this life span the glucocorticoid receptor species described by Hirota et al. (26) as "Peak C" was not detectable.
SUMMARY
The nucleoside analog 5-azacytidine is able to induce tyrosine aminotransferase several-fold in the liver of suckling rats. Bilateral adrenalectomy abolishes this inducing effect. The drug also decreases significantly the concentration of cytosolic glucocorticoid receptor accompanied by an increase of the glucocorticoid receptor concentration in the nuclei. The antiglucocorticoid RU 486 which abolishes the induction of tyrosine aminotransferase and serine dehydratase by dexamethasone very effectively is not able to inhibit either the induction of tyrosine aminotransferase or the translocation of the glucocorticoid receptor into the nuclei by 5-azacytidine. REFERENCES 1. O. GREENGARD, Enzymic differentiation in mammalian liver, Science 163, 891-895 (1969). 2. R. BITTNER, H.-J. BOHME, L. DIDT, W. GOLTZSCH, E. HOFMANN, M. J. LEVIN and G. SPARMANN, Developmental changes in the levels of hepatic enzymes and their relation to metabolic functions, Advan. Enzyme Regul. 17, 37-57 (1978). 3. W. DOERFLER, DNA methylation and gene activity, Ann. Rev. Biochem. 52, 93-124 (1983). 4. P. A. JONES and S. M. TAYLOR, Hemimethylated duplex DNAs prepared from 5-azacytidine treated cells, Nucleic Acid Res. 9, 2933-2947 (1981). 5. V. MAHARAJAN, L. TOSI, M. PRATIBHA and E. SCARANO, Analogues of 5-methyl cytosine and early embryonic development, Prog. Clin. Biol. Res. ~ A , 227-234 (1982). 6. S.M. TAYLOR and P. A. JONES, Multiple new phenotypes induced in 10 T1/2 and 3T3 cells treated with 5-azacytidine, Cell 17,771-779 (1979). 7. L. LUI, M. HARRINGTON and P. A. JONES, Characterization of myogenic cell lines derived by 5-azacytidine treatment, Dev. Biol. 117, 331-336 (1986). 8. P.G. CONSTANTINIDES, S. M. TAYLOR and P. A. JONES, Phenotypic conversion of cultured mouse embryonic cells by azapyrimidine nucleosides, Dev. Biol. 66, 57-71 (1978). 9. S. J. COMPERE and P. D. PALMITER, DNA methylation controls the inducibility of the mouse metallothionine-Igene in lymphoid cells, Cell 25, 233-240 (1981). I0. M. HARRIS, Induction of thymidine kinase in enzyme-deficient Chinese hamster cells, Cell 29,483--492 (1982). 11. J. de SIMONE, P. HELLER, L. HALL and D. ZWlERS, 5-Azacytidine stimulates fetal hemoglobin synthesis in anemic baboons, Proc. Natl. Acad. Sci. USA 79, 4428-4431 (1982).
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12. R.D. IVARIE and J. A. MORRIS, Induction of prolactin-deficient variants of GH3 rat pituitary tumor cells by ethyl methane sulfate: reversion by 5-azacytidine, a DNA methylation inhibitor, Proc. Natl. Acad. Sci. USA 79, 2967-2970 (1982). 13. R. ROTHROCK, S. T. PERRY, K. R. ISHAM, K.-L. LEE and F. T. KENNEY, Activation of tyrosine aminotransferase expression in fetal rat liver by 5-azacytidine, Biochem. Biophys. Res. Commun. 113, 645--649(1983). 14. H.-J. BOHME, D. BELAY, D. DETrMER, W. GOLTZSCH, E. HOFMANN, R. LANGE, C. SCHUBERT, E. SCHULZE, G. SPARMANN and E. WEISS, Interaction of adrenal and pancreatic hormones in the control of hepatic enzymes during development, Advan. Enzyme Regul. 26, 31-61 (1987). 15. R. ROTHROCK, K.-L. LEE, K. R. ISHAM and F. T. KENNEY, Changes in hepatic differentiation following treatment of rat fetuses with 5-azacytidine, Arch. Biochem. Biophys. 263,237-244 (1988). 16. M. BEATO and P. FEIGELSON, Glucocorticoid-binding proteins of rat liver cytosol, J. Biol. Chem. 247, 7890-7896 (1972). 17. Y.-L. YANG, J.-X. TAN and R.-B. XU, Down-regulation of giucocorticoid receptor and its relationship to the induction of rat liver tyrosine aminotransferase, J. Steroid Biochem. 32, 99-104 (1989). 18. J.J. OHISALO and J. P. PISPA, Heterogeneity of hepatic TAT. Separation of the multiple forms from rat and frog liver by isoelectric focusing and hydroxylapatite column chromatography and their partial characterization, Acta Chem. Scand. B30, 491-500 (1976). 19. M. SUDA and H. NAKAGAWA, L-Serine dehydratase (rat liver), Methods Enzymol. XVIIB, 346-351 (1971). 20. M.M. BRADFORD, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248-254 (1976). 21. K. BURTON, Determination of DNA concentration with diphenylamine, Methods Enzymol. XII, 163-166 (1968). 22. S.T. PERRY, R. ROTHROCK, K. R. ISHAM, K.-L. LEE and F. T. KENNEY, Development of tyrosine aminotransferase in perinatal rat: Changes in functional messenger RNA and the role of inducing hormones, J. Cell. Biochem. 21, 47-61 (1983). 23. E.-E. BAULIEU, Steroid hormone antagonists at the receptor level: A role for the heat-shock protein MW 90,000 (hsp90), J. Cell. Biochem. 35, 161-174 (1987). 24. B. SABLONNIERE, P. M. DANZE, P. FORMSTECHER, P. LEFEBVRE and M. DAUTREVAUX, Physical characterization of the activated and non-activated forms of the glucocorticoid-receptor complex bound to the steroid antagonist [3H]RU486, J. Steroid. Biochem. 25, 605--614(1986). 25. D. PHILIBERT, RU 38486: An original multifaceted antihormone in vivo, pp. 77-101 in Adrenal Steroid Antagonbm (M. K. AGARWAL, ed.), Walter de Gruyter & Co., Berlin (1984). 26. T. HIROTA, K. I-IIROTA, Y. SANNO and T. TANAKA, Precocious induction of tryptophan dioxygenase by glucocorticoid in suckling rats and correlation with change in giucocorticoid receptor, Biochim. Biophys. Acta 842, 195-201 (1985).
INTRODUCTION
Perinatal development of rat liver is associated with major changes in morphology, notably loss of the hematopoietic cells with a concomitant increase in size and number of parenehymal liver cells and in metabolic capacity. The hepatocytes attain much of their mature functional abilities during this life period. Characteristic enzymes expressed in adult liver have been shown to be absent throughout most of intrauterine life and to appear at definite times just before or after birth (1, 2). There is now ample evidence that the basic mechanisms underlying this developmental regulation of gene expression act primarily at the transcriptional level. Methylation and demethylation of specific cytosine residues in DNA is one of the mechanisms controlling the responsiveness of eukaryotic genes towards transcriptional activators. Transcriptionally active genes are often less methylated than silent genes (for a review see Ref. (3)). 5-Azacytidine has been found to be a useful tool in working out the critical role of DNA methylation in cellular differentiation. When 5-azacytidine is incorporated into DNA it inhibits its enzymic methylation at position 5 of cytosine. In recent years, the growing interest in the role of enzymic DNA methylation in gene regulation has prompted studies on the effects of 5-azacytidine on cell differentiation and cell functions. It has been demonstrated that 5-azacytidine incorporation into genomic DNA leads to DNA hypomethylation (4, 5). Experimental studies have demonstrated the differentiation of muscle cells, adipocytes and chondrocytes in C3H 10T1/2 mouse embryonic fibroblasts exposed to 5-azacytidine for a short period of time (6-8). This differentiation never occurs in culture unless it is exposed to 5-azacytidine. Moreover, it has been shown in several systems that 5-azacytidine-indueed hypomethylation of genomic DNA is associated with specific activation of previously silent genes such as: metallothionine-1 inducibility in mice (9), cellular thymidine kinase (EC 2.7.1.21) in Chinese hamsters (10), fetal hemoglobin in baboon 247
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and human cells (11), prolactin (12) and tyrosine aminotransferase (TAT, L-tyrosine : 2-oxoglutarate aminotransferase, EC 2.6.1.5) (13-15) in fetal rats. The present paper deals with the action of 5-azacytidine on the inducibility of tyrosine aminotransferase in the liver of suckling rats. Dexamethasone multiples the 5-azacytidine effect, while adrenalectomy prevents the induction by the drug. These results point to an involvement of glucocorticoids. On the other hand, the antiglucocorticoid RU 486, being able to suppress the dexamethasone-induced accumulation of tyrosine aminotransferase very effectively, does not abolish the action of 5-azacytidine. In order to clarify the role of glucocorticoids in this mechanism, the influence of 5-azacytidine on the concentration of the glucocorticoid receptor, its binding affinity as well as its chromatographic behavior on an anion exchanger, were investigated. MATERIALS AND METHODS Chemicals. Substrates and 5-azacytidine were obtained from Boehringer Mannheim GmbH (FRG). Dexamethasone was purchased from KRKA, Novo Mesto (Yugoslavia), estradiol and progesterone from VEB Jenapharm, Jena (FRG). The antiglucocorticoid RU 486 was a generous gift from the Central Institute of Microbiology and Experimental Therapy, Jena. All other chemicals were of the highest purity commercially available. Animals. Rats of a Wistar strain bred in this Institute were used. 5-Azacytidine was administered by i.p. injection in saline immediately after preparation of the solution because of its relative instability. The animals were sacrificed by exsanguination under light ether anesthesia. Determination of glucocorticoid receptor. Livers were homogenized in 6 vol of ice-cold 50 mM Tris/HC1 buffer pH 7.5 containing 50 mM KCI, 20 mM 2-mercaptoethanol, 10 mM sodium molybdate and 20% (v/v) glycerol (buffer A). For the determination of the cytosolic receptor the homogenate was then centrifuged at 90,000 x g for 60 min at 4°C. The resulting supernatant was aspirated and used as cytosol. Aliquots of the cytosol were incubated with 100 nM [1,2,4-(n)-3H]dexamethasone (sp. act., 45 Ci/mmol, Amersham, Buckinghamshire (UK)) in the absence or presence of a 1000-fold excess of unlabelled hormone at 0°C for 4 hr. Unbound dexamethasone was removed by incubation with 5 vol of 2% (w/v) dextran-coated charcoal as described in (16) and subsequent centrifugation for 5 min at 11,000 x g. Aliquots of the supernatant were assayed for radioactivity. For the determination of the nuclear glucocorticoid receptor a modi-
5-AZACYTIDINE AND CONTROL OF HEPATICTAT
249
fication of the procedure described in (17) was used. The liver homogenate was centrifuged at 900 x g for 10 rain. The pellet was suspended in 20 mM Tris/HCl buffer pH 7.5, 2.2 M sucrose and 3 mM CaCI 2 (TSS-buffer) and centrifuged at 40,000 x g for 30 min. The nuclei pellet was washed with TSS-Buffer and resuspended in the same buffer. Aliquots were incubated with [3H]-labeUed-dexamethasone as described above in the absence or presence of a 1000-fold excess of unlabelled hormone for 30 min at room temperature. The nuclei were then sedimented by centrifugation at 11,000 x g for 1 rain and the supernatant was carefully removed. The pellet was washed once with 0.25% (w/v) Triton X-100 in TSS-buffer in order to remove the outer nuclear membrane, which binds dexamethasone unspecifically, and three times with TSS-buffer. The final pellet was taken up in concentrated formic acid and transferred to scintillation vials containing 4 ml Bray solution.
High-performance ion-exchange chromatography. Cytosol was incubated for 4 hr at 4°C with 100 nM [1,2,4-(n)-aH]dexamethasone. After removal of unbound steroid by chromatography on Sephadex G-25, the samples were applied to a Mono Q H R 5/5 column connected to a Pharmacia FPLC system. The column was washed with buffer A at a flow rate of 1 ml/min. Elution was carried out with a linear 0-0.4 M NaC1 gradient in the equilibration buffer. Other assays. Tyrosine aminotransferase activity was assayed as described in (18) and serine dehydratase (SDH, L-serine hydrolyase, EC 4.2.1.13) by a modification of the procedure described in (19). Protein was measured using the dye-binding assay by Bradford (20) with human serum albumin as standard and D N A concentrations were estimated according to (21). RESULTS AND DISCUSSION
Induction of Tyrosine Aminotransferase Activity by 5-Azacytidine The nucleoside analogue 5-azacytidine is able to regulate hepatic gene expression in a highly targeted gene-specific manner. Only a subset of liver-specific genes, including tyrosine aminotransferase (13-15), phosphoenolpyruvate carboxykinase (EC 4.1.1.32) (15) and a gene of unknown specificity (gene 33) (15), is induced by the action of this compound in the liver of fetal rats (13, 14). These target genes share additional characteristics of regulation. They are all inducible via the cAMP pathway and by glucocorticoids. On the other hand, a number of enzymes, such as phosphofructokinase-2 (EC 2.7.1.105) (14), glucose 6phosphatase (EC 3.1.3.9) and tryptophan 2,3-dioxygenase (EC 1.13.11.11)
250
A. SC-~LZE, a d .
(15), regulated by the same hormonal stimuli, are insensitive against 5-azacytidine. Treatment of fetal rats with 5-azacytidine also changes hepatic morphology. The hepatocytes appear larger, more basophilic and better aligned in sinusoidal array, wherees the erythroid cell population is little affected by the drug (15). These results gave rise to the conclusion that in fetal rat liver 5-azacytidine administration causes a drug-induced advancement of processes normally programmed to occur later. Moreover, because the potent antiglucocorticoid 5-ot-pregnane-3,20-dione abolishes the "overshoot" in tyrosine aminotransferase expression occurring about 12 hr after birth, but does not influence either the transition from the fetal to the adult level of tyrosine aminotransferase expression (22) or the precocious induction of tyrosine aminotransferase by 5-azacytidine (15), it has been concluded that glucocorticoids are not involved either in the normal or in the 5-azacytidine-induced activation of tyrosine aminotransferase expression (15). In other words, 5-azacytidine is believed to cause an activation of selected genes similar to those occurring during normal development. This activation elicits glucocorticoid responsiveness, and as the levels of these steroids are high in the neonatal rat the maturation appears to be amplified by glucocorticoid induction causing the typical "overshoot" production of tyrosine aminotransferase.
400
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FIG. 1. Time dependence of the induction of tyrosine aminotransferase by 5-azacytidine an¢ dexamethasone. A n i m a l s were injected with 15 ~ g 5-~acytidine/g body weight and 10 ~ d e x a m e t h a s o n e / g body weight. T h e controls obtained the same vol saline. Results are giver as m e a n s of 5--8 animals per group + S.E.M. C) - - - O control. Q - - - • d e x a m e t h a s o n e ..~7=,-,,t,n,,,,~. V __ _
5-AZACYTIDINE AND CONTROL OF HEPATIC TAT
251
However, this concept does not explain the induction of tyrosine aminotransferase activity in suckling rat liver by a single injection of 5-azacytidine (15/zg/g body weight) which is clearly detectable already after 5 hr and reaches maximum values approximately 12 hr after the application of the drug (Fig. 1). In comparison to the induction by dexamethasone (10/~g/g body weight), the accumulation of tyrosine aminotransferase activity caused by 5-azacytidine occurs with a lower rate but reaches higher maximum values. Dexamethasone stimulates tyrosine aminotransferase within 5 hr about 4- to 5-fold, while in 5-azacytidine-treated animals only a 2- to 3-fold induction of tyrosine aminotransferase is observable at this time. Twelve hr after the injection, however, an 8- to 10-fold induction of tyrosine aminotransferase by dexamethasone and a 10- to 20-fold induction of the enzyme by 5-azacytidine is found. 5-Azacytidine also acts synergistically with progesterone which is a weak inducer of tyrosine aminotransferase in suckling rat liver under the conditions applied: enzyme levels 12 hr after treatment were 110.1 _+ 32.0 units/g protein in progesterone-treated animals, and 474.0 _+ 40.4 units/g protein in those treated with 5-azacytidine as well. The induction by estradiol of tyrosine aminotransferase is also slightly increased when the hormone is injected together with the drug, however, at a statistically not significant level (Table 1).
T A B L E 1. INFLUENCE OF 5-AZACYTIDINE ON THE INDUCTION OF TYROSINE AMINOTRANSFERASE AND SERINE D E H Y D R A T A S E IN THE LIVER OF SUCKLING RATS Activity (units/g protein) Treatment Saline 5-Azacytidine Dexamethasone Dexamethasone + 5-azacytidine Estradiol Estradiol + 5-azacytidine Progesterone Progesterone + 5-azacytidine
TAT
SDH
34.1 + 2.8 431.2 + 38.7* 327.5 _+ 50.9*
15.1 + 6.2 14.1 + 4.3 41.3 _+ 4.0*
625.9 + 73.5",t 224.3 + 34.0*
12.2 + 3.1t 29.3 + 3.8*
329.3 + 42.6* 110.1 + 32.0*
11.9 + 1.2t 25.0 + 4.5*
474.0 + 46.4",t
13.9 + 1.7t
Results are given as means of 5--6 animals per group + S.E.M. The hormones were applied to 21-day-old rats using the following dosages: 20 ~tg progesterone/g body weight; 20/zg estradiol/g body weight; 5 ~zg dexamethasone/g body weight; 15/zg 5-azacytidine/g body weight. The animals were killed 12 hr later. *Means significantly different from the control at the 0.05 level. tMeans significantly different at the 0.05 level from the group of animals treated with the respective hormone only.
252
A. SCHULZE, et al. TABLE 2. INFLUENCE OF 5-AZACYTIDINE ON TYROSINE AMINOTRANSFERASE ACTIVITY ON ADRENALECTOMIZED SUCKLING RATS Treatment
TAT activity (units/g protein)
Control 5-Azacytidine Dexamethasone
133.8 _+ 9.5 (4) 156.1 _+ 33.6 (4) 702.9 ± 296.2 (4)
Bilateral adrenalectomy was performed via the dorsolateral route 3 days before the experiment at the 22rid day of life. The animals were supplemented by daily injection of 0.05 ~g deoxycorticosterone/g body weight. The animals obtained intraperitoneally 10/zg/g body weight dexamethasone or 10/z/g body weight 5-azacytidine and were killed 20 hr later. Control animals obtained the same vol saline. Results are given as mean -+ S.E.M. with the number of animals in parenthesis.
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FIG. 2. Influence of the antiglucocorticoid RU 486 on the induction of tyrosine aminotransferas¢ (A) and serine dehydratase (B) by dexamethasone. Dexamethasone (2/~g/g body weight) was injected i.p. 1 hr after RU 486. The animals were sacrificed 15 hr later. Results are given as means of 6-9 animals per group ± S.E.M.
5-AZACYTIDINE AND CONTROL OF HEPATIC TAT
253
Serine dehydratase which behaves very similarly to tyrosine aminotransferase in its developmental activation is not stimulated by 5-azacytidine under these conditions. On the contrary, the drug abolishes the induction of the enzyme by all three steroid hormones tested (Table 1). Bilateral adrenalectomy renders the liver unresponsive towards the stimulating effect of 5-azacytidine (Table 2). These results point to an involvement of glucocorticoids in the mechanism by which 5-azacytidine activates the tyrosine aminotransferase expression in suckling rat liver. This implication was explored further by studying the influence of the antiglucocorticoid RU 486 on the induction of tyrosine aminotransferase by 5-azacytidine. Administration of this antagonist at a dosage (20/~g/g body weight) which inhibits the induction of tyrosine aminotransferase and serine dehydratase by dexamethasone very effectively (Fig. 2) has no influence on the stimulation of tyrosine aminotransferase activity by 5-azacytidine (Fig. 3).
Influence of 5-Azacytidine on the Glucocorticoid Receptor The binding affinities of the glucocorticoid receptor for radioactive labelled dexamethasone were found to be similar in 5-azacytidine-
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FIG. 3. Ineffectiveness of RU 486 to inhibit induction of tyrosine aminotransferase by
5-azacytidine.Results are givenas means _+S.E.M. of 6-7 animalsper group.
254
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FIG. 4. Saturation curve and Scatchard plot for the binding of [3H]dexamethasone by rat liver cytosol from control (A) and 5-azacytidine(B)-treated rats. Cytosol was incubated with increasing amounts of labelled dexamethasone at 4°C for 4 hr.
and saline-treated animals (Ka = 3.5 x 10-9 versus 4.5 x 10-9, Fig. 4). However, the number of cytosolic glucocorticoid receptors is decreased after the administration of 5-azacytidine (Table 3). The decline of cytosolic glucocorticoid receptor occurs very rapidly after treatment with 5-azacytidine. Apparently, this is not an experimental artifact caused by a competition of the drug with the labelled dexamethasone in the binding assay because it was found that 5-azacytidine (up to 0.25 mM)
TABLE 3. INFLUENCE OF 5-AZACYTIDINE ON THE CYTOSOLIC GLUCOCORTICOID RECEPTOR Time after injection (hr) 0.5 1 5 12 24 Control (without injection)
Glucocorticoid receptor (pmol/mg protein) Control 5-Azacytidine Dexamethasone 0.056 0.098 0.069 0.060 0.083
+ + + + +
0.012 0.004 0.012 0.008 0.012
0.046 0.041 0.036 0.029 0.039
+ + + + +
0.006 0.006 0.008 0.005 0.006
0.005 + 0.002 0.001 + 0.001 n.d. 0.002 + 0.001 0.018 + 0.004
0.079 + 0.008
n.d. - - not detectable. The animals (3 weeks old) were injected with 10/~g dexamethasone/g body weight or 15 ~g 5-azacytidine/g body weight. The control group received the same vol of the vehicle fluid only. The results are given as means + S.E.M. of 5-9 animals per group.
5-AZACYTIDINE AND CONTROL OF HEPATIC TAT
255
does not interfere with the receptor determination. The slight decrease of the cytosolic glucocorticoid receptor in the control animals detectable 30 rain after injection very likely reflects a stress effect due to the handling of the animals. In the 5-azacytidine-treated group, the concentration of cytosolic glucocorticoid receptor was found to be approximately 50% of the control even 24 hr after the application of the drug. The decrease of the glucocorticoid receptor concentration is comparable with the down-regulation of the glucocorticoid receptor by dexamethasone but smaller in magnitude and longer lasting. In the dexamethasone group, partial regeneration of the cytosolic glucocorticoid receptor can be seen about 24 hr after the injection (Table 3). As shown in Table 4, the fast decrease of cytosolic glucocorticoid receptor is obviously accompanied by an increase of the glucocorticoid receptor concentration in the nuclei. In all three groups of animals the increase of the glucocorticoid receptor in the nuclei is significantly different from the control group (untreated animals) with p < 0.05. In the saline-treated controls and in the dexamethasone group the level of nuclear glucocorticoid receptor returns within 12 hr to the normal value. In the 5-azacytidine group the concentration of nuclear glucocorticoid receptor remains elevated for this period of time; however, because of the high variation observed, only the 5 hr-value is statistically different from the respective saline-treated control. The antiglucocorticoid RU 486, which is unable to inhibit the induction by 5-azacytidine of tyrosine aminotransferase, is also not capable of preventing the decline of cytosolic glucocorticoid receptor in 5-azacytidine-treated animals. On the contrary, the glucocorticoid receptor concentration was found to be lower in animals which obtained the antiglucocorticoid along with 5-azacytidine (results not shown). However, it cannot be excluded
T A B L E 4. INFLUENCE OF D E X A M E T H A S O N E AND 5-AZACYTIDINE ON THE CONCENTRATION OF THE N U C L E A R GLUCOCORTICOID RECEPTOR Time (hr) 0.5 5.0 12.0 Control (without injection)
Glucocorticoid receptor (pmol/mg DNA) Saline Dexamethasone 5-Azacytidine 0.783 _+ 0.221 0.407 _+ 0.222 0.100 _+ 0.024
1.120 + 0.326 0.987 + 0.402 0.083 _+ 0.026
0.973 _+ 0.159 1.063 + 0.063* 0.417 + 0.125
0.040 _+ 0.012
The 21-day-old rats were injected with I0/~g dexamethasone/g body weight or with 15/tg 5-azacytidine/g body weight. The control group obtained the same vol saline. Results a r e given as means + S.E.M. of 4--8 animals per group. *Statistically different from the respective saline-injected animals at the 0.05 level.
256
A . S C H U L Z E , et al.
that this result is artificial, because RU 486 possesses a high affinity to the glucocorticoid receptor (25) and interferes with the receptor assay. The results point to an influence of 5-azacytidine on the activation of glucocorticoid receptor and cannot be explained in terms of alterations in the degree of DNA methylation since this process depends on de n o v o synthesis of DNA which takes place with a much slower rate than the observed induction of tyrosine aminotransferase and translocation of the glucocorticoid receptor into the nuclei. In the absence of steroid hormone a very large fraction of the glucocorticoid receptor is found in the cytosol and sediments as heterooligomer (designated as 8S-R) with an apparent mol. wt. of 300 k (24). In addition to the hormone-binding receptor species the fraction contains the heat-shock protein hsp 90 which is believed to be a "cap" for the receptor interacting with its DNA-binding site. Upon hormone binding, the 8S-receptor is physiologically transformed (activated). This change of structure is responsible for the release of hsp 90 from the hetero-oligomeric form of the receptor, forming the 4S-receptor species. Its putative DNA binding site may then interact with glucocorticoid responsive elements (GRE) of DNA. RU 486, a 19-norsteroid, stabilizes the 8S form of glucocorticoid receptor as shown by a number of studies in different mammalian tissues including rat liver (25). Therefore, the 5-azacytidine effect on the glucocorticoid receptor represents very likely not an influence of the drug on the transformation of the 8S- into the 4S-receptor species but may be a consequence of acting on a later step of glucocorticoid receptor activation such as translocation of the 4S-species into the nuclei.
-- 0 5
0.4
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.~
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~
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I0
20 Froction
F I G . 5. C h r o m a t o g r a p h y o f l i v e r c y t o s o l l a b e l l e d in vitro w i t h d e x a m e t h a s o n e o n a M o n o Q H R 5/5 c o l u m n . • - - - • c o n t r o l . © - - - O 5 - a z a c y t i d i n e (15 g/g b o d y w e i g h t ) . [] - - - [] d e x a m e t h a s o n e (10 p.g/g b o d y w e i g h t ) .
5-AZACYTIDINE AND CONTROL OF HEPATIC TAT
257
Anion Exchange Chromatographyof GlucocorticoidReceptor Chromatography of suckling rat liver cytosol on a Mono Q column in the presence of molybdate revealed independent of the treatment of the animals only the presence of the "classic" glucocorticoid receptor (designated as binder II) (Fig. 5). In this life span the glucocorticoid receptor species described by Hirota et al. (26) as "Peak C" was not detectable.
SUMMARY
The nucleoside analog 5-azacytidine is able to induce tyrosine aminotransferase several-fold in the liver of suckling rats. Bilateral adrenalectomy abolishes this inducing effect. The drug also decreases significantly the concentration of cytosolic glucocorticoid receptor accompanied by an increase of the glucocorticoid receptor concentration in the nuclei. The antiglucocorticoid RU 486 which abolishes the induction of tyrosine aminotransferase and serine dehydratase by dexamethasone very effectively is not able to inhibit either the induction of tyrosine aminotransferase or the translocation of the glucocorticoid receptor into the nuclei by 5-azacytidine. REFERENCES 1. O. GREENGARD, Enzymic differentiation in mammalian liver, Science 163, 891-895 (1969). 2. R. BITTNER, H.-J. BOHME, L. DIDT, W. GOLTZSCH, E. HOFMANN, M. J. LEVIN and G. SPARMANN, Developmental changes in the levels of hepatic enzymes and their relation to metabolic functions, Advan. Enzyme Regul. 17, 37-57 (1978). 3. W. DOERFLER, DNA methylation and gene activity, Ann. Rev. Biochem. 52, 93-124 (1983). 4. P. A. JONES and S. M. TAYLOR, Hemimethylated duplex DNAs prepared from 5-azacytidine treated cells, Nucleic Acid Res. 9, 2933-2947 (1981). 5. V. MAHARAJAN, L. TOSI, M. PRATIBHA and E. SCARANO, Analogues of 5-methyl cytosine and early embryonic development, Prog. Clin. Biol. Res. ~ A , 227-234 (1982). 6. S.M. TAYLOR and P. A. JONES, Multiple new phenotypes induced in 10 T1/2 and 3T3 cells treated with 5-azacytidine, Cell 17,771-779 (1979). 7. L. LUI, M. HARRINGTON and P. A. JONES, Characterization of myogenic cell lines derived by 5-azacytidine treatment, Dev. Biol. 117, 331-336 (1986). 8. P.G. CONSTANTINIDES, S. M. TAYLOR and P. A. JONES, Phenotypic conversion of cultured mouse embryonic cells by azapyrimidine nucleosides, Dev. Biol. 66, 57-71 (1978). 9. S. J. COMPERE and P. D. PALMITER, DNA methylation controls the inducibility of the mouse metallothionine-Igene in lymphoid cells, Cell 25, 233-240 (1981). I0. M. HARRIS, Induction of thymidine kinase in enzyme-deficient Chinese hamster cells, Cell 29,483--492 (1982). 11. J. de SIMONE, P. HELLER, L. HALL and D. ZWlERS, 5-Azacytidine stimulates fetal hemoglobin synthesis in anemic baboons, Proc. Natl. Acad. Sci. USA 79, 4428-4431 (1982).
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12. R.D. IVARIE and J. A. MORRIS, Induction of prolactin-deficient variants of GH3 rat pituitary tumor cells by ethyl methane sulfate: reversion by 5-azacytidine, a DNA methylation inhibitor, Proc. Natl. Acad. Sci. USA 79, 2967-2970 (1982). 13. R. ROTHROCK, S. T. PERRY, K. R. ISHAM, K.-L. LEE and F. T. KENNEY, Activation of tyrosine aminotransferase expression in fetal rat liver by 5-azacytidine, Biochem. Biophys. Res. Commun. 113, 645--649(1983). 14. H.-J. BOHME, D. BELAY, D. DETrMER, W. GOLTZSCH, E. HOFMANN, R. LANGE, C. SCHUBERT, E. SCHULZE, G. SPARMANN and E. WEISS, Interaction of adrenal and pancreatic hormones in the control of hepatic enzymes during development, Advan. Enzyme Regul. 26, 31-61 (1987). 15. R. ROTHROCK, K.-L. LEE, K. R. ISHAM and F. T. KENNEY, Changes in hepatic differentiation following treatment of rat fetuses with 5-azacytidine, Arch. Biochem. Biophys. 263,237-244 (1988). 16. M. BEATO and P. FEIGELSON, Glucocorticoid-binding proteins of rat liver cytosol, J. Biol. Chem. 247, 7890-7896 (1972). 17. Y.-L. YANG, J.-X. TAN and R.-B. XU, Down-regulation of giucocorticoid receptor and its relationship to the induction of rat liver tyrosine aminotransferase, J. Steroid Biochem. 32, 99-104 (1989). 18. J.J. OHISALO and J. P. PISPA, Heterogeneity of hepatic TAT. Separation of the multiple forms from rat and frog liver by isoelectric focusing and hydroxylapatite column chromatography and their partial characterization, Acta Chem. Scand. B30, 491-500 (1976). 19. M. SUDA and H. NAKAGAWA, L-Serine dehydratase (rat liver), Methods Enzymol. XVIIB, 346-351 (1971). 20. M.M. BRADFORD, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248-254 (1976). 21. K. BURTON, Determination of DNA concentration with diphenylamine, Methods Enzymol. XII, 163-166 (1968). 22. S.T. PERRY, R. ROTHROCK, K. R. ISHAM, K.-L. LEE and F. T. KENNEY, Development of tyrosine aminotransferase in perinatal rat: Changes in functional messenger RNA and the role of inducing hormones, J. Cell. Biochem. 21, 47-61 (1983). 23. E.-E. BAULIEU, Steroid hormone antagonists at the receptor level: A role for the heat-shock protein MW 90,000 (hsp90), J. Cell. Biochem. 35, 161-174 (1987). 24. B. SABLONNIERE, P. M. DANZE, P. FORMSTECHER, P. LEFEBVRE and M. DAUTREVAUX, Physical characterization of the activated and non-activated forms of the glucocorticoid-receptor complex bound to the steroid antagonist [3H]RU486, J. Steroid. Biochem. 25, 605--614(1986). 25. D. PHILIBERT, RU 38486: An original multifaceted antihormone in vivo, pp. 77-101 in Adrenal Steroid Antagonbm (M. K. AGARWAL, ed.), Walter de Gruyter & Co., Berlin (1984). 26. T. HIROTA, K. I-IIROTA, Y. SANNO and T. TANAKA, Precocious induction of tryptophan dioxygenase by glucocorticoid in suckling rats and correlation with change in giucocorticoid receptor, Biochim. Biophys. Acta 842, 195-201 (1985).