REGULATION OF TYROSINE AMINOTRANSFERASE mRNA IN HTC CELLS*

REGULATION OF TYROSINE AMINOTRANSFERASE mRNA IN HTC CELLS*

Daryl Granner, Pamela Olson, Sarah Seifert, Charlotte Block, Martin Diesterhaft, James Hargrove and Tamio Noguchi

Departments of Internal Medicine and Biochemistry The University of Iowa College of Medicine and VA Hospital

Iowa City, Iowa 52242

INTRODUCTION

Induction by glucocorticoid hormones of the hepatic enzyme tyrosine aminotransferase (TAT; ~-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.6) has served as a valuable model for studying the gluconeogenic action of this class of hormones. This induction has been shown to be due to an increased rate of synthesis of the enzyme' and considerable data suggest that glucocorticoid hormones act to enhance the rate of transcription of s cific enes which results in an increase in the amount of a few specific mRNAs."-'Rece:tly, several groups have shown that functional mRNATAT, the messenger RNA that codes for tyrosine aminotransferase, is increased in liver by glucocorticoid hormonesM and within the past year several groups, including our own, have been able to quantitate mRNATAT activity isolated from HTC Although an increase in transcription would appear to be the predom- inant effect, there is some evidence that steroid hormones may also act at post- transcriptional sites."-I6 How either transcriptional or translational processes could be affected by steroid hormones remains to be determined.

Hormones that act through cyclic AMP, or active derivatives of this compound, also induce TAT in adult rat liver,I6 fetal liver in organ ~u l tu re , ' ~ and in some hepatic-derived tissue culture lines." This induction is also the result of an increased rate of synthesis of TAT. Several studies indicate that CAMP-mediated induction of TAT is not maximal unless glucocorticoid hormones are also present. Adrenalectomy decreases the inductive effect of CAMP analogs in rat liver"; combinations of BtzcAMP and glucocorticoids give synergistic TAT induction in rat liver,I6 and in fetal liver organ culture"; hydrocortisone potentiates the induction of TAT by BtzcAMP in Reuber H35 cellsm; and finally, dexamethasone markedly augments TAT induction by Bt2cAMP in some lines of hepatoma tissue culture cells (HTC)." TAT induction is thus an example of the so-called "permissive effect" of glucocorticoid hormones postulated by Ingle in 1954,22 an important biological process that is poorly understood.

In contrast to the glucocorticoids, it has been hypothesized that cyclic nucleotides act by enhancing the rate of translation of specific rnRNAslg and recent1 Ro er and Wicks have shown that BtzcAMP decreases the transit time for TAT. That functional mRNA for TATz4* 25 and phosphoenolpyruvate carboxykinaseZ6 is increased by Bt2cAMP in liver suggests that translational control cannot completely explain the induction of TAT by cyclic nucleotides.

* This work was performed in the Iowa Diabetes and Endocrinology Research Center (P30- AM25295) and was supported by USPHS grant AM24037, by Veterans Administration research funds, and by funds from the National and Iowa Affiliates of the American Diabetes Associa- tion. D. K. G . is a Veterans Administration Medical Investigator.

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184 Annals New York Academy of Sciences

These studies have been done using a number of different systems, most of which are not amenable to more detailed analysis. HTC cells permit one to study the induction of tyrosine aminotransferase in a controlled environment wherein the effects of glucocorticoids and cyclic nucleotides can be studied independently and in concert. Advances in the techniques of extraction and isolation of mRNA, and the development of systems for cell-free protein synthesis now allow for the study of the regulation of eukaryotic mRNAs that are present in very small quantities in cells. In this paper we report on several aspects of the regulation of mRNATAT by glucocorticoid hormones and cyclic nucleotides in cultured hepatoma cells.

MATERIALS AND METHODS

Cell Culture

HTC cells were grown in suspension culture in modified Swim's S77 (S77S) medium as described previously?' For induction experiments the cells were removed from growth medium and were resuspended for two days in medium which differs from S77S in that it contains 2% dialyzed bovine serum (v/v) rather than 5% each of fetal calf and bovine sera (v/v).

Enzyme Assays

The specific activity of TAT was determined as described by Granner et a1.:' and is expressed as milliunits per mg of protein.

Antibody Production

TAT was purified by a modification of the procedure of Valeriote et a1.,2n to a specific activity of greater than 500 U/mg protein. Injection of this into New Zealand rabbits resulted in the formation of a highly specific antibody that was partially purified by precipitation with ammonium sulfate at 40% saturation.

In Vivo Rate of Synthesis of TA T

The rate of TAT synthesis in intact HTC cells was quantitated by determining the radioactivity incorporated into immunoprecipitable TAT by a modification of the procedure described previously." Aliquots of HTC cells (100 ml, 10' cells/ml) were concentrated by centrifugation and resuspended in 2 ml of the original medium. The samples were incubated with 80 pCi of [3H] leucine for 30 minutes at 37OC, and were then washed two times with cold phosphate buffered saline containing 2 mM unlabeled leucine. The cells were resuspended in 1.5 ml NET buffer [I50 mM NaC1, 5 mM EDTA, 50 mM Tris, and 0.02% NaN3 containing 0.05% Nonidet P 40 (NP 40) and 2 mM unlabeled leucine] and sonicated. TAT was immunoprecipitated using killed cells of Staphylococcus aureus, Cowan strain I (SAC) according to the procedure of Kessler.%

Extraction of Poly(A)+RNA

Pellets containing 10' cells were extracted using equal volumes of 50 mM Tris buffer, pH 9.0 (containing 2 mM EDTA, 1% SDS and phenol-chloroform). After

Granner et al.: Tyrosine Aminotransferase mRNA 185

reextraction, the aqueous phases were combined and the nucleic acids were precipi- tated by the addition of 0.5 M NaCl and 2 volumes of absolute alcohol. The precipitate was treated with proteinase K, reprecipitated and extracted with 3 M NaAc, pH 6.0, and chromatographed on oligo(dT)-cellulose according to the procedure of Krystosek et aLW

Cell- Free Protein Synthesis and Detection of TA T

Messenger RNA-dependent rabbit reticulocyte lysates were prepared according to established procedure^.^' The translation assay incubation consisted of 430 pl of nuclease-treated lysate which contained added amino acids, MgC12, KCI and creatine

containing polyadenylate residues at the 3'terminus-and distilled water to a total volume of 500 pl. After a 90 min incubation, the reaction mixture was diluted with 0.65 ml of 100 mM Tris/HCI (pH 7.5) and the incorporation of ["HI leucine into released polypeptide chains was determined. To detect the TAT synthesized 100 pl of buffer containing 0.5% NP-40 and 100 mU carrier TAT were added to the released chains. Antibody against TAT was added, the immune complex was adsorbed with SAC and the resulting pellet was washed. TAT was dissociated from the antibody by heating to 95" in Laemmli gel buffer. Electrophoresis was conducted on 10 cm, 10% SDS polyacrylamide gels, according to the procedure of Laemmli.32 Gels were cut, dissolved in 30% H202 and the radioactivity in the TAT peak area was determined.' This value divided by the radioactivity incorporated into total released chains is a representation of mRNATAT activity as a percentage of total mRNA activity.

phosphate), 50 pCi of [ $ ' H] leucine (60 Ci/mmole), 5-20 p1 of poly(A)+RNA-RNA

RESULTS AND DISCUSSION

Characterization of the Translational System

We adapted the techniques used to quantitate mRNATAT in rat liver for use in studies of HTC cells. Using the microccocal nuclease-treated reticulocyte lysate system,31 [3H]-leucine incorporation into total trichloroacetic acid insoluble material is linear with HTC cell poly(A)+RNA concentrations between 5 and 40 @mI, and increases for at least 90 min. A single peak of radioactivity is detected when the released protein Chains from the cell-free translation reaction are immunoprecipitated with an antibody directed against highly purified tyrosine aminotransferase and then electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels (FIGURE I ) . The ["HI-leucine labelled TAT that is synthesized in cell-free systems supplemented with poly(A)'RNA isolated from either uninduced or dexamethasone-treated HTC cells migrate identically on SDS gels. They also comigrate with tyrosine aminotransferase, which has been labeled in vivo, then partially purified from HTC cells, and with tyrosine aminotransferase, which has been purified from rat liver, and labelled with the fluorescent compound 2-methoxy-2,4-diphenyl 3 (ZH)-furanone. This experiment thus shows that HTC cell mRNA added to a rabbit reticulocyte lysate directs the synthesis of TAT which is indistinguishable from that made in intact HTC cells or in rat liver. This is the basic system used to obtain all the data reported in this Paper.

Quantitation and SpeciJcity of the Increase in mRNA TAT

An examination of FIGURE 1 reveals that addition of an AZm amount of poly(A)+RNA isolated from Dex-induced HTC cells results in the production of


Gel Slice Number FIGURE I . Characterization of tyrosine aminotransferase synthesized in v im. Poly (A)+

RNA isolated from uninduced (0) and dexamethasone treated (0) HTC cells was translated in the cell-free system, and the released chains were immunoprecipitated with antibody directed against tyrosine aminotransferase. Purified, in vivo labelled ["CI-tyrosine aminotransferase was added to the released chains and was coimmunoprecipitated with the uninduced sample (A). After immunoprecipitation, fluorescent-labelled tyrosine aminotransferase was added to the induced cell immunoprecipitation product, and both samples were electrophoresed on compan- ion gels. Migration of the fluorescent tyrosine aminotransferase was located by UV light, and is represented by the arrow. The gels were sliced, dissolved in 30% Hz02, and the radioactivity in the slices was determined.

4275 cpm of TAT as compared to the 601 cpm found in TAT in assays using the same amount of poly(A)'RNA isolated from control cells. This sevenfold increase in functional mRNATAT activity, when compared to the sevenfold increase in enzyme catalytic activity noted in the same cells, suggests that the induction is largely, if not entirely, due to an increase in mRNATAT activity.

The specificity of this increase is shown in TABLE 1, experiment I . The total protein synthesized by a given amount of poly(A)+RNA, and the percentage of released chains obtained, are unaffected by Dex. These results, similar to those found using livers from uninduced and hydrocortisone-treated rats as the sources of exogenous poly(A)'RNA? allow us to conclude that the addition of dexamethasone to HTC cells does not cause a general increase in total mRNA activity. Other studies, in which the total translation products are analyzed on the gel system, support this conclusion since the steroid causes no change in the overall gel profile. TABLE 1 illustrates several other points. First, it again shows that the increase in functional mRNATAT activity following Dex treatment is closely proportional to the increase in TAT catalytic activity. Also, the relative amount of mRNATAT in both the basal and induced states is very low compared to total poly(A)'RNA activity, being about

Granner et al.: Tyrosine Aminotransferase mRNA

TABLE I EFFECTS OF DEXAMETHASONE A N D BtSAMP ON mRNA ACTIVITY IN HTC CELLS*

Protein Synthesized in vilro

(cpm x lo-')

Experiment Condition Total Chains of Total (mU/mg)

187

Released mRNATAT as % TAT Activity

I Control 1,569 1,224 0.05% 16.2 BtycAMP 1,638 1,250 0.06% 16.9

I I Control 1,519 1,226 0.04% 8.0 Dexamethasone 1,330 1,165 0.18% 30.3

* Dexamethasone (5 x IO-'M) or Bt,cAMP (ImM) was added to HTC cell cultures and incubated at 37" C for four hours. At that time, samples were taken, and in v i m protein synthesis (total and released chains), mRNATAT activity and TAT catalytic activity, were determined as described in Materials and Methods.

0.04% and 0.18% respectively. Finally, this experiment is representative of many others done in our laboratory in recent years in that our HTC cells do not increase TAT activity in response to N6,02'-dibutyryl cyclic AMP (BtsAMP). Not surprising is the fact that mRNATAT activity is not changed by this nucleotide, nor is there any measurable effect of this agent on total mRNA activity.

Dexamethasone Concentration and mRNATAT Activity

Tyrosine aminotransferase catalytic activity increases in proportion to the amount of Dex in the culture medium until the maximally effective level is reached. As is seen in FIGURE 2, lo-'' M dexamethasone has no effect on TAT activity whereas larger concentrations result in progressively greater degrees of induction, with optimal induction being reached between M to 5 X lo-' M concentrations. As TAT catalytic activity increases with higher concentrations of dexamethasone there is a proportionate increase in the in vivo rate of synthesis of TAT and in functional mRNA activity for TAT. These measurements were all made in the same aliquot of cells and, in this experiment, all three functions increased by 10 fold at the maximal level. The concentration of dexamethasone that gave half maximal induction of these three functions is essentially identical, being 6-9 X lo-' which is very similar to the estimated Kd of the glucocorticoid receptor for dexamethasone.=

Kinetics of Induction

The results obtained in the experiments shown in Table I and FIGURE 2 suggest that the prime determinant of TAT synthesis is the amount of mRNATAT in the cell. According to current concepts, changes in the activity (? amount) of this mRNA should precede the increase in the in vivo rate of synthesis of TAT. A cultured cell system, such as HTC cells, leads itself ideally to this kind of experiment since aliquots of cells can be removed from the same culture at selected times, thus mRNA activity, the in vivo rate of synthesis, and enzymic activity can all be determined on the same cell aliquots. We had previously shown that the rate of synthesis of TAT is increased within an hour after steroid addition whereas changes in enzymic activity occur at about 2 hoursz7 The data shown in FIGURE 3 confirm this, and, in addition, show


DMPMETHPSONE [M]

FIGURE 2. Induction of tyrosine aminotransferase by various concentrations of dexamethasone. The various concentrations of dexamethasone indicated were added to HTC cells for 12 hours. At that time tyrosine aminotransferase catalytic activity (0), the in vivo rate of synthesis (O), and functional mRNATAT activity (0) were determined on the same aliquots of HTC cells. The initial value for catalytic activity of 8 mU/mg protein increased to 90 mU/mg with Dex, the rate of synthesis increased from 0.02% to 0.19% of the total protein synthesized; and functional mRNATAT activity increased from 0.04% to 0.4% of total rnRNA. The data are expressed as the percent of the maximal response over the control value for each function.

that mRNATAT activity is increased within 30 min after Dex addition. Thus, the sequence found in HTC cells is the one expected if there is a "precursor-product" relationship between mRNATAT and TAT synthesis and enzymic activity.

The effect of the steroid inducer is extremely rapid, since an increase in functional mRNATAT is seen at 30 min, the earliest time at which samples were examined, and it may be shorter than this in view of the relative lack of sensitivity of in vitro translation assays. The only glucocorticoid response that appears to be more rapid than this is the induction of mouse mammary tumor virus in GR cells by dexamethasone, which is maximal within 15 min of steroid addition as detected by a very sensitive hybridization te~hnique.~ This experiment also suggests that the lag between the increase in mRNATAT activity and the in vivo rate of TAT synthesis exceeds the 8-10 min ribosomal transit time estimated for TAT.23 Further studies will have to exclude several potential technical explanations before it can be decided whether this lag is real, and if so, what it is due to.

Effect of Inhibitors of Protein and RNA Synthesis on mRNATAT

Initial support for the concept that TAT induction involved both new protein and RNA synthesis came from experiments in which these processes were prevented by appropriate inhibitors. Evidence for the necessity of ongoing RNA synthesis in the Dex-mediated increase in mRNATAT activity is presented in TABLE 2. Two inhibitors of RNA synthesis were tested. Actinomycin D had little effect on tyrosine aminotransferase enzyme or mRNATAT activity in uninduced cultures. The simultaneous addition of dexamethasone and the inhibitor to an HTC cell culture followed

Granner et al. : Tyrosine Amhotransferase mRNA 189

by a four hour incubation resulted in proportionate decreases in mRNATAT and enzyme activity, indicating that in HTC cells, as in liver, ongoing RNA synthesis is required for the increase in mRNATAT seen after dexamethasone addition. This is, at best, indirect evidence for a transcriptional action of the glucocorticoid and must be followed by more direct experiments.

Inhibitors of protein synthesis result in quite different effects. Cycloheximide and emetine decrease TAT enzyme activity from 8 mU/mg protein to 3 mU/mg protein when incubated with uninduced cells for four hours (TABLE 2). This is expected if TAT degradation continues to occur in the absence of ongoing protein synthesis. Tyrosine aminotransferase mRNA activity levels, however, were not affected by either cycloheximide or emetine during this time period, even though the methods used are capable of detectin a decrease from the basal level. Thus, maintenance of

period of time. When dexamethasone is added to HTC cell cultures for four hours, there is a

threefold induction of TAT enzyme activity and a sixfold increase in mRNATAT activity. The difference in the extent of induction of enzyme and mRNATAT activities occurs because the new steady state has not been achieved at four hours (See FIGURE 3). The addition of cycloheximide or emetine at the same time as the dexamethasone prevents the increase of catalytic activity, yet mRNATAT activity levels increased as much as threefold.

In 1968, Peterkofsky and Tomkins showed that dexamethasone led to the accumulation of a product that was sensitive to inhibition of RNA synthesis but not to inhibition of protein synthesis and they postulated that this substance was mRNATAT.% The studies reported in this paper offer direct confirmation of this hypothesis in that mRNATAT accumulates in the presence of either cycloheximide or emetine (TABLE 2). There might be some dependence of RNA synthesis, processing, transport, or turnover on protein synthesisa*x since functional mRNATAT did not increase to the same extent as it did in cultures treated with dexamethasone alone. Furthermore, if the inhibitor is added two hours after the dexamethasone, greater increases in mRNATAT occur (data not shown).

The inhibitors did not increase mRNATAT by themselves, an observation that is of interest because of a recent report that protein synthesis inhibitors increase functional tyrosine aminotransferase mRNA in untreated rats.37 It was suggested that

the basal level of mRNATA $ does not require ongoing translation, at least for this

TABLE 2 EFFECT OF INHIBITORS ON mRNATAT AND ENZYME INDUCTION IN HTC CELLS*

mRNATAT as 70 of Treatment total TAT Activity (mU/mg)

~

Control 0.03% 8.3 + Cycloheximide, 0. I mM 0.03 3.0 + Emetine, 0.06 m M 0.03 2.8

Dexamethasone 0.18 21.5 + Cycloheximide, 0. I mM 0.10 2.5 + Emetine, 0.06 mM 0.09 2.8 + Cordycepin, 10 pg/ml 0.07 14.4

+ Actinomycin D, I pg/ml 0.02 8.8

+ Actinomycin D, I pg/ml 0.04 11.0

* The inhibitors, at the concentrations shown, were added to HTC cells either alone (control) or with the simultaneous addition of 5 X lo-' M-dexamethasone. After a four hour incubation at 37" C, tyrosine aminotransferase enzyme and mRNATAT activities were determined as described in Materials and Methods.


HOURS AFTER E X ADDITION

FIGURE 3. Induction of tyrosine aminotransferase by dexamethasone. Dexamethasone was added to HTC cells and, at the times indicated, assays for tyrosine arninotransferase catalytic activity (0), the in vivo rate of tyrosine aminotransferase synthesis (O), and functional mRNATAT activity (0) were performed. The data are expressed as the percent of the maximal response over the control value for each function.

short lived proteins may regulate the activity of aminotransferase mRNAs or that there is an increase in mRNA half-life in the presence of cycloheximide, i.e., that ongoing translation is needed for degradation. These experiments were performed using intact animals, thus the inhibitor may be causing the release of one or more other inducers of TAT such as glucocorticoids, catecholamines, or glucagon through a general stress response.

Functional mRNA TAT Activity During TA T Reinduction

As mentioned above, although both glucocorticoids and cyclic AMP can induce TAT, maximal inductjon of this enzyme in liver and liver-derived tissues requires the presence of both of these inducers. Our line of HTC cells has minimal ability to respond to BtzcAMP alone. Although dexamethasone results in a typical induction of TAT, the addition of 1 mM BtZcAMP to the control cultures results in little additional increase in TAT activity and no change in mRNATAT activity, (TABLES I and 3, Experiment I). On the other hand, the addition of the same concentration of Bt2cAMP to cells grown in dexamethasone results in an increase in TAT catalytic activity that is several times greater than the basal value. We have previously shown that this process, which we have called reinduction or the restoration of an apparently lost function of HTC cells, is due to an increased rate of synthesis of TAT and is specific to Bt2cAMP.2' Thus, it would be interesting to know if this increase, like the increase caused by Dex itself, was associated with an increase in mRNATAT activity in the HTC cells. TABLE 3 presents an analysis of two experiments in which we quantitated mRNATAT as a function of time under reinduction conditions. In experiment I the mRNATAT level in cells cultured for 24 hr in Dex was 0.15% of the total poly(A)+RNA activity isolated from HTC cells, or abmt four times greater than the basal level of about 0.04%. By 60 min this value had reached 0.24% and enzymic activity was beginning to increase (see FIGURE 3). In experiment 11, mRNATAT

Granner et al.: Tyrosine Aminotransferase mRNA TABLE 3

EFFECT OF BtpcAMP ON mRNATAT DURING THE EARLY TIME COURSE OF REINDUCTION OF TYROSINE AMINOTRANSFERSE I N HTC CELLS*

191

mRNATAT as % of TAT Activity Experiment Condition total (mU/mg)

Control 0.04% 10 I + BtpcAMP 0.04 12

Dexamethasone 0.15 I I9 + BtzcAMP 0.24 138

Dexamethasone 0.17% 45 I1 + BtsAMP (15 min) 0.34 43

+ BtpcAMP (30 min) 0.35 51 + Bt2cAMP (60 min) 0.40 60

HTC Cells were grown in the presence or absence of 5 X M-dexamethasone. Twent four hours later I mM BtpcAMP was added to the control and induced cells, and mRNAT and TAT catalytic activities were determined afler an additional incubation time of one hour in experiment I and for the time indicated in experiment 11. The small changes in TAT specific activity are due to the fact that the new steady state for this function has not been reached in I hr. These conditions customarily result in a doubling of TAT activity.

1-

activity was examined at shorter intervals after the addition of the cyclic nucleotide. Within 15 min of the addition of the Bt2cAMP this activity had doubled to a value of 0.34%, and by 1 hr mRNATAT represented 0.4% of the total. Although at this time enzymic activity had only increased from 45 to 60 mU/mg protein, this is expected since the kinetics of this change are slower. We typically see a doubling of enzymic activity at steady state, so the mRNATAT changes are quite proportional. In several other reinduction experiments we have shown that the change in mRNATAT activity at steady state parallels the change in the rate of TAT synthesis and of enzymic activity. Since the increase of mRNATAT can also be prevented by adding actinomycin D to the cells at the same time as the BtZcAMP is added, the requirement for ongoing RNA synthesis is as apparent in the reinduction of TAT as it is in glucocorticoid induction.

CONCLUSION

Studies using cultured hepatoma cells have revealed aspects of specific protein regulation that could never have been achieved using a whole animal. Nonetheless, a number of the features of cyclic AMP and glucocorticoid hormone action, partic- ularly those involving the role of mRNA, had to be inferred from indirect studies that required the use of inhibitors of protein or RNA synthesis. In recent years it has become possible to quantitate the activity of specific mRNAs using a variety of techniques. These procedures are now sensitive enough to assay scarce mRNAs like that for TAT even when isolated from a few million cultured cells. The combination of cultured cells and direct assays of mRNA activity can thus be used to test a number of hypotheses based on the earlier, indirect studies.

Since inhibitors of RNA synthesis block the induction mediated by glucocorticoid hormones it was postulated that an RNA intermediate, presumably mRNA, was involved.3 Subsequent studies of the induction of tryptophan oxygenase’ and tyrosine aminotransferase,6” and this paper, have clearly demonstrated that each is associated with a proportionate increase in specific mRNA activity, thus confirming the general feature of this hypothesis. Several additional points can be made from the data presented in this paper. There cannot be a significant effect of Dex on translation


since the increase in mRNATAT activity is always sufficient to account for the increases noted in TAT synthesis and/or catalytic activity (TABLES I and 2, FIGURES 2 and 3). In fact, the increase in mRNATA1 always precedes the increases in synthesis and enzymic activity, suggesting a precursor relationship for the former (FIGURE 3). The glucocorticoid effect is extremely rapid (-30 min), requires RNA synthesis but not protein synthesis (FIGURE 3, TABLE 2). hence the steroid effect may be exerted directly at the gene level.

Since actinomycin D failed to block cyclic nucleotide induction of specific proteins, it was thought that this effect involved some enhancement of the rate of translation of a static amount of specific mRNA.'S.'3 In a test of this hypothesis, the direct assays of mRNA activity gave an unexpected result. The mRNAs for both phosphoenolpyruvate carboxykinaseZ6 and tyrosine amin~transferase"~ 25 are increased by BtncAMP in proportion to the increase in catalytic activity, and this can completely account for the induction process. Thus the mechanism of action of BtzcAMP must be reevaluated.

Twenty five years ago, Ingle postulated that the general role of glucocorticoid hormones, and perhaps their single most important action, is to support ("permit") the capacity of tissues to attain maximal rates of metabolic processes when such are required to maintain homeostasis." Numerous reactions or processes can be cited and, interestingly, many of these involve hormones whose action is mediated by cyclic AMP.% The biochemical mechanism of the interaction of steroid hormones and cyclic nucleotides is not well understood at all. Tyrosine aminotransferase is clearly induced by both agents yet maximal induction clearly requires that both be present. Since our line of HTC cells essentially fails to respond to BtgAMP unless the cells have first been treated with Dex" (TABLES I and 3) this would appear to be an excellent system in which to study the "permissive effect."

As in the other conditions described, the combination of Dex followed by BtZcAMP, which we call reinduction, is characterized by parallel increases in mRNATAT, the rate of synthesis of TAT and in aminotransferase enzymic activity (TABLE 3). In this case the increase in mRNATAT is extraordinarily rapid, as it occurs by 15 min. The implications of this vis a vis the primary effect of dexamethasone remains to be elucidated.

Studies such as these extend understanding by at least another level, but much remains to be done. It is not clear whether increased mRNA activity in a translational assay means that there is an increased mass amount of this mRNA. If so, is this increase due to an .increased rate of mRNA transcription, to a decreased rate of breakdown, or to some combination? To answer such questions, and to localize the sites of action of glucocorticoids and cyclic nucleotides, more sophisticated tools are required. It is clear that cultured hepatoma cells will be a system of choice when such studies are done.

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REGULATION OF TYROSINE AMINOTRANSFERASE mRNA IN HTC CELLS*

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