Relation of steroid structure to enzyme induction in hepatoma tissue culture cells

J. Mol. Biol. (1970) 52, 57-74

Relation of Steroid Structure to Enzyme Induction in Hepatoma Tissue Culture Cells HERBERT

H. SAMUELS? AND

GORDON

M. TOMKINS$

Laboratory of Molecular Biology, National Institute of Arthritis and Metabolic Diseases, National Institute of Health Bethesda, Mel 20014, U.S.A. (Received 3 July 1969, and in revised form 22 April 1970) Glucocorticoid hormones induce an increase in the rate of synthesis of the enzyme tyrosine aminotransferase in hepatoma tissue culture cells. On the basis of an examination of steroid concentration and structure with regard to induction of enzyme, we have classified a wide variety of steroids into four groups: (1) optimal inducers (e.g. cortisol, dexamethasone and corticosterone) which induce the enzyme to a maximal level; (2) sub-optimal inducers (e.g. 11/3-OH progesterone, 11-deoxycortisol, and deoxycorticosterone) which induce the enzyme to a sub-maximal but characteristic level and competitively inhibit induction by optimal inducers; (3) anti-inducers (e.g. testosterone, 17a-methyltestosterone) which cannot induce the enzyme, but can competitively inhibit induction by optimaland suboptimal inducers; and (4) inactive steroids which neither induce nor inhibit enzyme induction. These differences in steroid behaviour do not appear to result from differences in steroid uptake or metabolism, or from an effect on general cell protein synthesis or degradation of enzyme. We interpret our findings in terms of the interaction of these

steroids with an allosteric receptor system concerned with the regulation of synthesis of enzyme.

1. Introduction The glucocorticoid induction of the enzyme tyrosine aminotransferase in hepatoma tissue culture cells has been studied as a model to investigate the mechanism of steroid regulation of gene expression. In hepatoma cells, glucocorticoids induce a 5- to 15-fold increase in Tyr-aminotransferaseg activity and in its rate of synthesis (Granner, Hayashi, Thompson & Tomkins, 1968). The induction requires the continuous presence of the steroid, concomitant RNA synthesis, and the accumulation of a specific RNA fraction (Peterkofsky & Tomkins, 1967; Tomkins et al., 1966). The adenyl cyclase-cyclic-AMP system does not appear to mediate Tyr-aminotransferase induction in HTC cells (Granter, Chase, Aurbach & Tomkins, 1968). t Present address: New York University School of Medicine, Department of Medicine, New York, N.Y., U.S.A. $ Present address: University of California Medical Center, Department of Biochemistry, San Francisco, Calif., U.S.A. f Abbreviation used: HTC, hepatoma tissue culture; Tyr-aminotransferase, tyrosine aminotransferase. 57

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In the present studies we have examined the relationship between steroid structure and enzyme induction to gain a better understanding of the role of the steroid in the induction process. On the basis of these experiments, we have classified a wide variety of steroids into four groups which we have defined as optimal inducers, sub-optimal inducers, anti-inducers, and inactive compounds. We interpret these differences in behaviour to re0ect the nature of the interaction of the steroid with an allosteric receptor system concerned with the regulation of tyrosine aminotransferase synthesis.

2. Materials and Methods (a) Materials Dexamethasone was obtained through the courtesy of Merck and Co., Inc. Zcc-methylcortisol, Gee-methyl-ll&OH-progesterone, and 9a(-F-11/3-OH-progesterone were gifts from the Upjohn Co. All other steroids were purchased from the Mann Research Laboratories. The purity of all steroids was checked by thin-layer and gas chromatography and where necessary were further purified by preparative thin-layer chromatography. Stock steroid solutions were prepared in absolute ethanol at concentrations of either 5 x 10Y3 or

5X10-'M. Cycloheximide, puromycin, pyridoxal phosphate and cc-ketoglutarate were obtained from the Sigma Chemical Co. Chromatographic grade chloroform, ethyl acetate, methylene chloride and acetone were obtained from Fisher Scientific Co. Swim’s S77 powder, trypan blue (0.4% solution in Hanks’ BSS), fetal calf and bovine sera were obtained from the Grand Island Biological Co. Tyrosine (A grade) and tricine were obtained from the Calbiochemical Corporation. Bovine serum albumin was obtained from the Armour Pharmaceutical Co. [14C]Inulin [3H]cortisol (57 C/m-mole), [3H]deoxycorticosterone (40 o/m-mole), (297p4d.h and deoxycortisol (each at 4 c/m-mole) and a 3H-labeled 17~OH-progesterone [3H]amino acid mix (no. NET 250) (1 me/ml.) were obtained from the New England Nuclear Corporation. [3H]Dexamethasone (4 c/m-mole) was obtained from Schwarz Bioresearch Corporation. Liquifluor was obtained from Nuclear Chicago Corporation, and butyl-PBD and BBS-3 solubilizer from the Beckman Instrument Corporation. (b) Growth

of cells and induction

conditions

One clone (C2) of HTC cells was used for all experiments. The cells were grown at 37°C in suspension culture in Swim’s X77 medium modified to contain 5% fetal calf serum, 5% bovine serum, and 3 g glucose/l. The medium was buffered with 0.5 g NaHCO,/l., and 0.05 M-trioine, adjusted to pH 7.6 at 37°C (Gardener &Tomkins, 1969). Inthismedimn, the cells have a generation time of 22 to 24 hr, remain in the log phase of growth between a cell density of 2 to 7 x lo5 cells/ml., and reach the stationary phase at a density between 1 to 1.5 X lo6 cells/ml. For all experiments cells were centrifuged from growth media while in log phase (usually at a cell density of 6 x 105/ml.) and suspended at the same density in serum-free “induction media” which was otherwise identical to growth media. This was done to eliminate possible consequences of serum protein-steroid interaction at low steroid concentrations. Steroids were added to cell cultures in 20 pl. of the ethanol stock solution per 10 ml. of cell suspension. Equivalent amounts of ethanol were always added to the control samples. HTC cells synthesize Tyr-aminotransferase and remain viable by the criterion of trypan blue exclusion (Merchant, Kahn, & Murphy, 1964) for at least 24 hr in the absence of serum and in the presence of up to O*S”h ethanol. All incubations were carried out in tightly capped bottles at 37°C in a Metabolyte gyrotory shaker at a speed of 100 rev./min. (e) Tyrosine

amilzotransferase

assay

For enzyme assay, 2- to 5-ml. samples of the cell culture were removed and centrifuged. The cell pellets were suspended in chilled phosphate-buffered saline (0.05 M-sodium phosphate-O.1 M-SO&Urn chloride, pH 7.6) and centrifuged again. The resulting pellet was

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drained and frozen at -20°C. The frozen cell pellets were thawed, and suspended in 05 to 1.0 ml. of chilled 0.05 M-potassium phosphate buffer, pH 7.6. The samples were then either sonically disrupted for 10 see with a Branson Sonifier at a setting of 2 A, or else agitated gently, and centrifuged at 800 g for 2 mm in a refrigerated centrifuge. The supernatant layer was used for assay. Both techniques gave identical specific, enzymio activities. The enzyme was assayed at 37’C by a modification of the method of Diamondstone (1966) as described by Hayashi, Granner & Tomkins (1967). One unit of activity represents the formation of 1 pmole of p-OH-phenylpyruvate/min. Protein was assayed by the method of Lowry, Rosebrough, Farr & Randall (1951) using bovine serum albumin as standard. Enzyme-specific activity is expressed as milliunits of Tyr-aminotransferase/mg of cell protein. All enzyme activity referred to in the Results section represents specific activity. Where used, the relative Tyr-aminotransferase activity was calculated using the induction by 1 x 10M5 M-cortisol to represent full induction. (d) Measurement

of amino

acid incorporation

The [3H]amino acid mixture was added to cell suspensions in induction media at 37°C (final activity 0.2 &ml.) and samples were removed 1, 2 and 3 hr later. The cells were treated as described for the enzyme assay, frozen and thawed, suspended in chilled 5% trichloroacetic acid, heated at 95’C for 20 min, and cooled. The precipitates were centrifuged, the supernatant layers decanted and the pellets suspended and centrifuged twice in the chilled 5% trichloroacetic acid. The final pellets were dissolved in 05 ml. of 0.1 N-NaOH. Half the sample was used to assay for protein while 0.2 ml. of the remainder was added to 10 ml. of Liquifluor-ethanol-toluene (3 : 22 : 75, by vol.) and counted in a Nuclear Chicago scintillation counter. (e) Measurement

of cell-associated

steroid

Tritium labeled steroids were added to 30 ml. of cell suspension in induction medium and these were then incubated for 16 hr at 37°C. At that time 0.25 to 0.35 PC (0~5 to O-7 ml.) of [14C]inulin was added and allowed to incubate for 10 min. The sample was then centrifuged, and the supernatant layer carefully drained and saved. Each pellet was suspended in fresh induction medium to a total volume of 1 ml. and sonicated as described previously. Samples of the original supernatant layer and of the cell sonicate were used for double label scintillation counting in 10 ml. of butyl-PBD scintillation mixture (8 g butyl-PBD, 100 ml. BBS-3/l. toluene). The moles of steroid in the sonicated pellet which were associated with cells was calculated by subtracting the moles of steroid in the extracellular in&n space from the total moles of steroid in the pellet. The moles of steroid in the extracellular volume of the cell pellet was calculated by multiplying the [14C]~mlin &s/mm in the pellet by the ratio of the moles of steroid to inulin cts/min determined in the supernatant layer. The intracellular steroid concentration is expressed as moles of steroid per liter of ceh water based on the finding that HTC cells are 83% water by weight (Samuels, 1968, unpublished results). To check that inulin is limited to the extraoellular volume, its volume of distribution was compared to that of trypan blue which does not penetrate viable cells (Merchant et a.?., 1964) and found to be identical. (f ) Steroid

chromatography

Steroids were chromatographed at room temperature on Eastman no. 6060 silica ge thin-layer sheets in either of two solvent systems: (1) ethyl acetate-chloroform (50 : 50, v/v) or (2) methylene chloride-acetone (70 : 30, v/v). These systems easily separate eortisol from its known metabolites and the less polar progesterone derivatives. The possibility that HTC cells metabolize steroids was investigated by incubating the [3H]steroid at the desired concentration with a 50 ml. suspension of cells at induction conditions for 20 hr at 37°C. At the end of the incubation a 4-ml. sample was removed and prepared for enzyme assay. The remainder of the suspension was centrifuged. The supernatant layer was saved (supernatant l), and the cell pellet carefully suspended in 1 ml. of chilled isotonic phosphate-buffered saline and centrifuged. This latter supernatant layer (supernatant 2) was decanted and saved. The cell pellet was then frozen and thawed,

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suspended in 0.05 M-potassium phosphate, pH 7.6, and sonicated as described for the enzyme assay. Both supernatant fractions and the sonicated pellet fraction were extracted once with either 4 vol. of ethyl acetate or methylene chloride by agitation for 30 see on a vortex shaker. About 97% of the added radioactive steroid remained in the media (supernatant 1) while approximately 3% of the total was present in the second supernatant and the sonicated pellet. Approximately 2 to 3% of the radioactivity in the pellet could not be extracted into either solvent. This fraction was extracted, however, after heating the sample at 60°C for 20 min. In some experiments [14C]steroid was added to the suspended pellet before sonication as an internal control to test the possibility that steroid transformation might occur during the heating and the extraction procedure. The ethyl acetate or methylene chloride extracts were evaporated to dryness under a stream of lOOo/o nitrogen and redissolved in 30 ~1. of ethyl acetate. These samples were chromatographed and compared to a simultaneous chromatogram of the stock E3H]steroid. In all cases a small amount of unlabeled steroid was added to the sample before chromatography as an internal control. After development on the appropriate solvent system the unlabeled steroid was located with an ultraviolet lamp and the ohromatogram was then out into strips of 2.5 cm x 20 cm. Each strip was then cut into 20 l-cm pieces and each piece was counted in 10 ml. of Liquifluor-ethanol-toluene system (3 : 22 : 75 by vol.). There was virtually complete recovery of the radioactivity added to the chromatogram, and known quantities of radiothat the strip did not interfere with the activity added to a 1 cm x 2.5 cm strip indicating

counting efioiency.

3. Results (a) Kinetics of tyrosine aminotralzsferase induction Figure l(a) and (b) illustrates a series of experiments in which the time course of enzyme activity in suspensions of HTC cells was followed after the addition of various concentrations of either cortisol (Fig. l(a)) or dexamethasone (Fig. l(b)). Each induction curve shows a slight lag period, followed by a rapid rise in enzyme activity which, after approximately 12 to 16 hours, reaches a constant value. The plateau activities increase with increasing inducer concentration over the range of 2.5 x 10e8 M to 1 x 10V6 M for cortisol, and 25 x 10eQ M to 5 x lo-* M for dexamethasone. The inducer concentration required to induce the maximum plateau value varied somewhat from experiment to experiment, e.g. with cortisol this varied from 2 x 10m7M to 1 X1o-6

M.

The time course of enzyme induction seen with other inducing steroids follows a similar pattern, and differs only in the level of the maximum plateau and the concentrations at which the induction begins and the maximum plateau is reached. (b) Dose-response curves Prior studies using a specific radioimmunoprecipitation technique have shown that at the fully induced plateau level, the rate of Tyr-aminotransferase synthesis is maximal (Granner, Hayashi, Thompson & Tomkins, 1968). This plateau represents a steady-state the activity of which is determined by the rates of enzyme synthesis and degradation.? We demonstrate below that the rate constant for enzyme degradation does not change from one experiment to another. Thus a difference in steady-state t The rate of enzyme accumulation dE/dt, has been represented by the equation, dE/dt = kI - k, [El, where Ic, is the zero-order rate constant for synthesis, k, the first-order rate constant for degradation, and [E] the enzyme concentration (Segal & Kim, 1966). At the steady state dE/dt = 0 and therefore [I$] = k,/k,. Thus, the ratio of two different steady-state enzyme a&vities equals the ratio of their rates of synthesis provided that kZ is the same in both experiments.

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FIG. 1. Time-course of Tyr-aminotransferase induction as a function of cortisol (a) and dexamethasone HTC cells (6OO,OOO/ml.) were incubated at the steroid concentrations shown in the Figure. Samples for enzyme activity as described in Materials and Methods. The maximum induced Tyr-aminotransferase methasone and 127 m-units/mg for cortisol.

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Fra. 2. Steroid dose-response curve. HTC cell suspensions were incubated with each of the steroids at the concentrations listed in the Figure. Samples were removed between 14 to 24 hr and assayed for enzyme to determine the steady-state activity. The net increase in steady-state activity was determined by subtracting the basal activity from the induced steady-state value. The maximum net steady-state enzyme activity induced by cortisol (1 x 10d6 M) was 125 m-units/mg and WBS used to represent full induction.

enzyme concentrations is a measure of the difference in the rates of Tyr-aminotransferase synthesis. These considerations have been verified by experiments in which steady-state enzyme aotivities were shown to correlate linearly with the rates of synthesis determined by radioactive amino acid incorporation and specific immunoprecipitation of Tyr-aminotransferase (Granner, 1968, unpublished data). We have constructed a series of dose-response curves for a variety of different inducing steroids in which steady-state enzyme activities are plotted against the steroid concentration (Fig. 2). On the basis of these steady-state activities induced at high steroid concentrations we have arbitrarily classified inducers into two groups. The steroids (dexamethasone, cortisol and corticosterone), which induce the enzyme to a maximal level we refer to as optimal inducers. These compounds differ with respect to the lowest hormone concentration which can induce as well as the concentration required to induce to the maximum level. Table 1 lists examples of optimal inducers. TABLE

1

Optimal inducers Optimal

inducer

Dexamethasone Cortisol Corticosterone Aldosterone Zcr-Methyl-oortisol Bcr-Methyl-l l/3-OH-progesterone 9a-F-llfi-OH-progesterone

Concentrationt (M) 8x10-9 6~10’-~ 3 x10-T 4 x10-7 * * *

t The concentration is that which induces to 0.5 of the maximum steady-state Tyr-aminotransferase activity. *Detailed dose-response kinetics were not determined for these compounds.

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TABLE 2 Effect of sub-optimal

indu,cers on tyrosine aminotransferase induction Relative

Sub-optimal inducer

net steady-state

111

Sub-optimal

(1 X lo-5

inducer

enzyme activity

Sub-optimal

M)

(1 x 10 - 5 M)

+

cortisol None 2 1-Deoxycortisol Deoxycorticosterone 5wDihydrocortisol 1 l/f-OH-Progesterone Il-Deoxycortisol Progesterone 17wOH-Progesterone

PI

inducer

-

(1 x lo-’

Ma)

73 58 50 43 33 11 12

75 46 36 35 25 6 5

HTC cells were incubated at 37°C with each sub-optimal inducer alone (1 x low5 M) and in combination with cortisol(1 x IO-? M). Samples were removed between 14 to 22 hr to determine the net steady-state enzyme activity as described in the legend to Fig. 2. The maximum net steadystate enzyme activity induced by cortisol (1 x 10e5 M) was 105 m-units/mg.

Figure 2 illustrates examples of another group of steroids (ll/%OH progesterone, lldeoxycorticosterone, 21-deoxycortisol and progesterone) which, even at very high concentrations, cannot induce to the maximum enzyme activity seen with the optimal inducers. Each, however, induces a sub-maximal level characteristic for the steroid. We call this class of compounds sub-optimal inducers. Examples of sub-optimal inducers in decreasing order of effectiveness are listed in the first column of Table 2. (c) Competitive

steroid interactions

It seemed probable that both optimal- and sub-optimal inducers have the same site of interaction in HTC cells. We examined this possibility by studying the effect that sub-optimal inducers have on induction by optimal inducers. Figure 3(a) illustrates an example of such an induction experiment with the optimal inducer, cortisol at 1 x 10M7 M and the sub-optimal inducer ll/%OH progesterone at 1 x 10e5 M. When both steroids were added at the beginning of the experiment, and enzyme activity was followed for 20 hours, the steady-state activity was not the sum of the individual steady-state levels but was close to that induced by the sub-optimal inducer alone. At lower cortisol concentrations, 5 x lOma M, lip-OH-progesterone, 1 x 10T5 M, completely inhibited cortisol induction. The ability to interfere with cortisol induction was found for all the sub-optimal inducers examined (Table 2). In each case the resultant steady-state level was close to that observed with the suboptimal inducer alone. The weaker the sub-optimal inducer the greater its relative ability to inhibit cortisol induction. The inhibition could be completely reversed either by increasing the cortisol concentration or by decreasing the sub-optimal inducer concentration. These findings suggest that optimal- and sub-optimal inducers compete for the same steroid receptor system; and that the maximum effect of the receptor system on enzyme synthesis is determined by the chemical structure of the interacting steroid.

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HTC cells (550,00O/ml.) were incubated for 22 hr with (1) cortisol, FIG. 3(a) Effect of llg-OH-progesterone on Tyr-aminotransferase induction. 1 x 1O-7 M, (2) Il,!?-OH-progesterone, 1 x 10mGM, and (3) the combination of cortisol, 1 x 10m7 M, + llfi-OH-progesterone, 1 x 10e5 M, for 22 hr. Samples were removed at the times indicated, and assayed for enzyme activity. The maximum steady-state activity induced by 1 X 10d5 M cortisol in a control experiment was 122 m-units/mg. (b) Effect of sub-optimal inducers on Tyr-aminotransferase induction. HTC cell suspensions (500,000 cells/ml.) were incubated with either (1) oortisol, 1 x lo-” N, (2) 11/3-OH-progesterone, 1 x low5 M, + cortisol, 1 x 10V7 iv& or (3) progesterone, 1 x 10T5 M, + cortisol, 1 x 10m7 M. After 18 hr the suspension incubated with cortisol alone was divided into three equal fractions. Progesterone was added to one fraction and lip-OH-progesterone to a second fraction, each to a final concentration of 1 x lo-5 N. The third served as a control. The suspensions were incubated for an additional 7 to 10 hr. Samples were removed at the times indicated and assayed for enzyme activity. The maximum steady-state activity induced by cortisol, 1 x lo- 5 M, was 116 m-units/mg.

.& .z :: El 0 .5 $ ‘Z 5

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Previous experiments have shown that in preinduced cells, the enzyme activity and the rate of synthesis falls rapidly to the basal level when the concentration of the inducer is reduced to non-inducing levels (Samuels, 1968, unpublished data; Martin, Tomkins & Bresler, 1969). This suggests that the interaction of the steroid with the receptor system regulating Tyr-aminotransferase synthesis can be rapidly reversed. Figure 3(b) illustrates a set of experiments which were designed to study the kinetics of inhibition by sub-optimal inducers. When either llfl-OH progesterone or progesterone was added to a culture of cells preinduced by cortisol there was a rapid decrease in the steady-state enzyme activity to that attained when both cortisol and the suboptimal inducer were added at the beginning of the experiment. These rates of decline of activity were slightly less than the rate observed when the cortisol concentration was rapidly reduced to non-inducing levels. This was expected since the sub-optimal inducers do not reduce the rate of enzyme synthesis to that of the uninduced state. In each case, 15 to 30 minutes after the sub-optimal inducer was added the enzyme activity had begun to decline exponentially. This suggests that the rate of synthesis decreased to the new rate almost immediately after the sub-optimal inducer was added. The wide spectrum of steroid response suggested the possibility that a class of purely inhibitory steroids, anti-inducers, might exist. A number of non-inducing steroids was examined for their ability to inhibit induction by oortisol. Certain of these were found to have anti-inducer activity. These compounds are listed in Table 3. Figure 4 illustrates a set of typical experiments with two such anti-inducers. At a concentration of 1 x lo-= M neither 17c+methyltestosterone nor testosterone-induced Tyr-aminotransferase, however, when either steroid was added with 1 x 10e7 Mcortisol, enzyme induction was inhibited. At higher cortisol concentrations, e.g. 1 x lo-= M, or at lower anti-inducer concentrations, e.g. 1 x 10m7M, this inhibition could TABLET Effect of anti-inducers on tyrosine aminotransferase induction Relative Anti-inducer

None Testosterone 19-Nor testosterone 178-Estradiol d l-Testosterone Cortisone Fluoxymesteronet 17a-Methyltestosterone

net steady-state enzyme activity Anti-inducer (1 x 10m5 M) + cortisol (1 X lo-7 M) 68 45 40 39 35 33 12 8

Cell suspensions were incubated with cortisol alone and together with each anti-inducer st the concentrations indicated. Cells were also incubated with cortisol at 1 x lOWe M to determine the maximum steady-state enzyme activity. Samples were removed for assay after 14 to 20 hr of incubation and the net Tyr-aminotransferase steady-state activity was determined as described in the legend to Fig. 2. The maximum net steady-state enzyme motivity induced by 1 x 10-s Mcortisol was 112 m-units/mg. t 9a-F-11 j-OH-testosterone. 6

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@Testosterone

(?xIO-~

Time (hr)

Fm. 4. Effect of anti-inducers on Tyr-aminotransferase induction. HTC cell suspensions (600,000 cells/ml.) were incubated with either (1) cortisol, 1 x 10m7 M, (2) testosterone, 1 x 10e5 M, (3) 17x-methyltestosterone, 1 x 10M5 M, (4) testosterone, 1 x 10-a &r, and cortisol, 1 x lo-’ M, or (5) 17or-methyltestosterone, 1 X 1Om5 M, and cortisol, 1 x lo-’ in. After 16 hr the suspension incubated with oortisol alone at 1 x 10 - 7 M was divided into three equal fractions. Testosterone was added to one fraction to a ha1 concentration of 1 x 10e5 M, and 17~~ methyltestosterone to the second to a final concentration of 1 x 10m5 M. The third fraction served as a control. All the suspensions were incubated for an additional 8 hr. Samples were removed at the times indicated and assayed for Tyr-aminotransferase specific activity. The maximum steadystate activity induced by cortisol, 1 x 10e5 M, was 128 m-units/mg.

’ 17oc- Methylte&osterone

1 xix9

5:10-s

Cortisd

concn (M)

FIG. 5. Effect of 17cc-methyltestosterone on steady-state Tyr-aminotransferase activity. Pairs of HTC cell suspensions were incubated at the cortisol concentrations indicated. To one of each pair was added, l+i’cr-methyltestosterone, at concentrations indicated. Samples were removed between 14 to 22 hr to determine the enzyme steady-state activity. The net steady-state activity was calculated as described in the legend to Fig. 2. The maximum net steady-state enzyme activity induced by cortisol, 1 x 10-S M, was 108 m-units/mg.

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be completely reversed. As seen with the sub-optimal inducers, the addition of an antiinducer to pre-induced cells results in a prompt fall in enzyme activity to the level seen when cortisol and the anti-inducer were added at the beginning of the induction. Again, the kinetics of inhibition suggest that a readjustment to the new rate of enzyme synthesis occurs almost immediately after the addition of the anti-inducer. The inhibition was also studied by examining the effect of varying the anti-inducer concentration on the cortisol dose-response curve. Figure 5 shows a plot of relative Tyr-aminotransferase steady-state activity verse cortisol concentration in the presence of several concentrations of 17c~methyltestosterone. The curve in the absence of antiinducer appears to be slightly sigmoidal. This shape became more pronounced as the concentration of anti-inducer is increased from 1 x 10V6 BI to 1 x 10e5 M. Only a small number of the non-inducing steroids examined had anti-inducer activity. The remainder we have classified as inactive steroids. They are listed in Table 4. TABLET Inactive steroids Epicortisol (1 k-OH) 1 la-OH-Progesterone I lee-OH-17a-Methyltestosterone Epitestosterone (17ar-OH) Androstenedione 1 l/3-OH-Androstenedione

BOrr-OH-Cortisol SOP-OH-Co&sol 58, 3cr-Tetrahydroeortisol 5cr, 36-Tetrahydrocortisol 5a, 3a-Tetrahydrocortisol

We would like to be able to interpret our findings in terms of the interaction of these steroids with a specific receptor system. To eliminate the possibility that the observed differences in steroid behaviour are a result of other mechanisms, we examined the effect of these compounds on enzyme turnover, cell protein synthesis, and the intracellular steroid concentrations, and steroid metabolism. (d) Enzyme turnover A comparison of steady-state Tyr-aminotransferase activities reflects rates of enzyme synthesis provided that the rate constant of degradation remains unchanged.? Our findings that different steroids induce to different maximal steady-state levels, and that some steroids act only to inhibit induction raised the question of whether any of these compounds might accelerate the rate of enzyme degradation. Recent experiments indicate that the half-life of Tyr-amiuotransferase measured by loss of enzyme activity corresponds with that measured directly by the radioimmunoprecipitation technique (Martin et al., 1969). These experiments also indicate that neither cycloheximide nor actinomycin D inhibit enzyme degradation under usual experimental conditions. Therefore, the influence of sub-optimal- and anti-inducers on the kinetics of enzyme degradation was examined by inducing cells with cortisol at 1 x 10M7 M and at the steady state adding the sub-optimal inducer or anti-inducer in question at a concentration of 1 x 10m5M. After 20 to 30 minutes of additional incubation general protein synthesis was inhibited with either cyoloheximide, 2 ~10~~ M, or puromyoin, 1 x 10V3 M, and the decline of enzyme activity was followed for five to eight hours. 7 See footnote

on p. 60.

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Time (hr)

FIG. 6. Turnover of Tyr-aminotransferase. Two suspensions of HTC cells were incubated for 16 hr with either cortisol, 1 x 1O-5 M, or cortisol, 1 X 10-r M. The suspension induced with cortisol, 1 x 10e5 M, was divided into three equal fractions. Cycloheximide was added (final concentration 2 x 10m4 M) to one fraction, while puromycin was added (6nal concentration of 1 x 10m3 M) to a second fraction. The third served as a control. Each suspension was incubated for an additional 6 to 8 hr, and samples were removed at the times indicated and assayed for enzyme activity. The cell suspension incubated with cortisol, 1 x 10-r M, was divided into 7 fractions. To each of four of the fractions a different one of the following was added to a final concentration of 1 x 10e5 M, 17~methyltestosterone, llfi-OH-progesterone, deoxycortioosterone or progesterone. All seven fractions were incubated for an additional 30 min. At that time cycloheximide was added (6nal concentration of 2 x lo-* M) to each of the four above fractions as well as to a fifth fraction containing only oortisol, 1 x lo-* M. A sixth fraction was diluted 20-fold with fresh induction media, prewarmed to 37V. The seventh fraction served as a control. All suspensions were incubated for an additional 6 to 7 hr and samples were removed at the times indicated and assayed for enzyme. The activities for cells incubated with cortisol, 1 x 10e5 M, was 113 m-units/mg and 78 m-units/mg for cells incubated with cortisol 1 x 10e7 M. (A) Cortisol, 1 x 10e5 M + cycloheximide; (A) cortisol, 1 X 10m5 M + puromycin; (0) cortisol, 1 x 10-r M + cycloheximide; (0) cortisol, 1 x 10-r M diluted to cortisol, 5 X 10e9 M; (V) progeaterone, 1 x 10m5 M + oortisol, 1 X lo-? M + cyoloheximide; (V) deoxycortisol, 1 X 10s5 M + cortisol, 1 x 10-r M + oyoloheximide; (H) ll/3-OH-progesterone, 1 X low5 M + cotiisol, 1 X 10e7 M + cycloheximide; (0) 17cr-methyltestosterone, 1 x 10e5 M + oortisol, 1 X 10M7 M f cycIoheximide.

Sub-optimal inducers which induced Tyr-aminotransferase to specific activities greater than 30 m,u/mg, were also studied by the same method. The effect of different concentrations of oortisol on enzyme turnover were also examined in a similar manner. Figure 6 illustrates a semi-logarithmic plot of the data from these experiments. For each case under induction conditions the half-time for enzyme decay was 5 hours & 20 minutes. This half-life is in the same range determined in preinduced cells after the cortisol concentration is rapidly reduced to a non-inducing level (Samuels, 1968, unpublished data). We conclude that the differences in induction kinetics seen with optimal-, sub-optimal, or anti-inducers cannot be accounted for by alterations in enzyme turnover. (e) Effects of steroids on protein synthesis Certain progesterone and androgen derivatives inhibit oxidative metabolism in mammalian cells (Yielding & Tomkins, 1959; Stoppani, Brignone & Brignone, 1968). We therefore examined the possibility that the effects of sub-optimal-and anti-inducers

STEROID

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5

Effects of steroids on protein synthesis Steroids

(1) Cortisol (2) 17or-OH-Progesterone + cortiso1 (3) Progesterone + cortisol (4) Testosterone + cortisol (5) Fluoxymesterone + cortisol (6) &Dihydrocortisol + cortisol (7) Cortisone + oortisol (8) Ilg-OH-Progesterone + cortisol (9) Deoxyoorticosterone + cortisol (10) 11 -Deoxycortisol + cortisol (11) Cyoloheximide

Conon (M) 1 x10-7 1 x10-5 1x10-7 1x10-s 1 x10-7 1x10-s 1x10-7 1x10-s 1 x10-7 1 x10-6 1 x10-7 1 x10-5 1 x10-1 1x10-s 1 x10-7 1 x IO-6 1 x IO-7 1 x 10-6 1x10-7 2x10-4

cts/min/mg/hr

Enzyme

activity

34,940 33,125

62 20

33,087

19

32,065

42

32,094

22

32,081

44

33,660

31

30,459

37

33,910

49

35,082

33

630

17

HTC cells were incubated with cortisol at 1 x 10e7 M and after 16 hr the suspension was divided into 11 equal volumes. A different sub-optimal or anti-inducer was added to each of 9 of the was added to the tenth suspension to a final concentrasuspensions of 1 x 10e5 M. Cycloheximide tion of 2 x 20m4 M, and the last suspension served as a control. All suspensions were incubated for an additional 25 min. The [3H]amino acid mixture (final activity 0.2 +/ml.) was then added to each sample, and the incubation continued for an additional 8 hr. Portions were removed 1, 2 and 3 hr after the addition of isotope and assayed for incorporation of radioactivity into general cell protein, as described in Materials and Methods. The rate of incorporation was linear for 2 hr and decreased by 10% at 3 hr. An additional sample was removed from each suspension after 8 hr and assayed for tyrosine aminotransferase activity.

on Tyr-aminotransferase induction are secondary to a general inhibition of protein synthesis rather than to a specific effect on enzyme synthesis. Table 5 shows the results of this experiment. Each of the steroids tested produced the expected degree of inhibition of enzyme induction but in no case did they inhibit the rate of amino-acid incorporation into general ’ cell protein. Therefore, the effects of sub-optimal- and anti-inducers on Tyr-aminotransferase induction are not secondary to a general inhibition of protein synthesis. (f ) Cellulur accumulation of steroids We considered the possibility that differences in the magnitude of enq-me induction seen with optimal- and sub-optimal inducers reside not in their different affinities for a specific receptor system, but rather in differences in their intracellular concentrations. In like manner, compounds which inhibit induction by cortisol could do so by limiting cortisol uptake resulting in a lower intracellular oortisol concentration.

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Extracellular

AND

G. M. TOMKINS

steroid

concn (M)

FIG. 7. Steroid accumulation by HTC cells. Each of the following 3H-labeled steroids, deoxycorticosterone, 17~(-OH progesterone, dexamethasone and cortisol were separately incubated with HTC cells for 18 hr at the concentration indicated. The cellular steroid concentration was estimated as described in Materials and Methods,

To test the first possibility, suspensions of cells were incubated with various [3H]steroids at concentrations from 1 X lo-lo M to 1 X 10m5M. [l*C]Inulin was used to estimate the steroid content in the extracellular space of the cell pellet. The intracellular steroid concentration was calculated at each extracellular concentration and expressed as moles of steroid per liter of cell water (see Materials and Methods). Figure 7 shows that at each cortisol concentration, the intracellular concentration was 2.5 to 3.5 times greater than the extracellular value. The ratio of the intracellular to extracellular concentrations was approximately 6 for dexamethasone, 20 for 17~OHprogesterone and 50 for deoxycorticosterone. Though we do not know the physical state of the steroid in the cell, nor the mechanism of its accumulation, the total cell steroid concentration bears no relationship to the effectiveness of a compound as au inducer. To investigate the possibility that inhibition of enzyme induction occurs as a result of inhibition of optimal inducer accumulation, the intracellular cortisol concentration was measured in the presence of 11-deoxycortisol, 17~OH-progesterone, deoxycorticosterone and 17c+methyltestosterone. [3H]Cortisol was added to each of five cell suspensions to a final concentration of 1 x 10T7 M. To each of four of the suspensions one of the above inhibitors was added to a final concentration of 1 x 10d5 M, while the fifth served as a control. After 16 hours of incubation, the intracellular cortisol concentration as well as the enzyme activity was measured. In each case the intracellular cortisol concentration was approximately 3 X IO- 7M, (range 2.7 to 3.3 X lo- 7M) while enzyme induction was inhibited to the expected degree. This indicates that suboptimal- and anti-inducers do not inhibit enzyme induction by inhibiting cortisol accumulation.

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(g) Steroid metabolism In intact animals the liver functions as the major organ for steroid metabolism. Since our cells were derived from a rat hepatoma, it was important to verify that steroids incubated with them were not metabolically altered. In certain hormoneresponsive tissues a metabolite and not the added steroid may act as the true inducer (Bruchovsky & Wilson, 1968). Thus, steroids might differ in their effectiveness as inducers of Tyr-aminotransferase as a result of differences in their metabolism by HTC cells. Likewise, sub-optimal- and anti-inducers might inhibit enzyme induction by inhibiting the conversion of cortisol to a true inducer. Steroid metabolism was investigated by incubating [3H]cortisol at 1 x lo-?, 1 x lOA and 1 x~O-~ M and deoxycorticosterone and 11-deoxycortisol each at 1 x 10m5M with HTC cells under induction conditions for 20 hours. The steroids were then extracted as described in Materials and Methods. Thin-layer chromatography of the extracts of the two supernatants as well as an extract from the cell pellet showed no detectable metabolic conversion. In the experiment incubated with 1 x 10M5 Mcortisol a small fraction (2 to 3%) of the radioactivity in the pellet could not be extracted at room temperature (23”C), but was extracted after heating the pellet at 60°C for 20 minutes. This extract was chromatographed and 95 to 97% of the radioactivity migrated as cortisol. The remaining 3 to 5% chromatographed as a more polar metabolite. This metabolic conversion accounts for less than 0.1 o/0of the total cellular cortisol. Although this metabolite remains unidentified, its 11, in the two solvent systems was identical to that of the inactive tetrahydrocortisol derivatives. [14C]Cortisol added to the pellet before sonication showed no conversion indicating that the more polar 3H-labeled derivative is not an artifact of the sonication or heating procedure. Deoxycorticosterone and 11-deoxycortisol were extracted and chromatographed in the same manner; neither was found to be metabolized or converted to their respective ll,%OH derivatives, e.g. corticosterone and cortisol. Thus, except at very high cortisol concentrations no detectable steroid metabolism was observed. This suggests that the hormones function without requiring structural modification by the cell.

4. Discussion The data we have presented indicate that HTC cells respond to a variety of structurally different steroids which either induce tyrosine aminotransferase to characteristic levels and/or inhibit its induction. These different responses are not a result of different metabolic fates of the steroids, their intracellular distributions, effects on general synthesis or changes in the rate constant for enzyme inactivation. We conclude, therefore, that the responses of the cells evoked by the different steroids result from the nature of their interaction with a specific steroid receptor system which, in turn regulates the synthesis of enzyme. To account for our findings, we propose that the steroid receptor is an allosteric system and, in fact, previous studies had indicated that steroid hormones can act as allosteric ligands to modify protein structure and activity (Tomkins &Yielding, 1961). To analyze our data quantitatively, we could have used either the model developed by Koshland, Nemethy & Elmer (1966) or that proposed by Monod, Wyman & Changeux (1965). We have no experimental basis on which to decide which of the two treatments is more appropriate and we arbitrarily chose the latter to represent our results.

72

H.

0.1

H.

SAMUELS

AND

G. M.

100

IO

I.0

Relative

steroid

TOMKINS

1000

toncn

FIG. 8. Theoretical steroid dose-response curve. These curves were derived from the equations given by Monod et al. (1965). Since maximum induction (R = 1) results in an approximate lo-fold increase in the rate of enzyme synthesis, we assumed, in drawing the theoretical curves, that L, the allosteric equilibrium constant is equal to 10. Because of the co-operative dose-response kinetics, n (the number of steroid binding sites per molecule) > 1. For the curves above we assume that n = 4. KT was taken to be 16 for each curve, and KR was calculated

from the values of C

shown.

The proposed allosteric steroid receptor is assumed to equilibrate between two different conformational states, R and T, each having different activities with respect to enzyme induction. For the purpose of discussion, we assume that the rate of enzyme synthesis is directly proportional to the fraction of the allosteric system in the R form, designated as ii. According to this model, the behaviour of the different classes of steroids is explained by their ability to determine different values of 2 as a result of their different affinities for the R and T states, expressed as K, and KT, respectively (see Rubin & Changeux, 1966). Co-operative steroid binding is predicted by the allosteric model of Monod et al. (1965), provided there are multiple sites of interaction between the steroid ligand and the macromolecular receptor. The dose-response curves shown in Figure 5 are all Xshaped. We do not know if these kinetics are the result of co-operative steroid binding, or of co-operative interactions of the receptor (R state) with a rate-determining step in enzyme synthesis. If we assume that the steroid receptor is composed of subunits, the co-operativity might occur at the level of the steroid-receptor interaction. Theoretical plots (Fig. 8) of the relative rate of Tyr-aminotransferase synthesis, i.e. 8, vers’susrelative steroid concentrations for several values of K,/K, have been made and show that at high concentrations, steroids having values of K,/K, up to 0.25 shift the allosteric equilibrium so that the rate of enzyme synthesis becomes maximal (& approaches 1). However, as the value of KR/KT increases to 0.25 the steroid concentration required for maximum enzyme synthesis also increases. These curves are quite similar to the dose response curves of the different optimal inducers shown in Figure 2. According to the allosteric model, steroids which have values of K&/K, between 0.25 and 1.0 are sub-optimal inducers. In each case at high steroid concentration the rate of synthesis (A) reaches a plateau at a sub-maximal level determined by the ratio of KR to II,. Likewise, anti-inducers would have values of KR/KT equal to or slightly greater than 1.0. Figures 2 and 8 indicate that induction of the enzyme begins and reaches a plateau

STEROID

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level at concentrations characteristic for each steroid. According to the model the concentration at which induction begins is determined by the absolute value of En, while the concentration at which the plateau level is reached is determined by both the absolute value of KS and the ratio of KS to KT. Since the limiting rate of synthesis is a function of the ratio of KE to K, and not their absolute values, it is conceivable that all steroids have the same affinity for one of the conformational states (e.g. T), and differ only in their afkity for the other state (e.g. R). The steroid dose response curve (Fig. 2), illustrates that with the exception of 11/3-OH-progesterone, the higher the steroid concentration at which induction begins, the lower the limiting enzyme activity. This suggests that the different limiting rates of enzyme synthesis induced by a steroid may be determined only by the value of Ir,. This model also explains the inhibition of induction observed with sub-optimal- and anti-inducers. The rate of enzyme synthesis observed with any steroid combination is determined by individual steroid concentrations as well as their values of KR and their ratios of KR to KT. According to the model, inactive steroids interact with neither conformational state of the receptor. This treatment of our data is similar to that presented recently by Williams & Paigen (1968) for the induction of ,&galactosidase in E. co&. We examined the relationship of steroid structure to Tyr-aminotransferase induction with the hope of gaining some added information regarding the nature of the steroid receptor binding site. The simplest steroid which can induce this enzyme is progesterone, which has a planar A/B ring junction, and a two-carbon side chain at the 17/3-position with a keto group at C-20. The addition of either a 17a-OH, 21-OH, 6u-methyl or an lI@-OH group or a combination of these substituents to the basic progesterone structure results in compounds which are more effective inducers. The 11/3-OH group is not an absolute requirement for enzyme induction. All steroids tested which have optimal inducer activity have an 11/3-OH group, however, not all steroids with this functional group are optimal inducers, e.g. lip-OH progesterone. In addition to a relatively planar A/B ring junction the only absolute structural requirement for anti-inducer activity appears to be the 17j%OH group. For example, testosterone (C-17/I-OH) is an anti-inducer while androstenedione (C-17 ketone) and epitestosterone (C-17~0H) are inactive compounds. The introduction of an ll~r-OH group, the formation of an A/B cis ring junction, or the reduction of both the 4-S double bond and the 3-keto group or of only the 28keto group results in the formation of an inactive compound. According to the receptor model this is explained by the fact that these compounds do not interact with either conformational state of the receptor. There are similarities in the steroid specificity for the induction of glutamine synthetase in embryonic chick retina (Moscona & Piddington, 1967), thymocyte involution (Makman, Dvorkin & White, 1968), alkaline phosphatase induction in HeLa cells (Melnykovyoh & Bishop, 1969) and tyrosine aminotransferase induction in hepatoma tissue culture cells. This suggests that the steroid receptors in each system may be very similar and that glucocorticoids have the same fundamental action in each case. REFERENCES Bruchovsky,

N. & Wilson,

J. (1968). J. Biol.

Chem. 243, 5953.

Diamondstone, T. I. (1966). Analyt. Biochem. 16, 395. Gardener, R. 8. & Tomkins, G. M. (1969). J. Biol. Chem. 244, 4761. GIXMCX, D. K., Chase, L. R., Aurbach, G-.D. & Tomkins, 0. M. (1968). Xcience, 162, 1013.

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Granner, D. K., Hayashi, S., Thompson, E. B. & Tomkins, G. M. (1968). J. Mol. Biol. 25, 291. Hayashi, S., Granner, D. K. & Tomkins, G. M. (1967). J. Biol. Chem. 242, 3998. Koshland, D. E., Jr., NBmethy, G. & Filmer, D. (1966). Biochemistry, 5, 365 Lowry, 0. H., Rosebrough, N. J., Farr, A. C. & Randall, R. J. (1951). J. BioZ. Chem. 193, 256. Makman. M. H., Dvorkin, B. & White, A. (1968). J. BioZ. Chem. 243, 1485. Martin, D. W., Tomkins, G. M. & Bressler, M. (1969). P-roe. Nat. Acad. Sci., Wash. 63, 842. Melnykovych, G. & Bishop, C. F. (1969). Biochim. biophya. Acta, 177,579. Merchant, D. J., Kahn, R. H. &Murphy, W. H. (1964). Handbook ojCeZZ andOrganCuZture. Minneapolis, Minn: Burgess Publishing Co. Monod, J., Wyman, J. & Changeux, J. P. (1965). J. Mol. BioZ. 12, 88. Moscona, A. A. & Piddington, R. (1967). Science, 158, 496. Peterkofsky, B. & Tomkins, G. M. (1967). J. Mol. BioZ. 30, 49. Rubin, M. & Changeux, J. P. (1966). J. Mol. BioZ. 21, 265. Segal, H. L. & Kim, Y. S. (1966). J. CeZZ Camp. PhysioZ. 66, (suppl 1) 11. Stoppani, A. 0. M., Brignone, C. M. C. & Brignone, J. A. (1968). Arch. Biochem. Biophys.

127, 463. Tomkins, G. M., Thompson, E. B., Hayashi, S., Gelehrter, T. D., Granner, D. K. & Peterkofsky, B. (1966). Cold Spr. Harb. Symp. Qualzt. BtiZ. 31, 349. BioZ. 26, 331. Tomkins, G. M. & Yeilding, K. L. (1961). Cold Spr. Harb. Symp. Quant. Williams, B. & Paigen, K. (1968). J. Bact. 96, 1774. Yielding, K. L. & Tomkins, G. M. (1959). Proc. Nat. Acad. Sci., Wash. 45, 1730.