Inhibitory Effects of Cortisol and Antibiotics on Substrate Entry and Ribonucleic Acid Synthesis in Rat Thvmocvtes in Vitro*

Abstract Incubation of rat thymocytes with either puromycin, cycloheximide, or actinomycin D resulted in a progressive inhibition of the accumulation of radioactivity derived from amino acids and uridine in the trichloracetic acid-soluble fraction. No further inhibitory effects on incorporation into either the trichloracetic acid-soluble or trichloracetic acid-insoluble fraction were produced by 1 x 10-6 m cortisol when incubations were carried out for 3 hours in the presence of the antibiotic. However, with antibiotic added late in the incubation period, inhibitory effects of steroid on incorporation into both trichloracetic acid fractions, similar to those obtained without antibiotic, were still evident. Cortisol and puromycin inhibited incorporation of orotic acid into the trichloracetic acid-insoluble fraction, with no corresponding effects on the trichloracetic acid-soluble fraction. Moreover, in the presence of puromycin, an additional inhibitory effect of cortisol on orotic acid incorporation into RNA was obtained. An inhibitory effect of cortisol on incorporation of uridine into the trichloracetic acid-insoluble fraction occurred in the absence of significant effects on the trichloracetic acid-soluble fraction when cells were incubated in the presence of puromycin for 6 hours. Fractionation by sucrose gradient centrifugation of the total RNA obtained from thymocytes exposed to cortisol and labeled with uridine revealed no differential effects of the steroid on a specific species of RNA. Incubation of thymocytes with cortisol resulted in a progressive decrease of RNA polymerase activity as measured directly with either a nuclear or aggregate enzyme preparation. This inhibitory effect on polymerase activity was also produced by incubation of thymocytes with 6α, 9α-difluoro-11α,16α,17α,21-tetrahydroxy-1,4-pregnadiene-3,20-dione-16,17-acetonide (fluocinolone acetonide) (1 x 10-7 m) but not by incubation with progesterone (1 x 10-6 m). Incubation of the cells with puromycin also resulted in decreased polymerase activity; however, a further inhibitory effect of cortisol was evident in the presence of the antibiotic. It is concluded that continuing protein synthesis throughout the incubation period is required for amino acid transport, for uridine transport or conversion to UMP (or both), and for the manifestation of the steroid effects on these processes. In contrast, continuing protein synthesis is not required for the inhibitory effect of cortisol on RNA synthesis.

From the Departments of Biochemistry and Pharmacology, Albert Einstein College of Uedicine, Yeshiva University, New York, New York 10461 SUMMARY Incubation of rat thymocytes with either puromycin, cycloheximide, or actinomycin D resulted in a progressive inhibition of the accumulation of radioactivity derived from amino acids and uridine in the trichloracetic acid-soluble fraction. No further inhibitory effects on incorporation into either the trichloracetic acid-soluble or trichloracetic acid-insoluble fraction were produced by 1 X lOWfi M cortisol when incubations were carried out for 3 hours in the presence of the antibiotic.
However, with antibiotic added late in the incubation period, inhibitory effects of steroid on incorporation into both trichloracetic acid fractions, similar to those obtained without antibiotic, were still evident. Cortisol and puromycin inhibited incorporation of erotic acid into the trichloracetic acid-insoluble fraction, with no corresponding effects on the trichloracetic acid-soluble fraction. Moreover, in the presence of puromycin, an additional inhibitory effect of cortisol on erotic acid incorporation into RNA was obtained.
An inhibitory effect of cortisol on incorporation of uridine into the trichloracetic acid-insoluble fraction occurred in the absence of significant effects on the trichloracetic acidsoluble fraction when cells were incubated in the presence of puromycin for 6 hours.
Fractionation by sucrose gradient centrifugation of the total RNA obtained from thymocytes exposed to cortisol and labeled with uridine revealed no differential effects of the steroid on a specific species of RNA. Incubation of thymocytes with cortisol resulted in a progressive decrease of RNA polymerase activity as measured directly with either a nuclear or aggregate enzyme preparation.
This inhibitory effect on polymerase activity was also produced by incubation of thymocytes with 6a,9a-difluorollor,16a,17a,21-tetrahydroxy-l,4-pregnadiene-3,2O-dione-16,17-acetonide (fluocinolone acetonide) (1 X 10m7 M) but not by incubation with progesterone (1 X 1OV M). Incuba-* This investigation was aided by Grant P-681 from the American Cancer Society, Grants CA-07470 and AM-09213 from the National Institutes of Health, United States Public Health Service, and Grant GB-6616X from the National Science Foundation. Some of the data presented here were reported previously in a preliminary communication (1). 1 Career Development Awardee 5-K3-AM-25,889-04, National Institutes of Health, United States Public Health Service. tion of the cells with puromycin also resulted in decreased polymerase activity; however, a further inhibitory effect of cortisol was evident in the presence of the antibiotic.
It is concluded that continuing protein synthesis throughout the incubation period is required for amino acid transport, for uridine transport or conversion to UMP (or both), and for the manifestation of the steroid effects on these processes. In contrast, continuing protein synthesis is not required for the inhibitory effect of cortisol on RNA synthesis.
In previous publications from this laboratory it was shown that addition of physiological concentrations (lo-* M to lo-+ M) of a thymolytic steroid, e.g., cortisol, to lymphoid cells in vitro inhibited significantly the transport and further utilization of amino acids and of precursors of RNA and DNA4 (2)(3)(4).
These inhibitory effects appeared to be dependent both upon an energy source (3,4) and upon the synthesis of RNA or protein (or both) (1,3). In additional studies, the activity of thymic DNA-dependent RNA polymerase has also been shown to decrease following injection of cortisol (3,(5)(6)(7)(8).
The studies in the present paper permit a more definitive distinction between two proposed sites of cortisol action (3), inhibition of transport processes, and decreased rate of RNA synthesis, as well as partial clarification of the role of protein synthesis in these processes. Eagle's minimum essent,ial medium for spinner culture (without serum) (8) was purchased from General Biochemicals as a dry powder; NaH2P04 was added to reconstitute the complete medium. Phenol was purchased from Mallinckrodt and was redistilled and stored frozen prior to use. Sucrose was purchased from Merck Sharp and Dohme, and puromycin and cycloheximide were from Nutritional Biochemicals.
Actinomycin D was kindly provided by Merck Sharp and Dohme.
Other reagents were analytical grade and obtained from commercial sources.
Male rats of the Sprague-Dawley strain, 7 to 10 weeks of age, were purchased from Carworth Farms and maintained on Rockland rat chow and water ad lib&m for at least 1 week prior to use.
When indicated, rats were injected intraperitoneally either with 5 mg of cortisol (suspended in 0.85c/, NaCl solution, 15 mg per ml) per 100 g of body weight or with an equal volume of NaCl solution at the indicated time intervals prior to death. For each experiment rats were killed by decapitation and the thymi from five animals were placed in ice-chilled Eagle's spinner culture medium (9). The cell suspensions (2 x lo7 cells per ml) were prepared as described previously (4). This procedure was carried out with use of Eagle's spinner culture medium (without serum) or with the balanced spinner salt medium containing glucose, depending upon the final incubation to be used, as indicated in the text.
For measurement of trichloracetic acid-insoluble radioactivity, the cell pellets were resuspended and precipitated in cold 5% trichloracetic acid, and the precipitates were washed and collected on Millipore filters (0.45 p pore size). For separation and recovery of both intracellular trichloracetic acid-soluble and trichloracetic acid-insoluble fractions, cell pellets were washed in cold incubation medium, resuspended in trichloracetic acid, and centrifuged; aliquots of the supernatant fluids were removed for measurement of trichloracetic acid-soluble radioactivity and the precipitates resuspended and collected on Millipore filters for measurement of trichloracetic acid-insoluble radioactivity. Radioactivity was determined by liquid scintillation spectrometry as described previously (4).
For preparation of thymic nuclei for RNA polymerase assay, 20.ml aliquots of the cell suspensions were centrifuged at 120 x g for 4 min and washed once with 10 ml of ice-chilled spinner medium. Washed cells were resuspended in 2 ml of medium cont,aining 0.25 M sucrose, 0.075 M KCl, 0.01 M MgC&, and 0.035 M Tris-Cl (pH 7.8). Nuclei were isolated by sonic oscillation of the cells in a Branson Sonifier for 10 set (20 kcycles per set, 3 amps d.c.) and then by centrifugation at 700 X g for 5 min. The nuclear sediment was washed once with the above medium and resuspended in the same medium for RNA polymerase assay.
The aggregate polymerase preparation was obtained by lysis of the cells at a concentration of 1.6 X lo8 per ml in 0.05 M Tris-Cl buffer, pH 7.8. Following lysis, the MgCIZ concentration of the mixture was adjusted to 0.005 M and the nuclear gel was dispersed by homogenizing in a Waring Blendor with a microcontainer at the low speed setting for 30 sec. The cell lysate was centrifuged at 15,000 X g for 10 min. The supernatant fluid was discarded and the sediment was washedoncewitha 0.05 M Tris-Cl (pH 7.8)-0.005 M MgClz medium.
The washed pellet was resuspended in a known volume of the same medium (pellet from 5 x lo8 cells per ml of medium) and dispersed in a Waring Blendor at the high speed setting for 30 sec. This preparation was used for polymerase assay.
The 0.5-ml (final volume) incubation medium for RNA polymerase assay included 0.25 ml of either the nuclear suspension or the aggregate enzyme preparation, each containing approximately 0.5 mg of DNA.
Incubation was carried out at 37" for 10 min, during which period the reaction rate was linear.
Other details were as described previously (7,8). Concentrations of UTP and of other nucleoside triphosphates in the assay system were at saturation (7,8).
For sucrose gradient studies, separate aliquots of control and cortisol-treated cell suspensions were labeled either with uridine-6-3H or with uridine-2-Y" /. In some experiments, the cells were "chased" by the addition of unlabeled uridine at a suitable time after addition of labeled uridine.
The 3H-labeled cells exposed to cortisol were combined with 14C-labeled control cells, or vice versa, and the pooled cell suspensions were then extracted with phenol by the method of Warner et al. (IO), with the following modifications.
Extraction of lysed cells with phenol was con ducted at 50"; following phenol extraction, RNA was precipitated by adding 3.5 volumes of cold absolute ethanol (-20").
The mixture was kept at -20" overnight for complete precipitation of RNB.
After centrifugation, the RNA was redissolved at room temperature in 0.01 M Tris-Cl buffer (pH 7.5) containing 0.01 M EDTA, and 0.2% sodium dodecyl sulfate.
An aliquot of 0.1 to 0.6 ml of RNA solution was layered on a continuous gradient of 15 to 30% sucrose containing 0.1 M NaCl, 0.01 M Tris-Cl (pH 7.5), 0.001 M EDTA, and 0.5% sodium dodecyl sulfate.
Centrifugation was performed at 25" in a Spinco centrifuge, model L2-6513, with either a three-or six-place rotor as indicated in the results.
After centrifugation, successive gradient fractions were collected with continuous monitoring of optical density at 260 rnp. To each fraction was added trichloracetic acid (final concentration 5%) plus 3.5 ~1 of calf serum, and the precipitate was collected on a Millipore filter (0.9 ,.J pore size) for determination of trichloracetic acid-insoluble radioactivity.

RESULTS
In order to examine the possible influence of antibiotics on the response of thymocytes to cortisol, initial studies were conducted with antibiotics alone. The data in Table I show the influence of length of incubation and concentration of antibiotics on incorporation of precursors into protein and nucleic acids. Actinomycin D exerted an early inhibitory influence on RNA synthesis and a later action on incorporation of precursors into protein and DNA, in agreement with the generally accepted causal relationship of protein synthesis to continuing RNA format.ion.
In contrast, 6dimethylaminopurine, a substance without effects on protein metabolism but mimicking the action of puromycin on carbohydrate metabolism (II), had no influence on incorporation of any of the precursors studied.
Cycloheximide and puromycin, but not actinomycin D, exert early inhibitory effects on the incorporation of deoxyuridine-14C into an acid-insoluble form (Table I). Under these conditions of incubation, deoxyuridine is incorporated exclusively into DNA (2,4). The early inhibition of deoxyuridine incorporation into DNA produced by cycloheximide was not associated with any change in the acid-soluble fraction; incorporation of thymidine was influenced similarly (data not presented).
The effects of cycloheximide and puromycin on deoxyuridine incorporation into DNA at the earlier time period were appreciably greater than those on uridine incorporation into RNA. The data suggest a close relationship between continuing protein synthesis and DNA formation.
Sensitivity of DNA synthesis to puromycin (12, 13) and to cycloheximide (14) in other mammalian cells in vitro has been reported.
Inhibitory effects of cortisol on the accumulation of radioactive uridine, glycine, and AIB3 in the trichloracetic acid-soluble and -insoluble fractions of thymocytes incubated with and without inhibitors of protein and RNA synthesis are summarized in Tables II and III. Incubation of thymocytes with either puromycin, cycloheximide, or actinomycin D throughout the entire period of incubation resulted in decreased accumulation of uridine-3H in both the trichloracetic acid-soluble and -insoluble fractions of the cells (Table II).
The magnitude of the inhibition by these antibiotics increased with time of exposure of the cells. The data for the acid-soluble fraction suggest that each of the antibiotics inhibits transport or phosphorylation of nucleoside (or both) .* However, the antibiotics did not inhibit appreciably uridine transport or phosphorylation (or both) when added at a later time (2.5 hours) even though the antibiotics depressed incorporation into the trichloracetic acid-insoluble fraction (Table II).
When an antibiotic was present from the beginning of the incubation, no additional inhibitory influence of cortisol on uridine-3H accumulation was apparent.
In contrast, when protein or nucleic acid synthesis was depressed by late addition of antibiotic shortly before pulse labeling, the inhibitory influence of steroid on the accumulation of precursors into both trichloracetic acid fractions was still evident. In the presence of concentrations of actinomycin D which produced only partial inhibition of RNA synthesis (0.1 or 0.4 fig per ml), effects of cortisol on the incorporation of uridineJH into the acid-soluble and -insoluble fractions were still evident (Table II).
The data indicate that steroid action is not selective for one or more species of RNA whose synthesis is either more or less sensitive to actinomycin D (see below). The presence of puromycin at the beginning of the incubation depressed markedly the incorporation of glycine-14C into the acid-insoluble fraction and to a lesser degree into the acid-soluble fraction (Table III).
Addition of cortisol had no further inhibitory influence on the incorporation of glycine into either fraction.
We have previously shown that essentially all of the glycine incorporated into an acid-insoluble form under these conditions is present in the protein fraction of the cell (4). Essentially similar data were obtained when actinomycin D was present from the beginning of the incubation.
In contrast, addition of either puromycin or actinomycin D 2.5 hours after initiation of the incubation, although significantly inhibiting glycine incorporation, did not block the steroid effects. The previously observed inhibitory effect of cortisol on the entry of AIB (3,4) was not evident in the presence of either puromycin or actinomycin D (Table III). The steroid and puromycin appeared to exert a similar influence on a transport process. The failure of actinomycin D to depress AIB entry into the cells, in contrast to its effect on uridine and glycine accumulation, is not understood at this time. This result was unexpected, particularly in view of the absence of cortisol effects on AIB accumulation in the presence of actinomycin D. Except for this last point, the influence of the antibiotics on the accumulation of radioactivity in the acid-soluble fraction resembled that produced by cortisol.
Cortisol inhibited the incorporation of erotic acid-l% into the acid-insoluble fraction but had no effect on the accumulation of radioactivity in the acid-soluble fraction (Table IV). These results are in contrast to the above-described actions of the steroid on the incorporation of uridine into the acid-soluble and -insoluble fractions.
Moreover, inhibition of uridine incorporation into the acid-insoluble fraction was significantly greater than that for erotic acid. These results support the conclusion that cortisol exerts an action on RNA synthesis by thymocytes in vitro independent of possible steroid effects on transport or phosphorylation of nucleoside (or both). The data in Table IV also indicate that the effect of cortisol on uridine incorporation into the acid-soluble fraction must be localized at a step involving transport of uridine or formation of UMP from uridine, since the reactions leading to the conversion of UMP to UTP are identical with either uridine or erotic acid as precursor.
Experiments in which thymocytes were incubated with cortisol for longer periods of time are summarized in Table V. After 6 hours of incubation in the presence of puromycin, no steroid effects were evident on the incorporation of glycine into either the trichloracetic acid-soluble or -insoluble fraction. This finding is in agreement with data presented above for a-hour incubation periods (Table III). However, after 6-hour incubation in the presence of puromycin, inhibitory effects of steroid were evident on the incorporation of uridine-3H into the acidinsoluble but not into the acid-soluble fraction (Table V). With erotic acid-14C as precursor, the inhibitory effect of steroid on accumulation of radioactivity into the acid-insoluble fraction was greater when the incubation was extended from 3 hours (Table IV) to 6 hours (Table V). Even after 6-hour incubation with steroid, the accumulation of erotic acid in the acid-soluble fraction of the cells was only slightly decreased.
Furthermore, in the presence of puromycin, a significant action of cortisol on accumulation of erotic acid in the acid-insoluble fraction was still evident.
Also to be noted are the similar magnitudes of the inhibitory effects of cortisol on the incorporation of either uridine or erotic acid into the acid-insoluble fraction in the presence of puromycin.
This supports our earlier suggestion (1) of a direct effect of cortisol on the RNA-polymerase system (also see below).
Thymic nuclei obtained by sonic oscillation, as well as the aggregate enzyme fraction, both described under "Experimental Procedure," were found to be satisfactory for measurement of RNA polymerase activity.
The polymerase activities of these preparations were similar to one another and to those of intact nuclei and of the nuclear aggregate fractions prepared from whole thymus (data not presented).
The sonic disruption technique was developed because of difficulties encountered in homogenizing cell suspensions by the procedure previously described for whole  (1) -28 (1) -29 f 4 (2)b -8 + 4 (2) a Pulse labeling carried out after 1 hour of incubation of cells with and without cortisol (1 X 10m6 M) and with and without puromycin.
thymus (5). In studies involving incubation of thymocytes with labeled precursors for 30 min, it was found that the nuclear fraction prepared by sonic disruption contained approximately 50% of the labeled protein, 85% of the labeled RNA, and 98% of labeled DNA.
In the polymerase assay system, intact cells did not incorporate nucleoside triphosphates into RNA (data not presented).
The measurements of polymerase activity described in this paper were conducted with a medium containing Mg2+ and of low ionic strengt'h (4 X 10e3 M (NH4)$04).
The influence of Mg2+, Mn*+, and (NH&SO4 on thymic RNA polymerase activity of thymocytes exposed to cortisol have been described (8). Those studies showed that the lower RNA polymerase activity of thymocytes exposed to cortisol in viva was not influenced by changes in the divalent cation or the ionic strength of the assay medium.
The specificity of the response of the RNA polymerase activity of thymocytes to a thymolytic steroid added in vitro is illustrated by the data in Table VI. Incubation of thymocytes with either cortisol or another potent thymolytic steroid, fluocinolone acetonide, lowered the RNA polymerase activity.
In contrast, a nonthymolytic steroid, progesterone, was without significant effect on this enzymic activky under the same experimental conditions. The influence of time of exposure of thymocytes to cortisol and to puromycin in vitro on nuclear RNA polymerase activity is summarized in Fig. 1. After 1.5 hours of incubation of cells with cortisol, no significant effect on polymerase activity was apparent.
After 3 hours of incubation, however, the inhibitory action of the steroid was evident and was approximately twice as great after 6 hours. Incubation of cells with puromycin also resulted in a decreased polymerase activity.
Moreover, even in the presence of puromycin, an inhibitory effect of cortisol on RNA polymerase activity was still evident.
Thus, continuing protein synthesis was not required for this action of cortisol in vitro.
This observation is in agreement with the results presented above (Tables IV and V). Since the effect of steroid on polymerase activity was somewhat reduced in magnitude when antibiotic was present, it is possible that cortisol may have more than one action on the polymerase system (see "Discussion").
An inhibitory effect of cortisol was also evident in aggregate RNA polymerase preparations obtained from thymocytes incubated with cortisol for either 3 or 6 hours (Table VII). The degree of inhibition by steroid noted with the aggregate preparations was quite similar to that obtained kth the whole nuclei.
Studies have also been carried out to assess whether the inhibitory effects of cortisol on net incorporation of precursors into   were then washed, resuspended, and incubated in fresh medium. Cells labeled with phenylalanineJ4C or with uridine-3H were incubated, respectively, in the presence of puromycin or actinomycin D in order to prevent reutilization of labeled intracellular precursors.
Although a significant rate of protein degradation was evident (10% per hour), cortisol did not enhance the rate of degradation of either protein or RNA during incubation of labeled cells for 3 hours with antibiotic (data not presented).
These findings are in agreement with our previous studies (4) in which antibiotics were not utilized. The possibility that cortisol might influence the synthesis of a specific species of RNA was examined by exposing thymocytes to cortisol either in vitro or in viva and utilizing double labeling techniques with RNA precursors in vitro. These studies included gradient centrifugation of total cellular RNA obtained with and without a "chase" with nonradioactive precursor. Representative experiments are summarized in Fig. 2. In studies involving exposure of thymocytes to cortisol in vitro for either 30 min (A) or 3 hours (data not shown) prior to labeling of RNA, the distribution patterns were not altered. Also, the distribution patterns of labeled RNA from thymocytes obtained from control animals and from animals injected with cortisol 3 hours prior to death were identical (B and C). Alterations in the RNA pattern attributable to cortisol were not seen either without (B) or with (C) a 40-min chase. It is possible that small changes in messenger RNA might have been obscured by the larger amount of 45 S RNA present (also see "Discussion"). DISCUSSION We have found that inhibitors of protein and RNA synthesis added to rat thymocytes incubated in vitro produce a progressive inhibition of amino acid transport and nucleoside accumulation. Thus, processes involving substrate accumulation by the cells are markedly dependent upon continuing protein synthesis. Also, such inhibitory effects on substrate entry or accumulation must be considered in evaluation of the actual net inhibition of macromolecular synthesis caused by antibiotics as well as by other agents, including steroids. We have found that puromycin does not influence the rate of entry of cortisol into rat thymocytes, even though this antibiotic does inhibit the entry of other small molecules as described in this paper.
The data obtained do not, however, permit distinction between a direct effect of cortisol on a rate-limiting step in transport and an effect on the synthesis of macromolecules which influences the rate of transport. Inhibitory effects of adrenal steroids on amino acid transport in rat diaphragm (15) and on uridine accumulation in bone cells (16) have been found to be dependent upon continuing protein synthesis.
In other studies,5 we have found that cortisol has an inhibitory influence on the rate of uptake not only of glucose (17, 18), but also of 3-0-methylglucose, an analogue of glucose which is actively transported into the cell, but which is not phosphorylated.
In addition, these effects of cortisol on hexose uptake were not evident in the presence of cycloheximide, puromycin, or actinomycin D.5 In the presence of puromycin the inhibitory effect of cortisol added in vitro on RNA polymerase activity was less but still evident. A similar result was obtained when aggregate enzyme was prepared from thymocytes incubated with cortisol and puromycin or cycloheximide. Calculations from the data in Fig. 1 indicate that the fraction of RNA polymerase activity which is sensitive to puromycin is also highly sensitive to cortisol. Thus, it may be calculated that, because of the presence of cortisol, the puromycin-sensitive activity is decreased from 7.6 to 5.2 dpm per pg of DNA (32%) after a-hour incubation and from 7.3 to 4.2 dpm per pg of DNA (42%) after 6 hours. These results suggest that cortisol may exert more than one effect on the RNA polymerase system. These effects may perhaps be In this regard, it is of interest that the estrogen-stimulated portion of RNA polymerase activity in rat uterus has been reported to be extremely sensitive to treatment with either puromycin or cycloheximide, both in viva and in. vitro (19,20). Inhibition of protein synthesis not only prevented initiation of the stimulatory effect of estrogen on RNA polymerase activity, but also reversed the hormone effect after it had been initiated. Therefore, synthesis of a rapidly turning over protein has been suggested to be involved in the action of estrogen on the uterine RNA polymerase activity.