Proc. Nat. Acad. Sci. USAVol. 72, No. 2, pp. 523-527, February 1975

Testosterone Stimulation of a Rapidly Labeled, Low-Molecular-WeightRNA Fraction in Human Hepatic Erythroid Cells in Culture

(fetal liver cells/erythropoietin/RNA and DNA synthesis/globin mRNA)

L. FERNANDO CONGOTE AND SAMUEL SOLOMON

Departments of Biochemistry and Experimental Medicine, McGill University, and the University Clinic, Royal Victoria Hospital,Montreal, Canada

Communicated by Helen M. Ranney, November 15, 1974

ABSTRACT The addition of erythropoietin to cell cul-tures of erythroid cells of human fetal liver resulted in anincreased incorporation of thymidine, adenine, anduridine into trichloroacetic acid-insoluble cell fractionsand in an increased uptake of adenine and uridine into thecell. Although the effects of testosterone and erythropoie-tin on heme synthesis in these cells are known to be verysimilar, there was no effect of testosterone on the totalincorporation of radioactive precursors into DNA or RNA.The RNA synthesized after short pulses of radioactiveuridine, when analyzed on sucrose gradients containing1% sodium dodecyl sulfate, consisted of a homogeneouspeak sedimenting at 10 i 2 S, which is quite differentfrom the heterogeneous, high-molecular-weight RNAsynthesized under identical conditions in primary cul-tures of human fetal lung, kidney, or liver parenchymalcells. Addition of testosterone to liver erythroid cells incultures for 5 hr followed by a 1-hr uridine pulse resultedin a 3-fold increase of RNA species with an average sedi-mentation coefficient of 14 A4 3 S. The similarity with thesedimentation coefficient of the globin mRNA describedin other systems and the high degree of specialization ofthe erythroid cells suggest that this RNA may be a stableintermediate involved in the synthesis of hemoglobin.

One of the earliest actions of erythropoietin on its target cellsis the stimulation of RNA synthesis (1-6). We have found thatprimary cultures of erythroid cells from human fetal liverexhibited an enhanced synthesis of heme after addition oferythropoietin or testosterone and that the heme fractionassociated with hemoglobin showed the highest stimulation (7).At the present time it cannot be said with certainty if

testosterone itself is active in hematopoietic cells or whether itexerts its effects via a metabolite(s) with the 5,8-configuration.Several observations support a direct role of testosterone.Erythropoietic mouse spleen and rat bone marrow cells con-tain testosterone receptors, and there is no metabolism of thehormone in mouse spleen (8, 9). The effects of testosterone oncolony formation in hematopoietic cells of mice are seen withina few minutes after hormone administration (10) when exten-sive metabolism of the hormone is not to be expected. In thissystem testosterone and 5(3-metabolites are equally active incolony formation, whereas in human fetal erythroid cellstestosterone seems to be the most active of a large number ofsteroids tested to date (ref. 7, and our own unpublished re-sults). There are two pieces of evidence that suggest a role ofmetabolites with the 5(3-configuration. One is the high degreeof specificity of the action of 5,B-reduced steroids in humanbone marrow cells, where both heme and globin synthesis arestimulated (11). Another supporting piece of evidence is therapid metabolism of testosterone to androstenedione and

523

etiocholanolone in human fetal liver cells (7). Of these twometabolites, only etiocholanolone is active on the stimulationof hemoglobin-associated heme synthesis (Congote and Solo-mon, unpublished data). It, therefore, appears that bothtestosterone and etiocholanolone may be physiologicallyactive in erythropoiesis in the human midterm fetal liver.The mechanism of action of testosterone on erythropoietictissues is unknown. We have found that in human fetal livercells simultaneous administration of testosterone and actino-mycin D (or a-amanitin) blocked this steroid-mediatedenhancement of heme synthesis (7). The aim of this com-munication is to define the changes in nucleic acid synthesisthat are occurring in these cells after testosterone stimulationand to describe the general changes in the total RNA metabo-lism of the cells and some unusual characteristics of the rapidlylabeled RNA synthesized.

MATERIALS AND METHODS

All tissue culture supplies were purchased from Flow Labora-tories, Rockville, Md. Some batches of fetal calf serum werefrom the Grand Island Biological Co. [MIethyl-3H]Thymidine(18-22 Ci/mmol), [8-3H ]adenine (25.8 Ci/mmol), [5-3H ]-uridine (5 Ci/mmol), and uniformly labeled ['4C]uridine(450-540 mCi/mmol) were purchased from Amersham/Searle. Human erythropoietin, specific activity 36.3 units/mgof protein, was provided by the N.I.H. It was collected andconcentrated by the Department of Physiology, University ofthe Northeast, Corrientes, Argentina, and further processedand assayed by the Hematology Research Laboratories,Children's Hospital of Los Angeles under research grant HE-10880. The hormone solutions were prepared in L-15 medium,containing 50 U of penicillin and 50 U of streptomycin per mland supplemented with 10% fetal calf serum. The final con-centrations were 50 nM testosterone and 0.5 U/ml of erythro-poietin. The methods used in preparing these solutions havebeen described in detail (7).The methods for the preparation of primary cultures of

kidney, lung, and liver cells have been described (7, 12). Allexperiments with primary liver erythroid cells were done 1 dayafter plating. All incubations were carried out in a humidifiedincubator at 370 with 5% C02-95% air. The details for eachtype of experiment will be described.The total incorporation of radioactive uridine and/or

adenine into RNA was measured as described (12). The radio-active precursors for RNA were dissolved in the L-15 mediumat a concentration of 0.75,Ci/ml of [3H ]adenine or 0.1 ,uCi/ml

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FIG. 1. Effect of testosterone and erythropoietin on the totalDNA and RNA synthesis of liver erythroid cells. Primary cul-tures of human fetal liver cells were incubated 1 day after platingwith medium alone (@), with 50 nM testosterone (0), or with0.5 U/ml of erythropoietin (A), for the time periods indicated.After a 30-min pulse with [3H]thymidine and [14C]uridine, thecells were washed and the incorporation into trichloroacetic acid-insoluble material was followed. A, thymidine incorporation;B, uridine incorporation. The bars indicate the range of duplicateexperiments. The increase in thymidine incorporation aftererythropoietin treatment during a short-term (6 hr) and a long-term (24 hr) incubation period was statistically significant (P <0.02 and P < 0.002, respectively; n = 8); the increase in uridineincorporation after erythropoietin treatment was also significant(P < 0.01 for the 6-hr incubation, P < 0.001 for the 24-hr incuba-tion). The differences between controls and testosterone-treatedcultures were not significant.

of [14C]uridine. After a 30-min pulse at 370, the cells were

washed with Hank's balanced salt solution and the nucleicacids were precipitated with trichloroacetic acid. Aliquots fordetermination of radioactivity were taken from the cold-acid-soluble fraction (soluble precursors) and from the cold-acid-insoluble fraction (nucleic acids) (12). Thymidine was in-corporated into acid-insoluble material as indicated for RNAsynthesis, except that after the acid-insoluble material was

washed in 75mM trichloroacetic acid and ethanol (12), the pel-lets were dissolved in Nuclear Chicago Solubilizer and countedin 11 ml of Instagel (Packard). Labeled thymidine (2 MCi/ml)was added to the incubation medium. For the measurement ofthe degree of phosphorylation of uridine, the uridine anduridine nucleotides were recovered from the cold-acid-solublefraction with acid-washed Norit (13) and analyzed in a AG1column (X 10, 200-400 mesh), as indicated by Lindsay et al.(14).RNA newly synthesized by different cell cultures was

analyzed by sucrose density gradient centrifugation afterextraction with phenol and sodium dodecyl sulfate. A total of

2 X 106 cells from lung and kidney were plated in tissue culture

dishes of 9 cm inner diameter. An equal number of liver

TABLE 1. Effects of testosterone anl erythropoietin on theincorporation of thymidine, adenine, and uridine

Ratios (hormone-treated cells/controls)X 100 i SEM

Testosterone Erythropoietintreatment treatment

Cold-acid-soluble radioactivityThymidine 106 i 11 (10) N.S. 99 4 11 (8) N.S.Adenine 100 i 3 (8) N.S. 113 ± 4 (12) P < 0.01Uridine 101 i 4 (10) N.S. 126 i 3 (12) P < 0.001

Cold-acid-insoluble radioactivityThymidine 92 :1 5 (10) N.S. 117 i 3 (8) P < 0.02Adenine 100 i 6 (8) N.S. 111 i 3 (12) P < 0.01Uridine 101 i 6 (10) N.S. 126 i= 5 (12) P < 0.001

One day after plating, human liver cells were incubated withtestosterone (50 nM) or erythropoietin (0.5 U/ml) for 6 hr,followed by a 30-min pulse with labeled thymidine, adenine, oruridine. The amount of radioactivity present in the cold-tri-chloroacetic acid-soluble and -insoluble fractions was measuredas indicated in Materials and Methods. The number of experimentsis in parentheses. P values were calculated with the t-test. N.S. =not significant.

epithelial cells was also plated. The same sedimentation pro-files were observed over a wide range of cell densities. Thecultures in the L-15 medium were pulse-labeled for 1 hr at 370with 13.3 jiCi/ml of [3H]uridine or 1.33 MCi/ml of ['4C]-uridine. Then the cells were washed in Hank's balanced saltsolution and suspended and lysed in 3 ml of extraction buffer.This buffer is a slight modification of that used by Schutz et al.(15), and contained 0.1% sodium dodecyl sulfate in 0.14 MNaCl and 0.05 M sodium acetate, pH 5.0. The RNA wasextracted twice with the same amount of water-saturatedphenol at 250 for 10 min, and the phenol phase and the inter-phase were also extracted with the same volume of freshextraction buffer at 650. The RNA in the pooled water frac-tions was precipitated with 2.5 volumes of ethanol at -20'Covernight. Extracted RNA was analyzed by sucrose densitygradient centrifugation as described (12), with the exceptionthat denaturing conditions (1% sodium dodecyl sulfate in allsolutions) were used and centrifugation was at 200 instead of404.

RESULTS

The human fetal liver at midterm contains mainly erythroidcells. The ratio of these cells to nonhematopoietic elements isabout 100:1 (16). These cells predominate in the early cellcultures; this is manifested by a high rate of synthesis of hemeassociated with hemoglobin, a property that is rapidly lostafter 2-3 days in culture due to the detachment of the eryth-roid cells from the surface of the culture dish. In the studiesreported here, freshly prepared cultures will contain mainlyerythroid cells, whereas 3 to 4-day-old cultures contain mainlyparenchymal type cells. Freshly prepared primary cultures ofhuman fetal liver cells were incubated for different time peri-ods with testosterone and erythropoietin, followed by a 30-min pulse with [8H]thymidine and [14C]uridine. The incorpo-ration of both precursors into acid-insoluble materials was

followed as indicated in Materials and Methods. The results areshown in Fig. 1. Of the two hormones tested, only erythro-

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FIG. 2. Sucrose density gradient centrifugation of rapidlylabeled RNA prepared from kidney, lung, and liver erythroidcells. Primary cultures of liver, lung, and kidney cells were incu-bated 1 day after they were plated with [3H]uridine (13.3 pCi/ml)for 1 hr, and the RNA was extracted and analyzed on 15-30%sucrose gradients containing 1% sodium dodecyl sulfate as indi-cated in Materials and Methods. The gradients were run in a

Beckman L2-75B ultracentrifuge with a SW 56.2 rotor at 40,000rpm for 165 min at 200. After centrifugation the fractions were

collected and precipitated on cold 5% perchloric acid (12). Thearrows indicate the positions of the marker RNA. A, RNA fromliver erythroid cells; 0, RNA from kidney cells; 0, RNA fromlung cells.

poietin significantly increased the total DNA and RNAsynthesis in short- or long-time incubations. We were primar-ily interested in the relation between early effects of the hor-mones on nucleic acid synthesis and heme synthesis. For thisreason all the following experiments were carried out after a

short incubation time, namely 6 hr, with the hormones,followed by a 30-min pulse with radioactive precursors. Theresults of these studies are summarized in Table 1. Aftertestosterone treatment there was no significant change in theincorporation of thymidine, adenine, or uridine into nucleicacids. Erythropoietin stimulated the uptake of adenine anduridine into the cell and the incorporation of thymidine,adenine, and uridine into cold-acid-soluble materials.

In previous experiments we found that the stimulatoryeffect of testosterone on heme synthesis in fetal liver dependedon the gestational age of the fetuses used for the preparation ofthe cells. The maximum stimulation was observed in cellstaken from fetuses of 10-11 weeks of gestation (7). After theaddition of testosterone the incorporation of uridine into totalRNA remained unchanged in cells from fetuses of 10-15 weeksgestation. It is possible that testosterone may increase some

specific fractions of RNA without changing the total RNAsynthesis. The next step in this investigation, therefore, con-

sisted of the analysis of the newly synthesized RNA by sucrose

density gradient centrifugation by a double labeling tech-nique. [8H]Uridine was used as precursor for the control cellcultures and ['4C]uridine for the testosterone-treated cellsand vice versa, to prevent isotope effects and artificial results.Since the method required the use of large amounts of isotopes

TOP FRACTION NUMBER

FIG. 3. Simultaneous sucrose density gradient centrifugationof rapidly labeled RNA from erythroid cells and epithelial cellsof human fetal liver. Freshly isolated human fetal cells, containingmainly erythroid cells, were labeled with ['4C]uridine (1.33 uCi/ml) for 1 hr, and the RNA was extracted as indicated in Materialsand Methods. Cells of the same experiment were kept in culturefor 5 days and the medium was changed twice to remove all of theerythroid cells (7). The remaining cells, which are of epithelialmorphology, were incubated with [3H]uridine for 1 hr, and theRNA was extracted. The RNA fractions isolated from the epi-thelial cells and from the erythroid cells were pooled and cen-trifuged under the conditions described for Fig. 2. 0, RNA fromerythroid cell cultures; 0, RNA from liver epithelial cells.

of high specific activity (in particular, tritiated uridine), it wasimportant to test if the phosphorylation of uridine was a

limiting step in our cell culture system. The analysis (by ion-exchange chromatography) of phosphorylation of uridine incells incubated for 1 hr with this radioactive RNA precursorshowed that the amount of labeled uridine in the cells (3.4 i0.4%) was very low compared with the amount of labeleduridine nucleotides (69.9 0.3% monophosphate, 10.9 i 0.2%diphosphate, and 15.8 i 0.2% triphosphate; means i SEMof nine experiments). This result indicated a rapid and almostcomplete phosphorylation of the radioactive precursor. Thesame distribution of labeled uridine and phosphorylatedproducts was seen in cells incubated with ['4C]uridine with orwithout testosterone or erythropoietin present in the culturemedium. The 1-hr uridine pulse was the standard incubationtime used for the sucrose gradient analysis described below.

Fig. 2 shows the sedimentation characteristics of the RNAsynthesized in freshly prepared fetal liver cultures, which con-sist mainly of erythroid cells (7), and of RNA synthesized bycells of other tissues under identical conditions. The RNAsynthesized in kidney and lung cells after a 1-hr pulse withradioactive uridine is heterogeneous and consists mainly ofhigh-molecular-weight species. By contrast, RNA synthesizedby liver cultures had a low molecular weight and a sedimenta-tion coefficient of about 10 S. Fig. 3 shows the sedimentationproperties of RNA synthesized in liver erythroid cells com-

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FIG. 4. Sedimentation characteristics of labeled RNA fromliver erythroid cells after different incubation times with [3H] uri-dine. Human liver cells after 1 day in culture were labeled with[3H]uridine (26.7 ,Ci/ml) and the RNA extracted was analyzedon sucrose gradients, as indicated in Materials and Methods andfor Fig. 2.0, 5-min pulse; *, 10-min pulse; A, 30-min pulse.

pared with that synthesized in 5-day-old liver cell cultures,which contain mainly liver epithelial cells and are free of allerythroid cells. RNA sedimenting at 10 S was only observed inthe cultures consisting of erythroid cells, whereas liver epi-thelial cells synthesize a heterogeneous, high-molecular-weightRNA. A small shoulder at 10 S was observed in the RNAgradient of the latter cells. Fig. 4 shows the sedimentationproperties of RNA synthesized by erythroid cells after a 5-,10-, and 30-min pulse with [3H]uridine. The sedimentationprofiles look very similar to one another, with the main radio-activity peak appearing in the same position on the gradient.

One-day-old liver cell cultures were incubated for 5 hr withtestosterone followed by a 1-hr pulse with ['H]uridine. Acontrol culture was treated in the same way, with [14C]uridineas a precursor but with no testosterone in the L-15 medium.At the end of the incubation both cultures were pooled and theRNA was extracted and analyzed. The results are shown inFig. 5. The 'H/14C ratio in the RNA should remain constant inall of the gradient fractions if there were no specific stimula-tion or inhibition in any of the RNA species separated underthese conditions (12). This was not observed after testosteronetreatment. We detected an increased incorporation of [H I-uridine in a part of the gradient close to the main radioactivitypeak, sedimenting at 10-14 S (Fig. 5). The evidence that thisis not an artifact of the 'H/14C labeling technique was the factthat identical results were obtained when the 3H and 14C werereversed in the precursors used and the products formed wereanalyzed in an identical manner. A total of eight experimentsof this type were performed. In half of them we used ["4C]-uridine in the controls, and in the other half ['H]uridine wasused. The sedimentation coefficients were calculated, takingthe peaks of 18 S and 28 S as reference points, according to themethod of Martin and Ames (17). The average sedimentationcoefficient of the radioactivity peak -± standard error was10 + 2 S. The average sedimentation coefficient of the RNAfraction stimulated by testosterone treatment was 14 ± 3 S.The difference between these two coefffcients was not statisti-

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FIG. 5. Effect of testosterone on the sedimentation char-acteristics of rapidly labeled RNA isolated from erythroid cells ofhuman liver. Two tissue culture dishes of 9-cm inner diameter,containing liver erythroid cells plated under conditions indicatedin Materials and Methods, were incubated for 5 hr with L-15medium alone (controls) or with L-15 medium containing 50 nMtestosterone. A 1-hr pulse with ['4C]uridine (1.33 MCi/ml, con-trols) or [3H]uridine (13.3 JuCi/ml, testosterone-treated cells)was then done. At the end of the incubation the cells of bothdishes were pooled and the RNA was extracted and analyzed asindicated for Fig. 2. 0, controls; 0, testosterone-treated cells;A, ratio of testosterone/controls.

cally significant. The average increase of the testosterone-stimulated RNA fraction was of the order of 2.9 4- 0.4 (ratioof testosterone/controls i SEM). This difference to thecontrols was statistically significant (P < 0.002, t-test). Inonly two of the experiments could we observe an additionalpeak at 35 S that was previously not seen in the absence oftestosterone.

DISCUSSION

The incorporation of radioactive precursors into cold trichloro-acetic acid-insoluble materials can only detect very largechanges in the total nucleic acid synthesis of the cell, as isevident from the incorporation of labeled precursors into DNAand RNA of erythroid cells stimulated by the addition oferythropoietin (Fig. 1 and Table 1) and as has been reportedby Nicol et al. for liver cell cultures from fetal mice (3). In ourstudies testosterone did not stimulate total RNA or DNAsynthesis in erythroid cells (Fig. 1 and Table 1). The inductionof the rate-limiting enzyme of heme synthesis, 6-amino-levulinic acid synthetase, does not seem to require a totalincrease in RNA synthesis (18). Etiochlolanolone, an inducerof this enzyme, can stimulate the a-amanitin-sensitive RNApolymerase activity of chick liver, indicating a qualitativechange in the RNA synthesized (19). The inhibition byactinomycin D or a-amanitin of the heme synthesis stimu-lated by testosterone indicates that a requirement for RNAsynthesis exists at some step in the mechanism of action of thishormone (7). The marked stimulation by testosterone ofRNA sedimenting at about 14 S substantiates this dependencyon RNA synthesis (Fig. 5). The high degree of specializationof the erythroid cell and the similarity of the rapidly labeled

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Testosterone Stimulation of RNA Synthesis 527

RNA described here to the hemoglobin mRNA of othersystems, which sediments at about 9-10 S (20, 21), suggestthat this RNA may be involved in the synthesis of hemoglobinas a direct precursor of globin mRNA. It is improbable thatthe mRNA precursor coding for enzymes involved in hemesynthesis could be detected by this method of labeling. Theamounts of mRNA needed for globin chains are much largerthan those mRNAs needed for enzymes of heme synthesis.Each heme incorporated into hemoglobin requires a priorisynthesis of the globin chain. In our system of primarycultures the greatest stimulation observed with testosteronewas for the heme associated with hemoglobin.The data reported in this paper support the concept that the

stimulatory action of androgens on erythroid cells is by amechanism different from that of erythropoietin. The evidencefor this concept is as follows. Erythropoietin, but not testo-sterone, is capable of stimulating total DNA and RNAsynthesis in liver erythroid cells. Testosterone has its optimaleffect on the synthesis of heme associated with hemoglobin incells of fetuses of 10-11 weeks of gestation (7), whereas eryth-ropoietin has its optimal effect on cells from fetuses ofgestational age 14-18 weeks (22). Kinetic studies in tivo indi-cate that androgens such as 19-nortestosterone act on ery-throid cells to increase the size of the stem cell populationcapable of responding to erythropoietin (23). In all of thesediscussions it should be borne in mind that testosterone is notfound in the liver of the midterm human fetus but in theadrenals and gonads (24). As for cAMP involvement, it hasbeen shown that both testosterone and erythropoietin actionon hematopoietic cells are independent of this cyclic nucleo-tide (25, 26).RNA synthesis in human liver erythroid cells has unusual

characteristics. Even after extremely short uridine pulses themain peak of radioactivity sediments at 10 S (Fig. 4). Thissedimentation characteristic is completely different from theRNA synthesized by human fetal lung, kidney, or liver epi-thelial cells (Figs. 2 and 3) and also differs greatly from therapidly labeled, high-molecular-weight RNA that has beendescribed as a precursor of globin mRNA in other systems(20). It is possible that the sedimentation characteristics'observed are the end result of nuclease action in vivo on a high-molecular-weight RNA precursor or of artificial formationduring the isolation. The fact that degradation stops with anRNA sedimenting at 10 S instead of continuing to oligonucleo-tides strongly suggests a rather specialized maturation process.The experiments described do not differentiate between the

transcription of RNA or the processing of a high-molecular-weight RNA as the possible sites of action of testosterone. Thehormone could act at the level of RNA synthesis or at thelevel of RNA maturation; both processes of RNA metabolismare steps where a regulation of specific gene expression canoccur.

Studies on the regulation of human fetal liver erythropoiesisdescribed herein are of importance because they offer a uniquemodel for investigation of fetal hemoglobin synthesis. In thismodel the indicators of the action of an androgen can bedescribed in terms of stimulation of a definitive, readilyidentifiable, product, namely, fetal hemoglobin.

We thank Ms. P. Johnstone and A. Mirijello for skillfultechnical assistance. Human fetal tissues were obtained with thehelp of Dr. M. Gelfand of the Jewish General Hospital, Montreal.This work was supported by a grant from the Medical ResearchCouncil of Canada (MT-1658).

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