Transcriptional and Posttranscriptional Regulation of Human Expression by Androgen

Dieter A. Wolf*, Thomas Herrinaert. Heiko Hermeking*, Dorothea Blasdhke, and WolfraG H&t

Institute of Physiological Chemistry University of Munich D-8000 Munich 2, Germany

Autoregulation is a control mechanism common to INTRODUCTION several proteins of the /thyroid re- ceptor superfamily. In this work the effect of andro- The androgen receptor (AR) is a member of the steroid gens and on the expression of the and thyroid superfamily of - human androgen receptor (hAR) in prostate and inducible factors (for review see Ref. 1). cell lines was studied. Northern blot Cotransfection experiments of AR cDNA with a reporter analysis revealed a decrease in hAR steady state construct have shown that the AR mediates androgen- RNA levels in LNCaP cells by 3.3 nht of the synthetic regulated transcription (2, 3). By analogy with androgen . Maximal down-regulation of other steroid hormone receptors it is assumed that a hAR RNA to 30% of control levels occurred 48 h complex between hormone and receptor is formed after hormone addition. T47D breast cancer cells within the cell. A conformational change of this complex showed a similar effect with mibolerone, while hAR enables specific binding to recognition sequences near expression in normal skin fibroblasts did not re- the promoter of the target gene to be activated. Mod- spond to androgen treatment. As shown by nuclease ulation of the rate of transcription initiation is the ulti- Sl analysis, hAR transcripts initiate at three principal mate result (for review see Refs. 4-6). AR cDNAs have been cloned from various species start sites, all of which are equally sensitive to an- (7-l 0). Comparative sequence analysis has revealed drogen. Steroidal as well as antiandro- the typical domain structure (reviewed in Ref. 1l), which gens were capable of partially antagonizing andro- is highly conserved among the members of the steroid gen-mediated hAR RNA down-regulation in LNCaP hormone receptor family (4). Deletion mutagenesis has and T47D cells, while not exerting a significant effect identified three functionally important regions: an amino when administered alone. While hAR RNA stability terminal segment involved in transcriptional activation, was increased by hormone, nuclear run-on analysis a cysteine-rich DNA-binding domain, and a carboxy- revealed a 4-fold reduction of hAR gene transcrip terminal hormone-binding domain. A nuclear transloca- tion 98 h after androgen treatment. Although de- tion signal homologous to that found in the SV40 large creased hAR RNA levels did not coincide with a T antigen was recognized within the hinge region be- parallel decrease in AR protein levels, analysis of tween the DNA- and steroid-binding domains (12). androgen-inducible reporter constructs demon- The promoter regions of the human AR (hAR) and strated that prolonged androgen administration to rat AR (rAR) lack typical TATA and CAAT se- quence motifs but include a GC-rich region and contain ceils results in a progressively impaired sensitivity putative Spl binding sites characteristic of house- of the intracellular androgen response mechanism. keeping promoters. Sl nuclease protection analyses These results show that prolonged androgen expo- have demonstrated two major transcription initiation sure leads, besides its effect on hAR RNA levels, to sites separated by few nucleotides, approximately 1 .l functional inactivation of the AR. Thus, in viva, post- kilobases (kb) upstream of the initiator methionine of translational control of AR activity appears to be a the AR gene (13, 14). novel mechanism of negative autoregulation of an- Expression and regulation of the hAR and rAR genes drogen effects on . (Molecular En- have been investigated in cell lines and in animals. docrinology 7: 924-938 1993) Significant expression of hAR RNA was detected in malignant breast (13) liver (15) and prostate ceil lines 0666-6603/93/0924-0936$03.00/0 (16) as well as in primary genital skin fibroblasts (13). -Endouinology Copydght 0 1993 by The Endocrine Socmty In the rat ventral prostate (15, 17, 18) and in the human

924 Autobgous Regulation of the hAR 925

cell line LNCaP, expression of AR RNA probed with an hAR cDNA fragment. In LNCaP cells is regulated by (19, 20). Androgen with- three major hAR RNAs of 11 kb, 8 kb, and 4.7 kb are drawal increases and androgen administration de- detectable (Fig. 1A). While the 11-kb RNA represents creases AR steady state RNA as assayed by Northern the full-length transcript containing all exons, the 8-kb hybridization. In addition, in the human hepatoma cell RNA originates from the use of an alternative splice site line HepG2 and in the human mammary cancer cell line thus leading to the loss of 3 kb 3’untranslated se- MFM-223 autologous down-regulation of AR RNA co- quences (14). The identity of the 4.7-kb RNA remains incides with decreased AR protein levels (15,21). These uncertain. It was proposed that the 4.7-kb signal de observations have led to the concept of negative auto- rives from unspecific hybridization of 28s ribosomal regulation of AR expression in rat and human tissues RNA (17). As shown in Fig. 1A, all three hAR transcripts analogous to that observed for other steroid hormone continuously decrease with similar kinetics within 120 receptors (22-28). However, this hypothesis has re- h of hormone incubation. Since equal amounts of total mained controversial, since other studies, employing RNA were loaded on each gel lane, unspecific cross- ligand-binding assays, have demonstrated positive au- hybridization to 28s ribosomal RNA does not explain toregulation of AR protein levels in genital skin fibro- the disappearance of the 4.7-kb transcript after MIB blasts (29, 30). In contrast, by in situ hybridization and treatment. Therefore the 4.7-kb RNA probably repre- immunocytochemistry, Takeda et al. (18) have demon- sents a degradation product of the high molecular strated up-regulation of both AR RNA and AR protein weight forms of hAR RNAs (14). However, this mRNA in the prostate of androgen-treated rats and mice. An is still considerably longer than the protein coding part even more complicated scenario has emerged from of the hAR cDNA [2730 base pairs (bp)], and therefore studies in LNCaP cells where androgen-dependent we cannot rule out a possible coding capacity of the down-regulation of AR RNA appears to coincide with a 4.7-kb RNA. transient P-fold up-regulation of the AR protein (19). On Down-regulation of hAR RNA by MIB in LNCaP cells the other hand, we reported recently that rapid protein is down to 30% of control levels and is maximal after synthesis-independent androgen induction of prostate- 48 h as compared to RNA levels simultaneously deter- specific antigen (PSA) gene transcription in LNCaP cells mined in the absence of MIB (Fig. 18). Thus hAR RNA takes a transient course, thereby pointing to a negative down-regulation in LNCaP cells is a delayed hormone regulation of the androgen response pathway after effect. The RNA level of the gene for the glycolysis long-term hormone administration (31). enzyme glyceraldehyde phosphate dehydrogenase In this study we have thoroughly reevaluated the (GAPDH) is not significantly influenced by MIS, and the effects of androgen on AR steady state RNA and corresponding RNA can thus be used to correct for protein levels in LNCaP cells. Moreover, we have per- unequal loading of RNA per lane. formed experiments to study the hormonal regulation To explore whether autologous down-regulation of of AR expression with respect to hAR RNA metabolism, the hAR gene is a phenomenon restricted to trans- promoter usage, and transcriptional activity of the hAR formed LNCaP cells only, other normal and transformed gene, as well as subcellular localization and functional human cells were analyzed. Normal genital skin fibro- activity of the AR protein. We demonstrate that AR blasts (GSF) derived from infant foreskin as well as the transcription from all mRNA initiation sites is negatively human breast cancer cell line T47D express substantial regulated by androgens in human prostate and breast amounts of hAR RNA (Fig. 1C). In T47D cells and GSF cancer cell lines but not in normal human fibroblasts. the major RNA species of 11 kb and 8 kb are detectable. Although hAR RNA down-regulation apparently does The Northern blot in Fig. 1C moreover shows that, not coincide with reduced receptor protein levels or while hAR expression in T47D cells was equally sensi- altered subcellular localization, androgen administration tive to MIB, normal GSF showed no effect of MIB on leads to functional desensitization of the intracellular hAR steady state RNA (Fig. 1C). The kinetics of hAR hormone response mechanism in LNCaP and T47D down-regulation in T47D cells were similar to the kinet- cells as judged by the ability to activate androgen- ics observed in LNCaP cells (data not shown). responsive reporter gene constructs. Thus functional inactivation provides a novel mechanism of negative AR RNAs Starting from Different Transcription autoregulation of androgen action on gene expression. Initiation Sites are Equally Sensitive to Androgen

Expression of the hAR gene uses a common promoter RESULTS in diverse human tissues. Although, according to earlier reports (13, 14) the heterogeneity of transcription ini- Effect of Androgen on AR Steady State RNA Levels tiation described for a number of genes lacking canon- ical CAAT and TATA box sequences is also a property To document the effect of androgens on hAR steady of the hAR gene, two major transcription initiation sites state RNA levels, the AR-containing cell line LNCaP separated by few nucleotides have been recognized. (32) was treated for increasing periods of time with 3.3 Using a uniformly labeled single-stranded probe derived nM of the nonmetabolizable synthetic androgen mibol- from the hAR upstream region, we have analyzed the erone (MIB) (33). RNA was analyzed on Northern blots hormone sensitivity of the different hAR RNA initiation MOL ENDO. 1993 Vol7 No. 7 926

A C

I 002505 1 2 4 6 8 12 -----24 48 72 96 120 tlrs

I I

llkb T47D GSF Bkb ‘- ‘- MI6 4.7 kl

B

Fig. 1. Northem Blot Analysis of Total RNA of LNCaP, T47D, and GSF Cells after Treatment with the Synthetic Andmgen MIB A, LNCaPcells were cultivated in the presence (+) or absence (-) of 3.3 nM MIB for various periods from O-120 h. RNA was extracted and analyzed on Northem blots (20 Mg/lane) hybridized with an hAR EcoRI/Pstl cDNA probe labeled by random priming. Subsequently the probe was washed off, and the filter was rehybridized with a probe for the housekeeping enzyme GAPDH (62). B, The autoradiirams were scanned densitometrically, and the results of hAf? hybridization relative to GAPDH are shown schemaWIly in a block diagram. C, T47D cells and GSFs were treated with MIB for 96 h. Total cellular RNA was isolated, and filters were hybridized with the hAR o&e as in A. The ethidium bromide-stained gels before transfer are shown to document equal RNA loading per lane.

sites in a nuclease Sl protection assay. This seemed Since I3 was not recognized in previous reports, we reasonablesince the incompletedisappearance of hAR have mapped this start site relative to a sequencing RNA even after prolonged hormone administration reaction of this region starting from primer hAR 5. It could reflect a differential effect of MIB on individual turned out to be 4-6 nucleotidesdownstream of I2 (Fig. RNA start sites. We have recently observed differential 2A). As can be gathered from Fig. 28, the transcripts effects of MIB on the promoter usage of the c- initiating from the three mRNA start sites are equally protooncogenein LNCaPcells (34). sensitive to autdogous down-regulation by MIB in Figure 2A confirms the results of previous reports LNCaPand T47D cells but again not in GSF. Note that showingthat hAR gene expressionin several cell types the internal ratio of start site usage does not change uses two principal mRNA initiation sites located 1115 upon hormone addition (Fig. 28). These results show and 1126 nucleotidesin front of the ATG translation that II, 12, and I3 are subject to a common regulatory initiation codon. The upstream and downstream start mechanismby androgens. sites are referred to as I1 and 12, respectively. In un- treated LNCaP and T47D cells the relative usage of Effect of Antiandrogens on AR RNA Expmasion sitesI1 :I2 is 1.51 as determinedby densitometricscan- ning of the correspondingbands (data not shown). In Transductionof hormonalsignals into the nucleuslead- addition to the I1 and I2 bands, a third, smaller band, ing to modulation of gene expression is known to 13,is found which comprises20% of the total hAR RNA. involve the hormone receptor protein. To evaluate the Autologous Regulation of the hAR 927

A Figure 3 again shows autologous down-regulation of hAR RNA by MIB. However, the simultaneous presence of CA or Flu-OH for 96 h partially abolishes the effect of MIB on all species of hAR steady state RNA. Antian- drogens alone have no significant effect on hAR RNA expression in LNCaP cells. Identical results were ob- tained for hAR regulation by antiandrogens in T47D cells (data not shown). Quarmby et a/. (17) recently reported drastic hAR down-regulation in LNCaP cells in response to the CA. To substantiate our contradictory findings, we have studied the effect of CA on hAR expression under more rigorous conditions. After down- regulation of hAR RNA by treating LNCaP cells for 72 h with MIB, the culture medium was renewed, an appropriate amount of CA but no MIB was added, and cells were harvested for RNA preparation after different periods. As shown in Fig. 3C, CA is not in a position to maintain the low level of hAR expression induced by MIB pretreatment for 72 h. In contrast the presence of CA alone allows for the restoration of hAR RNA levels B prevailing in untreated control cells. The kinetics of this reversion effect are similar to those obtained for autol-

LNCaP T47D w ogous hAR down-regulation by MIB. Only 24-48 h after --- the addition of CA, pretreatment levels of hAR RNA are PMt-+-+-+ MIB achieved. These results clearly demonstrate that hAR down-regulation in our stock of LNCaP cells is strongly antagonized by antiandrogens and thus in principle reversible. We conclude that the AR protein is involved 160 - in the process of autologous hAR regulation. 147 - AR RNA Down-Regulation by MIB Occurs at the 122 - Level of Transcription initiation 110 - The AR is presumed to act as a transcription initiation Fig. 2. Androgen Sensitivity of AR RNA Start Sites modulator, but several in viva studies of known andro- A, Mapping of hAR RNA initiation sites by nuclease Sl gen target genes have suggested that andrcgens pri- protection assay with an antisense DNA probe derived from marily control RNA stability (37, 38). We therefore the hAR promoter region run in parallel with a sequencing measured the effect of MIB on hAR RNA half-life. reaction starting from primer hAR5. RNA derived from initiation LNCaP control cells and cells incubated with MIB were site 1 (II) protected fragments of 134-137 bases, from I2 treated with the RNA synthesis inhibitor actinomycin D fragments of 126-127 bases, and from I3 fragments of 121- (5 pg/ml) for different periods from O-24 h. As shown 122 bases. B, Several of the RNAs described in Fig. 1 were in Fig. 4A, the half-life of the hAR RNA in untreated subjected to nuclease Sl analysis. P, Labeled DNA probe; M, LNCaP cells is 3.8 h. Preinduction of cells with MIB for molecularweight standard; t, yeast tRNA. 72 h increased hAR RNA half-life to 6.4 h. Notably, an accelerated turnover of hAR RNA in the presence of MIB was not detectable in LNCaP cells (Fig. 4A), making possible participation of the AR protein in hAR RNA a posttranscriptional mechanism of hAR RNA down- down-regulation by MIB, we tried to antagonize the regulation in LNCaP cells unlikely. androgen effect by the simultaneous addition of AR The fact that the AR protein is apparently involved in blocking agents (antiandrogens). The steroidal antian- hAR RNA down-regulation without decreasing hAR drogen acetate (CA) as well as the nonste- RNA half-life prompted us to investigate the effect of roidal antiandrogen hydroxy (Flu-OH) were androgens on hAR transcription. Autologous down- recently shown to effectively prevent the inhibitory ac- regulation of the RNAs of various steroid hormone tion of MIB on proliferation and c-myc expression in receptors was shown to occur at the level of transcrip LNCaP cells (34, 35) while not exerting any effect when tion initiation (23, 26, 28). Run-on analysis was per- administered alone. Due to the relatively low affinity of formed in nuclei isolated from cells treated with MIB antiandrogens for the AR a several hundredfold molar and/or CA for 96 h. Nuclear transcripts were hybridized excess over the androgen is required for effective block- to strand-specific hAR cDNA probes. Probes for the age of androgen action (36). PSA gene and the gene encoding phosphoglycerate MOL ENDO. 1993 Vol7 No. 7 928

A C

-++--+- MIB MIB pretreatment (72h) --++-- CA ----++ Flu-OH o o 1 2 4 8 122448 CAWs) z’glkkbb f4.7 kb + 4.7 kb

GAPDH i GAPDH

L

B

Fig. 3. Northem Blot Analysis of RNA of LNCaP Cells Treated with the Synthetic Androgen MIB and the Antiandrogeh CA A, LNCaP cells were maintained in the presence (+) or absence (-) of 3.3 nM MIB and 1.8 MM CA or 2.6 PM Flu-OH. RNA was isolated and analyzed on Northern blots (20 pg/lane) with the hAR probe. Filters were reprobed with GAPDH to correct for unequal loading of RNA per lane. B, Densitometric analysis of the blot presented in A. C, Effect of antiandropen oh hAR RNA levels after preincubation with MIB. LNCaP cells were maintained in the presence of 3.3 nM MIB. After 72 h the medium was removed, and 1.8 PM of CA but no MIB were added. RNA was isolated after diierent Periods between 0 and 72 h and Probed on Northern blots with the hAR probe. kinase(PGK) were used as controls. As shown in Fig. protein (15, 17, 21). In contrast Krongrad et al. (19) 48, significant hAR transcription in LNCaPcells is only recently reported slightly increasedAR protein levels observedon the coding strand. Treatment with MIB but early after androgen administrationto LNCaPcells. not with the antiandrogen CA suppresseshAR tran- To see whether or not changesin hAR RNA result in scriptionby 75% comparedto untreated controls (scan- altered protein expressionunder our culture conditions, ning data not shown). Combined administration of MIB total cellular proteins of LNCaP cells, T47D cells, and + CA largely prevents down-regulation of hAR tran- GSF were probed on immunoblotswith the monoctonal scription. The transcription rate for the PSA and PGK antibody F39.4.1 (39) directed against the hAR. In genes after 96 h is influenced by neither hormone LNCaP cells the antibody recognized two proteins mi- treatment. grating with an apparent mol wt of about 11OK as expected for the hAR (40) (Fig. 5). In T47D cells and in Effect of Androgen on AR Protein Expression GSF the amount of receptor protein was obviously too low to be detected by this antibody. After 96 h of The results presented above demonstrate that andro- androgen treatment, i.e. at a time point at which hAR gens negatively regulate hAR RNA expression at the RNA down-regulationis maximal, the sameamount of level of transcription initiation. Autologous down-regu- AR protein was detectable in androgen-treatedvs. con- lation of progesterone and receptor RNAs trol samples of LNCaP cells. However, both protein was reportedto correspondto reduced receptor protein bands migrated with a slightly slower mobility, sug- levels(24-26,28). In the humancancer cell linesHepG2 gesting a posttranslational modification. This result was and MFM-233 as well as in the rat ventral prostate AR confirmedin two independentexperiments. RNA down-regulationalso leads to a decrease of AR In order to be able to directly follow the intracellular Autologous Regulation of the hAR 929

A B

+ MIB 0 3 6 1224 0 3 6 1224 hrs Act D

MIB+CA

Fig. 4. Posttranscriptional and Transcriptional Regulation of the hAR Gene by MIB A, Effect of MIB on hAR RNA half-life. Untreated LNCaP cells and cells treated with MIB for 72 h were incubated for various periods from O-24 h with actinomycin D (5 rg/ml), and RNA was isolated and subjected to Northern analysis by hybridization with an hAR cDNA probe. Autoradiograms were scanned, and hAR half-lives were calculated by regression analysis of absolute MR RNA levels at each time point after actinomycin-D treatment. The ethidium bromide-stained gels before transfer are shown below the autoradiograms. B, Transcriptional regulation of the hAR gene by androgen and antiandrogen in LNCaP cells. Ptasmids containing double-stranded and single-stranded DNA probes were transferred to nylon membranes by slot blot and hybridized with nuclear run-on RNA (10’ cpm/3 ml hybridization buffer) from cells treated without or with MIB and/or CA for 96 h. Probes detecting sense RNA are marked by +, those detecting antisense RNA by -.

dynamicsof the AR, we carried out immunocytochem- ical analyses of AR protein expression in LNCaP cells treated with androgen for different periods. LNCaP cells, when maintainedin the presenceof 10% unmod- ified fetal calf serum (FCS), which contains approxi- LNCaP T47D GSF mately 0.1 nM and androstendione(41) KD ‘- +‘I- +I’-- +’ [i.e. at least lOO-fold less than normal male sera (42)], 200 + showed predominantlynuclear staining of the AR (Fig. 6B). While a recombinantAR expressed in COS celb was reported to be enriched in the perinuclearregion of the before hormone addition (12, 43), 94 + nuclear staining in LNCaP cells was even preserved after maintenancein serum-freemedium for 48 h (Fig. 6A). Addition of physiologicalamounts of androgenfor various periods (3-96 h) resultedin neithera qualitative F@ 5. Western Blot Analysis of AR Protein Expression nor a significantquantitative changein AR staining(Fig. LNCaP cells, T47D cells, and GSFs were cultivated either in the presence (+) or absence (-) of 3.3 nhi MIB for 96 h, and 6, C-F). In the AR-negative prostate cancer cell line total cellular proteins were isolated. Proteins were separated DU145 (44) only slight background staining was ob- by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, served (Fig. 6H), as was the case with LNCaP control and immunologicaldetection was performedusing the mono- slidesnot incubated with the specific primary antibody ckmal antibody F39.4.1 as described in Materials and Methods. (Fig. 6G). These results confirm and extend those of AR protein expression in T47D cells and in GSF was too low Krongrad et al. (19), demonstrating that, at least in to be detected by this antibody. LNCaP cells, hAR RNA down-regulationdoes not nec- essarily coincide with reduced protein levels or altered subcellularlocalization. MOL ENDO. 1993 Vol7 No. 7 930

Oh 3h 24 h

48 h 96 h - F39.4.1 DU145

Fig. 6. lmmunocytochemical Analysis of AR Protein Expression LNCaP cells were maintained either in serum-free medium (A) or in medium containing 3.3 nM MIB, cultivated for 0 h (B), 3 h (C), 12 h (D), 48 h (E), and 96 h (F), fixed, and processed for indirect immunofluorescent labeling using the monoclonal antibody F39.4.1. The AR-negative prostate cancer cell line DU145 (H) and a sample of LNCaP cells not incubated with the specific primary antibody are used as controls (G)

Functional Inactivation of the AR after Prolonged To test this hypothesis, we have cloned the natural Hormone Administration PSA promoter in front of a promoterless chloramphen- icol acetyltransferase (CAT) reporter plasmid and as- In previous reports we demonstrated that MIB rapidly sayed for inducibility under various conditions of andro- and transiently increases PSA gene transcription in gen treatment. The construct PSACAT61 was trans- LNCaP cells (31, 34). Maximal induction occurred after fected by electroporation into LNCaP and T47D cells 3 h and slowly declined toward pretreatment levels pretreated with MIB for increasing periods of time. from 48 to 96 h. From these kinetics we concluded that Immediately after transfection duplicate dishes either androgen-dependent PSA induction is a direct hormone did or did not receive 3.3 nM MIB for 48 h. In cells not effect involving the binding of the AR to an element preincubated with MIB, a 7- to g-fold increase of CAT present in the PSA promoter, closely resembling the expression is found upon androgen stimulation (Fig. consensus sequence for hormone response elements 7A), showing that the PSA promoter contains an andro- (31). Moreover, we speculated that the transient char- gen-inducible enhancer. However, with increased times acter of PSA induction might be due to inactivation of of MIB preincubation in the range of 24-96 h, androgen the intracellular hormone response mechanism. With inducibility of PSACAT61 in both cell lines decreases regard to the parallel kinetics of PSA inducibility and as compared to control levels simultaneously deter- hAR RNA levels, it seems intuitively logical to consider mined in the absence of hormone (Fig. 7A). CAT induc- a numerical reduction of AR molecules per cell as a tion showed some variability during the first 48 h of consequence of hAR RNA down-regulation being re- hormone pretreatment but was consistently more than sponsible for this effect. However, the immunoblotting 5-fold reduced after 96 h. data presented above point to a functional inactivation Since the PSA promoter fragment used for construc- of the AR after prolonged hormone administration in tion of PSACAT61 has an approximate length of 5 kb, the presence of stable amounts of receptor proteins. we could not rule out the possibility that numerical or Autologous Regulation of the hAR 931

PSACAT61 LNCaP T47D MB pretreatment 0 24 40 96 0 24 48 96 hrs anlirogen ------mm stimulation -+ -+ -+ -+ -+ -+ -+ -+

B pG29GtkCAT LNCaP T47D . ..^ MIB pretreatment 0 24 48 96 0 24 48 96 hrs androgen ------stimulation -+ -+ -+ -+ -+ -+ -+ -+

Fig. 7. Effect of MIB Pretreatment on Androgen lndudbility of PSACAT61 (A) and pG29GtkCAT (B) in LNCaP and T47D Cells Duplicate cultures of cells pretreated with 3.3 nM MIB for increasing periods from O-96 h were transfected with 2.5 ag of the respective plasmids and either stimulated or not by the addition of androgen for 46 h. Protein extracts were prepared and assayed for CAT activity. TLC plates were autoradiographed, and autoradiograrns were scanned. Scanning data are presented in a block diagram. The factor of CAT induction was calculated as CAT activity in the presence of hormone divided by CAT activity without

functional reduction of factors other than the AR is DISCUSSION responsible for the self-limitation of androgen action on PSA transcription. We therefore performed control ex- The phenomenon of negative autoregulation of the periments with the plasmid G29GtkCAT which contains expressionof genes encoding membersof the steroid as sole androgen sensitive regulatory sequencestwo hormone receptor superfamily has previously been glucocorticoid/progesterone response elements demonstrated for the receptor (26, 27) the spaced by 29 nucleotidesdirectly in front of a thymidine progesteronereceptor (24,25, 28) and the glucocorti- kinase (tk) promoter-driven CAT construct (45). Again, coid receptor (GR) (23, 46, 47). In this study we have in both cell lines, LNCaP and T47Q progressively re- analyzed in detail the hormonal regulation of the hAR duced androgen inducibility was detectable depending with respect to promoter usage, RNA stability, rate of on the duration of MIB pretreatment (Fig 78). These transcription, and trans-activating function. results establishthat, in vim, prolonged androgen ad- Androgen Regulation of hAR RNA Expression ministration results in self-limitation of androgende- pendent &ins-activation of androgen-sensitive pro- The mechanismsinvolved in autoregulation of other moterswithout any change in AR protein levels. steroid receptor genes are diverse. While autologous MOL ENDO. 1993 Vol7 No. 7 932

down-regulation of the , the es- human glandular kallikrein-1 (31) and delayed suppres- tradiol receptor, and the GR genes occurs at the level sion of c-myc oncogene transcrfption in LNCaP cells by of transcription initiation in some cell types (23,26,28), MIB (34). While antiandrogens prevented the androgen the contribution of posttranscriptional mechanisms has effect on c-myc transcription, CA acts as an androgen been demonstrated in others (27). Negative autoregu- on kallikrein gene transcription (31) and con- latiin of the hAR gene in homogeneous cultures of sequently fails to antagonize androgen induction. Thus human prostate cancer cells (LNCaP) is primarily a antiandrogens act differentially on androgen-regulated consequence of reduced transcriptional initiation at all gene transcription in LNCaP cells. mRNA start sites. In view of available sequence data, As in the case of c-myc transcriptii, CA is now this finding is particularly interesting. Within 500 bp shown also to antagonize the effect of MIB on hAR upstream from the heterogeneous transcription initia- gene transcription and thus to behave as a classical tion sites the promoter region of the rAR gene contains antiandrogen, as does Flu-OH. This finding cannot be four repeats of the DNA-binding consensus sequence easily explained by the point residing in the for steroid hormone receptors [hormone response ele- hAR gene of LNCaP cells. Since the wild type AR of ment (HRE)] homologous to those found in other an- T47D cells also mediates androgen-antagonistic activity drogen-regulated genes (49). Given the high homology of CA on hAR gene expression (data not shown), it of the hAR and the rAR genes, the presence of HREs appears that the mutated LNCaP AR behaves normally in the hAR gene promoter is possible. Unfortunately the in transmitting CA signals on c-myc and hAR gene published sequence of the hAR promoter region (13) expression. does not extend to the region containing clustered Androgen regulation of c-myc and hAR gene tran- HREs in the rat homolog, and a sequence comparison scription has several features in common, which distin- is thus impossible at present. Whether negative auto- guishes it from kallikrein gene regulation: 1) c-myc and regulation of hAR gene transcription is mediated by hAR genes are negatively regulated by androgens, HREs, as is hormone-dependent rrans-activation (4) whereas kallikrein genes are positively regulated; and remains to be elucidated. 2) while kallikrein gene induction is a rapid event taking On the other hand, trans-repression of the gene less than 3 h until maximal induction has occurred, c- encoding the Fos is fundamentally myc and hAR suppression is slow (>48 h). These different from Fos-mediated trans-activation. The c-foe differences may provide the basis for the differential gene is subject to autorepression (50) but the target effect of CA on androgen-regulated genes within one site is not an APl binding motif but the serum response cell type. element (SRE) (51-53). Since repression via the SRE Although the number of genes investigated is limited, requires an intact Fos repeat but no functional it appears from our studies that CA antagonizes andro- DNA-binding site (52) Fos is presumed to interact with gen effects in LNCaP cells when slow negative regula- another protein and inhibit transcription by competing tion is executed. The pathways involved in this type of for a factor needed for activation of the SRE. Trans- regulation are expected to include additional, still uni- repression of the collagenase I gene by the GR has dentified mechanisms which, as a net result, mediate also been shown to be independent of receptor DNA androgen-antagonistic activity of CA. In contrast, rapid binding. In this case, &ens-repression is mediated by positive regulation, as in the case of kallikrein genes, direct protein-protein interaction between the DNA- appears to be a direct hormone effect with a significant binding domain of the GR and the DNA-binding region penetrance of the receptor leading to of a Jun monomer (54). Thus trans-repression by the androgen-agonistic activity of CA. According to the AR may also differ from frans-activation in that it might model presented above, in LNCaP cells, the alteration be independent of HRE binding but involve interaction of AR conformation induced by CA may be appropriate with another tissue-specific protein which is probably to allow binding to HREs and trans-activation but may lacking in genital skin fibroblasts. be insufficient to mediate interaction with other reguls Data in favor of this model are provided by the tory proteins necessary for Vans-repression. analysis of antiandrogen action on hAR expression. The effects of antiandrogens on gene expression in Androgen Regulation of hAR Protein Expression LNCaP cells are particularly interesting, since a point mutation in the steroid-binding domain of the LNCaP Direct action of androgen analogs on the transcription cell hAR gene results in an altered hormone-binding of the androgen-regulated PSA gene has been shown specificity (55). Specifically, this mutation was shown to be mediated via the HRE homologous sequence to cause both increased receptor binding of CA com- residing 150 bp upstream of the RNA initiation site (58). pared to the wild type receptor and trans-activation of Although androgen analogs are metabolically stable a reporter construct by CA via the mutated receptor over prolonged periods (59) PSA induction in LNCaP (56,57). cells is transient (48-96 h) (31, 34) indicatfng that During recent years we have studied regulation by androgen action is attenuated somewhere downstream androgens and antiandrogens of three other genes in of the ligand but upstream of the target sequence. LNCaP cells. We demonstrated rapid transcriptional From the parallel kinetics of hAR RNA down-regula- induction of kallikrein-like genes encoding PSA and tion and PSA inducibility one tentatively could infer that Autdcgous Regulation of the hAR 933

reduced RNA levels might result in decreased protein lead to nuclear exclusion and consequent impairment levels, thus accomplishing transience of PSA induction. of trans-activating function. As demonstrated by immunoblot analysis, this model, In conclusion, this study uncovers a novel level of AR which may hold true for most steroid receptors in other regulation and indicates that a complex pattern of tran- cell systems (15, 21, 24-26, 28), proved too simple in scriptional and posttranscriptional mechanisms oper- the case of the AR in LNCaP cells. Maximal down- ates in LNCaP cells to bring about fine tuning of the regulationof hAR RNA after exposure to androgen for homeostasis of receptor-mediated androgen effects on 96 h remainswithout a measurableeffect on AR protein gene expression. levels in LNCaP cells (Fig. 5). It thus appears that the principal aim of hAR gene regulation in LNCaP cells is to maintain constant AR protein levels over prolonged MATERIALS AND METHODS periodsof androgen exposure. Since androgen admin- istration is known to result in increasedstability of the Cell Culture and AR protein (43) the cell apparently has to activate a complex pattern of regulatory mechanisms including The human prostate cancer cell line LNCaP (32) was from the transcriptional shut-off of the hAR gene as well as Human Cancer Cell Laboratory, Sloan Kettering Institute for Cancer Research (Rye, NY). LNCaP cells between passages posttranscriptional stabilization of hAR RNA (Fig. 4A) in 70 and 80 were used for the experiments described. LNCaP order to keep AR protein levels constant. cells were maintained in RPM1 1840 as monolayers in the The question remains, however, why, despite the presenceof 10% FCS and 2 mM glutamine as described (35). presence of high amounts of receptor molecules as well The human breast cancer cell line T47D (81) was kindly provided by Dr. R. Renkawitz (University of Giessen, Giesaen, as receptor ligand, the transcriptionalinducibility of the Germany). T47D cells were cultivated in Dulbeccc’s modified androgen-regulatedPSA gene is transient. The results Eagle’s medium containing 10% FCS and 2 mM glutamine. presented in this study provide a key to understanding GSFs were isolated from primary cultures of human foreskin this process by the demonstrationthat, in viva, down- keratincqtes. Infant foreskins obtained by routine circumci- sion were freed from adjacent blood vessels and placed on a regulation of hAR RNA but not AR protein coincides trypsin/EDTA solution (0.25% trypsin/0.05% EDTA) in PBS with reduced inducibility of a reporter construct under devoid of Ca” and Mg2+ at 4 C for 21 h. Cells were separated the control of PSA regulatory sequences (Fig. 7A). from the upper side of the dermis and filtered through sterile Interestingly, a minimal promoter containing only two gauze into a 0.01% solution of soybean trypsin inhibitor in PBS. After centtifugation at 1000 rpm for 5 min, the cells were HREsis also subject to self-limitedinducibility depend- resuspended in serum-free keratinocyte medium (GIBCO, ing on the duration of hormone pretreatment (Fig. 7B), Grand Island, NY) containing 5 pg/liter epidermal growth fac- demonstrating that the AR protein is functionally im- tor, 50 mg/liter bovine pituitary extract and gentamicin, and paired in LNCaP and T47D cells treated with hormone seeded into tissue culture flasks. Upon reaching approximately 75% confluency, the medium was removed, and the cells were for prolonged periods. A similar observation was very washed twice with Ca2+/Mg2+-free PBS and incubated in 2 ml recently reported by Hackenberget al. (21) in another trvbsin/EDTA solution (0.025% trvbsin/0.02% EDTA) for 2 min human breast cancer cell line. Thus posttranslational ai d. During that period the fibroblasts detach fir& as they control may be the actual mechanism of self-limitation are less adherent to the substratum than the keratinocytes. Cells were aspirated and suspended in Dulbeccu’s modified of androgen effects on gene expression. Eagle’s medium containing 10% FCS and 2 mM glutamine. Within the particular signaltransduction pathway ac- For the preparation of seed stocks, cells were grown to 50- tivated by steroid hormones (reviewed in Refs. 4, 6), 75% confluency before use. Hormones were added 48 h after several explanations for the self-limitation of receptor- seeding as ethanol solutions. Final concentrations of the hor- mones were 3.3 nM for the synthetic andrcgen MIB (Upjohn, mediatedeffects on gene expressionare conceivable. Kalamazoo, Ml), 1.8 PM for the antiandrcgen CA (Schering Uponpassively entering the cell, the hormone binds to AG, Berlin, Germany) and 2.8 PM for the antiandrcgen Flu-OH its homologous receptor. As a consequence of hor- (Essex). Actinomycin D (Boehringer Mannheim, Mannheim, mone bindingthe receptor dissociatesfrom heat shock Germany) was used at a final concentration of 5 pg/ml. proteins, a process which enables homodimerization, RNA Extraction and Northern Blot Analysis heterooliaomerizationwith other proteins, and binding to target&!quences on the DNA, eventually resulting in Standard protocols were followed as described elsewhere tfaans-activationor tfans-repressionof responsive pro- (35). Autoradidgrams were scanned in an LKB (Bromma, moters. Constant levels of receptor proteins and ligand Sweden) Ultra&an XL Laser Densitometer. provided posttranslationalautoregulation of receptor activity is theoretically possible at several levels: nuclear Hybridization Probes and Plasmids translocation, interaction, receptor dimerization, oligomerization with other proteins, DNA 32P Labeling was performed with a random-primed labeling kit (Boehringer Mannheim) according to the recommendations of binding, and potential. While it has al- the supplier. The 450-bp EcoRI/Pstl cDNA fragment used as ready been shown that phosphorylation of the AR in an hAR probe for Northern analyses was generated by pulym- responseto hormone administrationis a potential form erase chain reaction from reverse-transcribed total cellular RNA isolated from LNCaP cells. The probe covers sequences of posttranslational modification (43, 60), our studies encoding parts of the homtcne-binding domain and the 3’- rule out the model that this modificationmight alter the untranslated region. The probe for the housekeeping enzyme subcellularlocalization of the AR, which in turn could GAPDH was a synthetic single-stranded digonudectide (100 MOL ENDO. 1993 Vol7 No. 7 934

bases) derived from the published sequence (62) and was end- ford, MA; Immobilon-P) using an electroblot apparatus oper- labeled with polynucleotide kinase. ating at 35 mA for 12 h. Membranes were blocked by two For the nuclear run-on transcription assays the plasmid sequential incubations in 5% low-fat powdered milk (dissolved pSVARo (kindly provided by J. Trapman, Erasmus University, in PBS) for 90 min each. Filters were incubated with a 1:lOO Rotterdam, The Netherlands) was digested with HindIll and dilution of the hAR antibody F39.4.1 (39) (Monosan, Uden, The Pstl (positions 1850-3002 according to Ref. 63), and the Netherlands) for 3 h at room temperature on a rocking platform resulting 1 .15-kb fragment was subcloned in both orientations and for an additional 8 h at 4 C without shaking. Unbound into Ml 3 mp18 and mpl9 phages. The probe for PSA was an antibody was removed by five washes, 10 min each, in PBS/ EcoRl cDNA fragment spanning the complete coding region 0.1% Tween 20 at room temperature. Incubation with a per- for the mature PSA protein (64). The probe for the gene oxidase-conjugated antimouse immunoglobulin G antibody encoding PGK was a 1.8-kb cDNA subcloned in pBR 328 (65). was performed at room temperature for 2 h on a rocking table. The template for the AR probe used in the nuclease Sl After five washes in PBS/O.l% Tween 20, immunoreactive protection assays was amplified from 1 pg genomic DNA from bands were visualized by incubation in 80 ml of a PBS solution LNCaP cells by the polymerase chain reaction. The primer containing 30 mg diaminobenzidine (Sigma, St. Louis, MO) hAR3 corresponded to nucleotides -300 to -278 and the and 30 ~1 H202. After achieving maximal band intensity filters primer hAR5 to nucleotides 119-l 39 relative to 12 (numbered were rinsed in distilled water and photographed. according to Ref. 13). The resulting 438-bp fragment, termed PAR1 , was cloned into Ml 3 mp18 (Ml 3PARl). The PSACAT61 plasmid used in DNA transfection assays lmmunocytochemistry was generated by inserting a genomic HindIll fragment con- taining the genuine PSA promoter in addition to 5 kb PSA LNCaP cells were seeded onto glass cover slips in the absence upstream sequences into the promoterless CAT reporter plas- or presence of FCS and/or 3.3 nM MIB. After different times mid pBLCAT6 (generously provided by G. Schlitz, Deutsches cells were washed twice in PBS and fixed for 30 min at 4 C in Krebs-Forschungzentrum, Heidelberg, Germany). The andro- 2% paraformaldehyde. Cells were washed and incubated in gen-inducible control plasmid G29GtkCAT contains two prc- PBS containing 0.01% saponine (Sigma) and 0.01% sodium gesterone/glucocorticoid response elements upstream from a azide for 15 min. Cells w&e incubated for 1 h at 37 C with a herpes simplex virus tk promoter-driven CAT construct (45) 1 :lO dilution of the monoclinal antiiv F39.4.1 in PBS/ and was kindly provided by R. Renkawitz. saponine, followed by three washes in bBS/saponine. The tetramethylrhodamine B isothiocyanate-labeled secondary an- Sl Nuclease Protection Assay timouse immunoglobulin G antibody was used at a 1:40 dilu- tion for 45 min at 37 C. After 2 washes in PBS/saponine and two washes in PBS alone, cover slips were prepared for A single-stranded uniformly labeled DNA probe was prepared fluorescent microscopy, and representative sections were by primer extension of M13PARl in the presence of [&*P] photographed at a 400-fold magnification. deoxy-ATP and [a-32P]deoxy-CTP. The extended products were digested with the restriction endonuclease BssHII, and the labeled probe was separated from the Ml3 template on a DNA Transfection and Assay for CAT Activity 5% denaturing polyacrylamide gel. The probe was detected by autoradiography and eluted with 0.5 M ammonium acetate, DNA transfection was performed by electroporation using a 10 mM acetate, and 10 mM EDTA (pH 8.0) for 8 h Bio-Rad (Richmond, CA) Gene Pulser operating at 300 V (960 at 37 C. Hybridization of labeled DNA fragments to total RNA microfarads). Cells were preincubated with 3.3 nM MIB for was carried out using a modification of the method of Berk various periods (time points indicated in Fig. 7). After the and Sharp (86). Hybridization mixtures of 20 ~1 containing 5 x preincubation period cell numbers were adjusted to 5 x lo6 10’ cpm of the labeled antisense probe, 30 pg (LNCaP) or 60 cells per sample suspended in 800 ~1 culture medium in an Pg (T47D and GSF) RNA, 80% formamide, 400 mM NaCI, 40 electroporation cuvette; 2.5 pg of each reporter plasmid were mM piperazin&,N’-bis(2ethanesulfonic acid), pH 6.5, and 1 added for transfection. To assure equal transfection efficien- mM EDTA were denatured at 90 C for 5 min and immediately cies, samples were devided into two equal parts (400 11 each) transferred to 52 C. After 15 h the hybridization was terminated after transfection and were seeded on two 1 O-cm dishes, one by addition of 180 ~1 icecold buffer containing 250 mM NaCI. of which received hormone (3.3 nM MIB) immediately after 30 mM Na-acetate, pH 4.5, 2 mM Zn-acetate, 5% glycerol, and seeding. Cells were harvested 48 h after transfection for 400 U nuclease Sl (Boehringer Mannheim). After incubating preparation of CAT extracts. the samples at 42 C for 1 h, the reaction was stopped by the Preparation of extracts and assay for CAT activity was addition of 50 ~1 4 M ammonium actetate, 50 mM EDTA, and performed according to Sambrook et al. (67). Briefly, cells 50 pg/ml yeast tRNA, and the nucleic acid was precipitated were scraped from the culture dish with a rubber policeman, with ethanol. Protected DNA fragments were separated on washed twice in PBS, and resuspended in 100 ~10.25 M Tris/ 7% polyacrylamide gels containing 7 M urea. Gels were dried HCI, pH 7.6. Cells were lysed by two repeated cycles of and exposed to Fuji (Tokyo, Japan) X-ray films for 8-12 h freezing (-80 C) and thawing (4 C), cell detritus was spun between intensifying screens at -80 C. down at 12,000 rpm for 10 min at 4 C, and the supernatant was transferred into a new tube. Fifty microliters of extract Nuclear Run-On Analysis were incubated at 55 C for 15 min and added to 50 ~1 1 M Tris/HCI, pH 7.4, 20 ~1 acetyl coenzyme A (3.5 mg/ml, freshly Preparation of cell nuclei and hybridization were performed as oreoared). and 10 ~1 I’“Clchloramphenicol (0.1 mCi/ml). and de&Wed (34). Filters were exposed to Fuji X-ray films be- ihe’ reacion was i&bat& at 37 ‘C for 2 h. One rriilliiter of tween DuPont (New England Nuclear, Boston, MA) Lightning ethyl acetate was added, mixed by vigorous vortexing, and Plus intensifying screens for 3 days. centrifuged for 5 min at room temperature. The supematant was evaporated under vacuum and reaction products were redissolved in 30 ~1 ethyl acetate, 20 ~1 of which were applied to the origin of a silica gel TLC plate. Plates were run in chloroform/methanol (95/5, vol/vol), dried, and exposed to Fuji Cellular proteins were isolated according to Sambrook et al. X-ray films for 12 h. The spots corresponding to the monoac- (67) and stored in aliquots at -80 C until use. Equal amounts etylated forms of chloramphenicol were measured with a thin- of protein (80 ag/lane) were passed through a 4% stacking layer scanner, and the total amount of [“Clacetyl&Warn- gel and separated in an 8% resolution gel. After elactropho- phenicol was calculated from the area under the resulting resis gels were blotted onto nylon membranes (Millipore, Bed- peak. Autologous Regulation of the hAR 935

Acknowledgments promoter in diverse human tissues and cell lines. J Biol Chem 265:13776-l 3781 14. Faber PW, van Rcoij HC, van der Korput HA, Baarends We thank Drs. G. Schutz, Ft. Renkawitz, and J. Trapman for WM. Brinkmann AO, Grootegoed JA, Trapman J 1991 providing plasmids and cell lines and Dr. D. Eick for carefully Characterization of the human androgen receptor tran- reading the manuscript. scription unit. J Biol Chem 266:10743-l 0749 15. Shan LX, Rodriguez MC, Janne OA 1990 Regulation of androgen receptor protein and mRNA concentrations by Received September 4, 1992. Rerevision received April 20, androgens in rat ventral prostate and seminal vesicles 1993. Accepted May 11,1993. and in human hepatoma cells. Mol Endocrinol 4:1636- Address requests for reprints to: Dieter A. Wolf, lnstitut fur 1646 Klinische Molekularbiologie und Tumorgenetik, Marchionin- 16. Tilley WD, Wilson CM, Marcelli M, McPhaul MJ 1990 strasse 25, D-8000 Munchen 70, Germany. Androgen receptor gene expression in human prostate This work was supported by the Deutsche Forschungsge- carcinoma cell lines. Cancer Res 50:5382-5386 meinschaft. 17. Quarmby VE, Yarbrough WG, Lubahn DB, French FS, Wilson EM 1990 Autologous down-regulation of androgen l l Present address: lnstitut fur Klinische Molekularbiologie und Tumoraenetik. GSF Forschunaszentrum fur Umwelt und receptor messenger ribonucleic acid. Mol Endocrinol Gesondhei{Marchioninistrasse 25-D-8000 Munchen 70, Ger- 4:22-28 many. 18. Takeda H, Nakamoto T, Kokontis J, Chodak GW, Chang T Present address: Dermatologische Klinik und Poliklinik der C 1991 Autoregulation of androgen receptor expression Universitat Munchen, Frauenlobstrasse 9-l 1, D-8000 in rodent prostate: immunohistochemical and in situ hy- bridization analysis. Biochem Biophys Res Commun Miinchen 2, Germany. 177:488-496 19. 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