Evaluation of the Tetracycline-RepressibleTransactivator System for Inducible Gene

Expression in Human Prostate Cancer Cell Lines

Jurgen E. Gschwend, William R. Fair, and C. Thomas Powell*

Urologic Oncology Research Laboratory and George M. O’Brien Urology Research Centerfor Prostate Cancer, Memorial Sloan-Kettering Cancer Center, New York, New York

BACKGROUND. Studies of genes that may inhibit growth or induce death of cells arefacilitated greatly by tightly controlled expression of those genes. A promising system forcontrol of transgene expression over a wide range is the tetracycline-repressible transactivator(tTA) system developed by Gossen and Bujard [Proc Natl Acad Sci USA 1992;89:5547–5551].We investigated the effectiveness of this system in three well-established human prostatecancer cell lines.METHODS. LNCaP, PC-3, and Tsu-Pr1 cells were transfected with a vector coding for thetTA protein and/or a luciferase reporter vector, and luciferase activity was measured in thepresence and absence of tetracycline or the tTA protein.RESULTS. In the absence of tetracycline, the tTA system yielded high levels of luciferaseactivity in all three cell lines. Background luciferase activity in the presence of tetracycline wasnearly undetectable in LNCaP cells, moderate in Tsu-Pr1 cells, and more than 20-fold higherin PC-3 than in Tsu-Pr1 cells. Similar background activity was observed in Tsu-Pr1 and PC-3cells, even in the absence of the transactivator protein.CONCLUSIONS. The tTA system should be useful for stable transfection of cytotoxic trans-genes in LNCaP cells and for control of transgene expression over a wide range in Tsu-Pr1and PC-3 cells. Prostate 33:166–176, 1997. © 1997 Wiley-Liss, Inc.

KEY WORDS: LNCaP; PC-3; Tsu-Pr1; death genes

INTRODUCTION

Prostate cancer (PC) has the highest incidence of allcancers among men and is one of the leading causes ofcancer deaths in the United States [1]. Androgen ab-lation is the only available treatment for locally ad-vanced or systemic disease. Because this treatment isnot curative and prostate cancer remains hormone-dependent for only a short time period, other treat-ment modalities are demanded. In many types of can-cer, understanding of tumor growth and progression,as well as of tumor suppression and programmed celldeath, at the molecular level has increased rapidly inrecent years. Studies of genes potentially involved insuppression of tumor growth or induction of celldeath are facilitated greatly by tightly controlled ex-pression of these genes in tumor cell lines. Becauseexpression of such genes might be cytotoxic to cells inculture [2–4], a system allowing complete suppres-

sion, as well as potent induction, of transgene expres-sion is desired. Examination of the consequences ofexpression of these genes and the signaling pathwaysinvolved can yield clues to the development of newtreatment modalities.

One system that allows tightly controlled indi-vidual gene expression over a wide range is the tetra-cycline-repressible transactivator system, developedand described by Gossen and Bujard and Gossen et al.[5,6]. The system utilizes the repressor protein of an E.coli tetracycline resistance operon (tet-repressor) andthe operator DNA sequence to which it binds. Two

*Correspondence to: C. Thomas Powell, Ph.D., Urologic Oncol-ogy Research Laboratory, Memorial Sloan-Kettering CancerCenter, 1275 York Avenue, Box 334, New York, NY 10021.E-mail: powellt@mskcc.orgReceived 23 July 1996; Accepted 28 January 1997

The Prostate 33:166–176 (1997)

© 1997 Wiley-Liss, Inc.

types of plasmid cassette were designed, one of whichyields constitutive expression of a chimeric transacti-vator protein (tTA) consisting of the tet-repressorfused to the C-terminal activation domain of the her-pes simplex virus virion protein 16 (VP16). VP16 canactivate transcription from certain viral promoters, in-cluding the human cytomegalovirus (hCMV) pro-moter. The second type of plasmid contains seven tet-operator sequences in a row and a minimal hCMVpromoter without enhancer, just upstream of a re-porter gene or multiple cloning site for a gene of in-terest. Addition of tetracycline (tet) prevents the tet-repressor portion of tTA from binding to the operatorsequences and the VP16 activation domain from acti-vating the hCMV promoter (Fig. 1). In the absence oftet, tTA can bind to the tet operators and greatlystimulate transcription from the hCMV promoter. Thissystem has already been shown to be a powerful toolin several eukaryotic cell lines and in transgenic mice[7–11]. However, in some cell lines, addition of tetfailed to downregulate completely expression fromthe minimal promoter [8,10]. So far, this system hasnot been investigated for controlled gene expression inprostatic cell lines. The purpose of this study was toinvestigate thoroughly the tet-responsive expressionsystem as a tool to temporally regulate transgene ex-pression in three different human prostate cancer celllines. The LNCaP cell line, which was originally de-rived from a lymph node metastasis of a prostatic ad-enocarcinoma, is androgen-responsive and resemblesthe biochemical features of clinical prostate cancermost closely among prostate cell lines [12]. The andro-

gen-independent cell lines PC-3 and Tsu-Pr1 were de-rived from bone and lymph node metastases, respec-tively, of human prostate cancer [13,14]. Using a sen-sitive luciferase reporter gene in the vector with thetet-repressible promoter, we observed high levels ofinducible expression in all three lines, with negligible,moderate, and high background activity in the pres-ence of tet in LNCaP, Tsu-Pr1, and PC-3 cells, respec-tively. These results imply that the tTA system shouldbe an excellent tool for studies of potentially cytotoxicgenes in LNCaP cells and possibly Tsu-Pr1, but that itis less promising for such studies in PC-3 cells.

MATERIALS AND METHODS

Cell Culture

Androgen-responsive LNCaP and androgen-insensitive PC-3 and Tsu-Pr1 human prostate cancercell lines were obtained from the American Type Cul-ture Collection (Rockville, MD). All cell lines weremaintained as monolayer cultures in RPMI-1640 (Me-morial Sloan-Kettering Cancer Center Media Prepara-tion Facility, New York, NY) supplemented with 10%fetal calf serum (FCS, Sigma Chemical Co., St. Louis,MO), 100 U/ml penicillin, 100 U/ml streptomycin,and 2 mM L-glutamine (complete medium), in a hu-midified atmosphere at 37°C, and 5% CO2.

Tet-Repressible System Plasmids

The plasmids pUHD 15-1 and pUHC 13-3 [5] werekindly provided by M. Gossen and H. Bujard (Center

Fig. 1. In eukaryotic cells, the tet-repressible transactivator system [5] al-lows control of gene expression by lowconcentrations of tet in the culture me-dium. In the presence of tet, the tet-repressor/VP16 activation domain fusionprotein (tet-repressible transactivator pro-tein, tTA) is prevented from binding to thetet-operator/hCMV promoter element anddoes not activate transcription of the geneof interest. In the absence of tet, the tet-repressor portion of tTA can bind to thetet-operator sequence and enable theVP16 activation domain to activate thehCMV promoter.

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for Molecular Biology, University of Heidelberg,Heidelberg, Germany). Plasmid pUHD 15-1 contains achimeric gene coding for the repressor protein of theE. coli Tn10-specified tet resistance operon fused to theC-terminal activation domain of the herpes simplexvirus virion protein 16 (tTA, Fig. 2a). This chimericgene is under the control of an hCMV promoter andenhancer that constitutively drive expression in mostmammalian cells. Plasmid pUHC 13-3 (firefly lucifer-ase reporter vector, Fig. 2b) contains seven operatorsequences from the tet resistance operon in a row, justupstream of an hCMV promoter without enhancer.This inducible operator/promoter element controlsexpression of a firefly luciferase reporter gene. Bothplasmids were transformed into competent E. coli(OneShot E. coli, Invitrogen Corporation, San Diego,CA), grown in large-scale cultures and purified byalkaline lysis followed by cesium chloride density cen-trifugation [15]. Prior to transfections, purified plas-mid DNA was ethanol-precipitated and dissolved insterile 10 mM Tris-Cl, pH 8.0, 0.1 mM EDTA.

tTA Vector Transfection and Clonal Selection

For transfection, LNCaP cells were plated at a den-sity of 2 × 106 cells in 100-mm culture dishes (CorningGlass Works, Corning, NY) in complete medium. PC-3and Tsu-Pr1 cells were plated at 5 × 105 cells in T25flasks. Transfections were performed 48 hr later forLNCaP cells and 24 hr later for PC-3 and Tsu-Pr1 cellswith the tTA vector pUHD 15-1 and a hygromycin-selectable vector, pgkhyg [16] (gift of M. Jasin, Sloan-Kettering Institute, New York, NY). Aliquots contain-ing 10 mg of pUHD 15-1 and 5 mg of pgkhyg werepreincubated with 30 ml of lipofectamine reagent(GIBCO BRL, Gaithersburg, MD) for 30 min in 200 mlserum-free RPMI, and then diluted to 2 ml in the same

medium. Cells were overlaid with the DNA/lipofec-tamine mixture and incubated at 37°C for 6 hr. Thetransfection medium was then replaced with completemedium and the cells were incubated an additional 48hr. At that time, PC-3 and Tsu-Pr1 cells were trypsin-ized and seeded in 10-mm plates at different dilutionsin complete medium containing 150 mg/ml hydromy-cin (Calbiochem, Cambridge, MA), whereas LNCaPcells were treated with 150 mg/ml hygromycin in theoriginal plates without trypsinizing. After 2–3 weeksof culture in hygromycin-selection medium, indi-vidual colonies were isolated on the plates using ster-ile cloning rings, trypsinized, and plated in fresh me-dium. Clones were maintained in medium containing150 mg/ml hygromycin + 1 mg/ml tet (Sigma Chemi-cal Co.).

Transient and Stable Luciferase ReporterVector Transfections

Hygromycin-resistant cells, cotransfected with thetTA vector pUHD 15-1 and pgkhyg, were transientlytransfected with the luciferase reporter vector pUHC13-3. Cells were plated in 6-well plates at a density of2 × 105 cells per well in complete medium containing150 mg/ml hygromycin + 1 mg/ml tet. Cells weretransfected 24 hr (PC-3, Tsu-Pr1) or 48 hr (LNCaP)after plating. For each well, 100 ng of pUHC 13-3 DNAwere preincubated with 1 ml of lipofectamine reagentfor 30 min in 200 ml serum-free RPMI, and then themixture was diluted to 1 ml with serum-free RPMI +tet (final concentration, 1 mg/ml). Cells were overlaidwith the mixture and incubated for 6 hr, and then themedium was replaced with complete medium ± 1 mg/ml tet. The cells were incubated an additional 48 hr, atwhich time luciferase assays were carried out as de-scribed below.

In order to assess endogenous activation of lucifer-

Fig. 2. a: The tTA vector pUHD 15-1 [5] contains an hCMV enhancer/promoter element driving constitutive expression of the tTAprotein (tet-repressor fused to the C-terminal activation domain of the herpes simplex virus VP16 protein). b: The luciferase reportervector pUHC-13-3 [5] contains seven tet-operator sequences in a row next to an hCMV promoter without enhancer, just upstream of afirefly luciferase reporter gene.

168 Gschwend et al.

ase expression from pUHC 13-3, previously untrans-fected cells were transiently transfected with pUHC13-3 alone. Transfection efficiencies were estimated bytransfecting parental cells with pRL-CMV alone (Pro-mega Corp., Madison, WI), a constitutive reporter vec-tor that contains a Renilla reniformis luciferase genecontrolled by an hCMV promoter and enhancer. Thesetransient transfections were carried out as above, ex-cept that tet and hygromycin were omitted.

One LNCaP and one PC-3 clone stably expressingtTA and hygromycin resistance were each further sta-bly transfected with the luciferase reporter vectorpUHC 13-3 as described in the previous section, withthe following differences: 1) tTA-expressing cloneswere plated and transfected in media without tetracy-cline or hygromycin; 2) pUHC 13-3 was cotransfectedwith the G418-selectable vector pcDNA3 (InvitrogenCorporation, San Diego, CA); and 3) clones were se-lected and maintained in complete medium supple-mented with 150 mg/ml hygromycin, 500 mg/ml G418(Geneticin, GIBCO BRL), and 1 mg/ml tet. For lucifer-ase assays of double stable-transfected clones, 2 × 105

cells were plated per well in 6-well plates in completemedium containing 1 mg/ml tet. Twenty-four to 72 hrlater, cells were washed twice with phosphate-buffered saline and fresh medium was added contain-ing tet, doxycycline (Sigma Chemical Co.), verapamil(Sigma Chemical Co.), tet + verapamil, or no drug.Cells were harvested and luciferase assays were per-formed 4 days after plating.

Cell Lysates and Luciferase Assays

Luciferase assays of transiently and stably trans-fected cells were performed using the Promega lucif-erase assay system (Promega Corporation). Briefly,cells in 6-well plates were washed once with 2 mlice-cold phosphate-buffered saline and immediatelyoverlaid with 250 ml of ice-cold 1 × lysis buffer. Fol-lowing incubation for 15 min, cells were scraped andtransferred to a microfuge tube. Samples were spunfor 5 sec, and luciferase activity of 1–5-ml aliquots ofthe supernatants was measured in an automated lu-minometer (Lumat 9501, Berthold Inc., Nashua, NH).Luciferase activity was calculated as relative lightunits/mg cellular protein. Protein concentrations ofcell lysates were determined as described [17]. Cellswere plated in triplicate for luciferase assays, and datawere plotted with error bars showing SE. All experi-ments were performed at least twice with similar re-sults.

RNA Extraction and RNase Protection Assays fortTA and Luciferase mRNA

For assays of tTA RNA, stably transfected or paren-tal cells were grown to near-confluence in 75-cm2

flasks in complete medium without drugs. For assaysof luciferase RNA, cells were transiently transfected asdescribed above and harvested 2 days later. Total cel-lular RNA was isolated by acid guanidinium thiocya-nate-phenol-chloroform extraction [18] (RNAzol B,Tel-Test, Inc., Friendswood, TX). Fragments of the tTAcDNA from pUHD 15-1 and the luciferase cDNA frompUHC 13-3 were subcloned into pBluescript vectors(Stratagene, La Jolla, CA) and used as templates forsynthesis of high specific activity, 32P-labeled anti-sense RNA probes as described [19], using T3 or SP6RNA polymerase (Promega Corporation) and equi-molar amounts (35 pmole) of [a-32P] CTP (3,000 Ci/mmole) and unlabelled CTP. A subclone of humanacidic ribosomal phosphoprotein PO (HARP) cDNA[20] (gift of J. Laborda, Georgetown University, Wash-ington, DC) was used to generate a high amount oflow specific activity RNA probe as previously de-scribed [21], for control of RNA quality and loading.Several nucleotides at the 58 end and, in most cases,the 38 end of each probe consist of pBluescript se-quences that are not protected from RNase digestionby eukaryotic RNA, thus providing an internal controlfor each assay. RNase protection assays were carriedout as described [18], using 20-mg aliquots of totalcellular RNA, 1 × 106 cpm of antisense tTA or lucifer-ase RNA probe, and 4 × 104 cpm of HARP RNA probe.Samples were resolved on 5% denaturing polyacryl-amide/7 M urea gels and exposed to X-ray film (Hy-perfilm, Amersham, Arlington Heights, IL). Protectedprobe fragments were quantified by scanning the gelson a Fuji Phosphorimager (Fuji Photofilm Co., Tokyo,Japan).

RESULTS

Characterization of tTA-Transfected ProstateCancer Cell Lines

Hygromycin-resistant clones cotransfected with thetTA and pgkhyg vectors were screened for expressionof tTA by transiently transfecting the clones with theluciferase reporter vector pUHC 13-3 and measuringluciferase activity. Most LNCaP clones expressed verylittle luciferase activity above the instrumental back-ground when maintained, transfected, and assayed inthe presence of 1 mg/ml tet, yet removal of tet for 48 hrafter transfection yielded abundant activity. TwoLNCaP clones that were among the highest expressersof luciferase activity are shown in Figure 3a. Althoughthe fold induction is difficult to assess with such low

Inducible Gene Expression in PC Cell Lines 169

background, it appeared to be between 103–104. Sev-eral tTA-transfected PC-3 and Tsu-Pr1 clones, whentransfected transiently with pUHC 13-3, also exhibitedhigh luciferase activity 48 hr after removal of tet. How-ever, all PC-3 and Tsu-Pr1 clones expressed back-ground activity in the presence of tet, with the back-ground in PC-3 clones averaging 20-fold higher thanin Tsu-Pr1 clones (Fig. 3b,c). Because of the back-grounds, inducibility of luciferase by removal of tet

was limited to about 9- and 200-fold in PC-3 and Tsu-Pr1 clones, respectively (Fig 3b,c).

Expression of tTA mRNA in Transfected HumanProstate Cancer Cell Lines

The levels of tTA mRNA in the stable tTA-transfected LNCaP, PC-3, and Tsu-Pr1 clones shownin Figure 3 were measured by RNase protection analy-

Fig. 3. Cell clones cotransfected with the tTA and pgkhygvectors and resistant to 150 µg/ml hygromycin were transientlytransfected with the luciferase reporter vector pUHC 13-3, andluciferase activity per µg of cellular protein was determined asdescribed in Materials and Methods. a: LNCaP tTA/pgkhygclones LNGK4 and LNGK9. b: PC-3 tTA/pgkhyg clones PC-3GK11 and PC-3GK16. c: Tsu-Pr1 tTA/pgkhyg clones TsuGK23and TsuGK27. Cells were plated and transfected with pUHC13-3 in the presence of 1 µg/ml tet and assayed in triplicate inthe continued presence (+ tet) or 48 hr after removal (− tet) oftet. Background activity was measured in untransfected cells(Untrans) and in a blank containing lysis buffer only (No lysate,instrumental background).

170 Gschwend et al.

sis (Fig. 4a). The amounts were somewhat variable,but did not correlate with the levels of luciferase ac-tivity in the presence of tet in the different cell lines(Fig. 4b). The PC-3 clones expressed slightly less tTARNA than the LNCaP clones, indicating that the highbackground luciferase activity in PC-3 clones was notdue to higher expression of tTA mRNA.

Transfection of Parental Cell Lines With pUHC13-3 and pRL-CMV Luciferase Reporter Vectors

Parental LNCaP, PC-3, and Tsu-Pr1 cells were tran-siently transfected with the tTA inducible reporterplasmid pUHC 13-3 and, in a separate experiment,with a vector, pRL-CMV, that should express lucifer-ase constitutively in most cells. Transfection of pUHC13-3 into cells that did not express tTA yielded barelydetectable, very high (182 × untransfected) and mod-erate (10.7 × untransfected) luciferase activity inLNCaP, PC-3, and Tsu-Pr1 cells, respectively (Fig. 5a).After subtracting untransfected background, the rela-tive levels of luciferase activity in LNCaP:PC-3:Tsu-Pr1 were 1:1,365:80. The accuracy of the level inLNCaP cells for comparison to PC-3 and Tsu-Pr1 maybe limited by its near equality to the instrumentalbackground. The amounts of luciferase mRNA insimilarly transfected cells (Fig. 6) roughly reflected lu-ciferase activity levels, although the difference in lu-ciferase mRNA levels among the transfectants wasless than the difference in luciferase activity levels

(compare Fig. 6b with Fig. 5a; see Discussion). Tran-sient transfection of parental cells with pRL-CMVyielded similar, high levels of luciferase activity inLNCaP, PC-3, and Tsu-Pr1 cells (Fig. 5b). Southernblot analysis of plasmid DNA extracted from parentalcell lines transiently transfected with pUHC 13-3 re-vealed a slightly higher number of plasmid copies inPC-3 compared to LNCaP and Tsu-Pr1 cells, but thosedifferences were minor compared to the backgroundluciferase activity found in PC-3 cells (data notshown).

Characterization of Double Stable-TransfectedLNCaP and PC-3 Cells

To determine the influence of stable genomic incor-poration of the tTA-inducible luciferase reporter vec-tor on background and on inducibility of luciferaseactivity, selected LNCaP and PC-3 clones stably ex-pressing tTA were transfected in a second step withpUHC 13-3 and the G418-selectable vector pcDNA3.Double stable-transfected clones were selected andmaintained in complete medium supplemented withtet, G418, and hygromycin. Among LNCaP clones,levels of background luciferase activity in the presenceof tet were variable, but some clones expressed barelydetectable background, yet with high luciferase activ-ity in the absence of tet. A representative LNCaP clonewith minimal background is shown in Figure 7. Maxi-mal luciferase activity was obtained in this clone

Fig. 4. Expression of tTA mRNA in stable pUHD 15-1-transfected LNCaP, PC-3, and Tsu-Pr1 cell clones. Twenty micro-grams of total RNA from each cell clone were incubated withantisense RNA probes corresponding to tTA and HARP mRNAs.RNase protection assay was performed as described in Materialsand Methods. a: Lane 1, untransfected LNCaP; lane 2, pgkhyg-transfected LNCaP; lanes 3 and 4, pUHD 15-1-transfectedLNCaP clones LNGK4 and LNGK9; lane 5, untransfected PC-3;

lanes 6 and 7, pUHD 15-1-transfected PC-3 clones PC-3GK11and PC-3GK16; lane 8, untransfected Tsu-Pr1; lanes 9 and 10,pUHD 15-1-transfected Tsu-Pr1 clones TsuGK23 and TsuGK27;lane 11, yeast transfer RNA; lane 12, undigested tTA probe;lane 13, undigested HARP probe. Sizes of 1-kb DNA ladder frag-ments (GIBCO BRL) are indicated at right. b: Phosphorimager-quantified tTA RNA levels divided by corresponding HARP RNAlevels.

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within 48 hr after removal of tet (Fig. 7a) and appearedto be >104 fold higher than in the presence of tet (Fig.7b.).

Double stable-transfected PC-3 clones expressedsubstantial background luciferase activity in the pres-ence of 1 mg/ml tet, but the background in someclones was lower than in the tTA-expressing PC-3 cellstransiently transfected with pUHC 13-3. In one clone,luciferase activity was induced about 16-fold 3 daysafter removal of tet (Fig. 8a). Increasing the concentra-tion of tet to 3 or 10mg/ml did not lower the back-ground much further (maximal induction 16.6-fold,Fig. 8a), and 10 mg/ml tet inhibited PC-3 cell growthabout 18% (data not shown). In addition, tet was re-placed with 1–10 mg/ml of doxycycline, a derivativeof tet that is 100 times more potent than tet in activat-ing a mutated, reverse tet-controlled transactivator[22]. Doxycycline was no more effective than tet inreducing background luciferase activity (Fig. 8a), and10 mg/ml doxycycline inhibited PC-3 growth substan-tially (47%, data not shown).

Others have reported that multidrug-resistant cellscan extrude tet and that this effect can be reversedby treatment with verapamil [23], which binds top-glycoprotein. Another group reported that PC-3

cells exhibited verapamil-sensitive drug efflux, whileLNCaP cells did not [24]. To determine if expulsion oftet contributed to the high background luciferase ac-tivity in double stable-transfected PC-3 clones, we in-cubated the cells with 3–30 mM verapamil in the pres-ence of 3 mg/ml tet for 3 days before performing lu-ciferase assays. Coincubation with tet and verapamildid not lower the background luciferase activity morethan did tet alone. However, those results may havebeen obscured by the finding that incubation of thecells with verapamil alone increased luciferase activityin a dose-dependent manner (Fig. 8b).

DISCUSSION

We have investigated the efficacy of a tet-repressible system for regulating transcription oftransgenes in three human prostate cancer cell lines.Using a luciferase reporter gene, we found similar in-duction of luciferase activity in all three cell lines afterremoval of tet, but different levels of background ex-pression among the cell lines in the presence of tet orin the absence of the transactivator protein tTA. Anessential feature of any inducible system is low pro-

Fig. 5. Luciferase activity in parental LNCaP, PC-3, and Tsu-Pr1 cells transiently transfected with pUHC 13-3 (a) or pRL-CMV (b).Transfections of previously untransfected cells were carried out in the absence of tet, and luciferase assays were performed 2 days lateras described in Materials and Methods.

172 Gschwend et al.

moter activity in the repressed state. In LNCaP cellsexpressing tTA, background luciferase activity in thepresence of tet was almost indistinguishable from in-strumental background, especially in some clones sta-bly transfected with the tTA-inducible reporter plas-mid pUHC 13-3 (Fig. 7b). Yet removal of tet yieldedvery high luciferase activity in tTA-expressing LNCaPcells transiently or stably transfected with pUHC 13-3.These results are similar to the initial observations ofGossen and Bujard [5] in Hela cells. The concentrationof tet that yielded maximal inhibition of luciferasegene transcription in LNCaP cells, 1mg/ml, had noeffect on cell growth (data not shown). Others havereported that mice given 200 mg/ml tet in their drink-ing water exhibited no apparent distress [22]. Similarrepression and inducibility of the tet-repressible pro-moter was observed in the double stable-transfectedLNCaP clones after several months in culture. Thesefeatures make the tTA system an attractive tool forgene expression studies in LNCaP cells in vitro as wellas in vivo.

Several clones of PC-3 and Tsu-Pr1 cells that stablyexpressed the transactivator protein were obtained.When transiently transfected with pUHC 13-3, allPC-3 and Tsu-Pr1 clones exhibited much more back-

ground luciferase activity in the presence of 1mg/mltet than did LNCaP cells. Among the clones with thelowest background, Tsu-Pr1 clones expressed about 20times more background than LNCaP clones, and PC-3clones expressed about 400 and 20 times more back-ground than LNCaP and Tsu-Pr1 clones, respectively(Fig. 3). The high backgrounds limited induction ofexpression by removal of tet to 9.5-fold in PC-3 cells(clone PC-3GK16) and 237-fold in Tsu-Pr1 cells (cloneTsuGK27).

It has been shown previously that use of high con-centrations of tTA-inducible reporter plasmids in tran-sient transfection experiments can cause elevatedbackground activity [25]. Stable transfection of boththe tTA plasmid, pUHD 15-1, and pUHC 13-3 yieldeda somewhat lower background than transient trans-fection of pUHC 13-3 in LNCaP and PC-3 cells, butinduction of luciferase activity was still limited toabout 16-fold in PC-3 clones (Fig. 8a, compare to Fig.3b). That the background was not reduced further bystable incorporation, relative to transient transfection,of pUHC 13-3 may be due to the fact that we used lowamounts of pUHC 13-3 in transient transfections.

We examined several parameters that might influ-ence background activity in different cell lines.

Fig. 6. Expression of luciferase mRNA in parental LNCaP, PC-3,and Tsu-Pr1 cells transiently transfected with the tTA-induciblereporter plasmid pUHC 13-3. Transfections of previously untrans-fected cells were carried out in the absence of tet, and totalcellular RNA was isolated 2 days later as described in Materialsand Methods. Twenty micrograms of total RNA from each cellclone were incubated with antisense RNA probes correspondingto luciferase and HARP mRNAs. RNase protection assay was per-formed as described in Materials and Methods. a: Lanes 1–3 areuntransfected LNCaP, PC-3, and Tsu-Pr1 cells, respectively.

Lanes 4–6 are LNCaP, PC-3, and Tsu-Pr1 cells, respectively, tran-siently transfected with pUHC 13-3. Lane 7, yeast transfer RNA(negative control); lane 8, undigested luciferase probe; lane 9,undigested HARP probe. Sizes of 1-kb ladder fragments (GIBCOBRL) are indicated at right. b: Phosphorimager-quantified lucifer-ase RNA levels divided by corresponding HARP RNA levels. Num-bers were normalized by setting the value for pUHC 13-3-transfected PC-3 cells (lane 5) equal to 41.0, for comparison withFigure 5a. ND, not detectable.

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Among the tTA-transfected clones shown in Figure 3,no correlation was observed between tTA mRNA lev-els and, when transfected with pUHC 13-3, luciferaseactivity in the presence of tet (Fig. 4). Thus, the higherbackground in PC-3 and Tsu-Pr1 cells is not likely dueto higher expression of the transactivator protein inthose cells. Increasing the concentrations of tet ordoxycycline did not reduce further the backgroundluciferase activity in double stable-transfected PC-3cells, although the antibiotic doses were limited bytoxicity to the cells (Fig. 8a). In fact, 10mg/ml of doxy-cycline, which inhibited cell growth by about 47%,yielded higher luciferase activity per mg protein thanthe lower doses. Since this dose also resulted in alower amount of protein per cell, the higher luciferaseactivity may reflect greater stability of luciferase activ-ity relative to the predominant proteins in PC-3 cells.Coincubating tet with verapamil, which may inhibitp-glycoprotein-mediated extrusion of tet from PC-3cells, also did not lower luciferase activity in thesecells more than did tet alone. The results with vera-pamil are difficult to interpret, since treatment withverapamil alone resulted in increased luciferase activ-ity in double stable-transfected PC-3 cells. Further ex-

periments are necessary to determine if verapamilstimulates expression from the tet-repressible pro-moter, or if it exerts a specific effect on the luciferaseenzyme. Nevertheless, these data together suggestthat inability to repress tTA was not a major cause ofthe high background in tTA- and double stable-transfected PC-3 cells. This is supported further by thefinding that background luciferase activity in PC-3and Tsu-Pr1 cells transfected with the tTA-induciblereporter plasmid pUHC 13-3 did not depend on thepresence of the transactivator protein tTA. Rather, pre-viously untransfected PC-3 and Tsu-Pr1 cells, whentransiently transfected with pUHC 13-3, expressed atleast as much luciferase activity as the tet-repressedtTA transfected clones (Fig. 5a, compare to + tet barsof Fig. 3). The relative luciferase activities amongpUHC 13-3-transfected LNCaP, PC-3, and Tsu-Pr1 inFigure 5a are similar to those of Figure 3 (+ tet bars),except for the lower difference between pUHC 13-3-transfected and -untransfected LNCaP in Figure 5acompared to Figure 3. It is not likely that the differentluciferase activities shown in Figure 5a are due to dif-ferences in transient transfection efficiencies, sincetransient transfection with the hCMV promoter/

Fig. 7. Induction of luciferase activity in double stable-transfected LNCaP cells. LNCaP cells stably transfected with tTAplasmid pUHD 15-1 and the tTA-inducible reporter plasmidpUHC 13-3 were maintained in complete medium containingG418, hygromycin, and 1 µg/ml tet. Cells (2 × 105 per well) wereplated in 6-well plates in complete medium containing 1 µg/ml tet.Beginning 24 hr later, tet was removed at different times by wash-

ing the cells twice and adding complete medium without tet (threewells per time point). Cells were lysed 5 days after plating, andluciferase assays were carried out as described in Materials andMethods. a: Time course of induction of luciferase activity aftersubtracting instrument background. Dashed lines indicate times toreach 10% and 50% of maximal activity. b: Bar graph of instrumentbackground and time points 0 (+ tet) and 72 hr (− tet) from a.

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enhancer-controlled reporter vector pRL-CMVyielded similar levels of luciferase activity in all threecell lines (Fig. 5b). The data presented in Figure 5a,bprovide strong evidence, supported by the data in Fig-ures 4 and 8, that most or all of the background lucif-erase activity from pUHC 13-3 in PC-3 and Tsu-Pr1cells is independent of tTA.

The differences in luciferase activity among thethree cell lines transiently transfected with pUHC 13-3in the absence of tTA were reflected, for the most part,by differences in luciferase mRNA levels (Fig. 6, com-pare to Fig. 5a). However, the differences in luciferaseRNA levels were lower than the differences in lucif-erase activity levels by 4.6-fold (PC-3/Tsu-Pr1), 17-fold (PC-3/LNCaP), and 3.7-fold (Tsu-Pr1/LNCaP).Thus, firefly luciferase mRNA may be more efficientlytranslated, or the luciferase protein may be morestable in PC-3 cells than in LNCaP and Tsu-Pr1 cells.However, the higher background in PC-3 and Tsu-Pr1cells appears to be due mainly to either higher trans-activator-independent transcription from the induc-ible promoter or higher stability of the luciferasemRNA in those cells relative to LNCaP. The formerhypothesis seems more likely and could be due to thepresence in PC-3 and possibly Tsu-Pr1 cells of endog-

enous transactivating elements that can stimulatetranscription from the hCMV promoter without its en-hancer. Although such transactivators could be pri-marily present in PC-3 and Tsu-Pr1 cells, it is alsopossible that they were incorporated via viral trans-formation at a later time. Such viral transformationswith E1A and E1B adenoviral elements were sug-gested for human embryonic kidney (HEK 293) cells[10], which showed elevated background activitywhen transfected with this system. In this regard, theobserved differences also suggest profound geneticand molecular differences between LNCaP, PC-3, andTsu-Pr1 cells. Whether these differences are due todifferent origins of the cell lines or to genetic transfor-mations that have occurred since the establishment ofthese lines remains to be determined.

CONCLUSIONS

These studies demonstrate the efficiency of a tet-repressible promoter system in human prostate can-cer cell lines. The establishment of tight regulation oftransgene expression in the LNCaP human pros-tate cancer cell line will facilitate studies on func-tions and interactions of genes involved in cell-death

Fig. 8. Luciferase activity in double stable-transfected PC-3 cells.PC-3 cells stably transfected with tTA plasmid pUHD 15-1 and thetTA-inducible reporter plasmid pUHC 13-3 were maintained incomplete medium containing G418, hygromycin, and 1 µg/ml tet.Cells (2 × 105 per well) were plated in 6-well plates in completemedium containing 1 µg/ml tet. Twenty-four hours later, all cells

were washed twice with drug-free medium, and the medium wasreplaced with complete medium containing the indicated amountsof (a) tet or doxycycline (dox), or (b) verapamil or tet + verap-amil (three wells per treatment). Cells were lysed 3 days after drugadditions, and luciferase assays were carried out as described inMaterials and Methods.

Inducible Gene Expression in PC Cell Lines 175

pathways (death genes, antisense RNAs to growth-stimulatory genes). In contrast, incomplete suppres-sion of promoter activity resulting in elevated back-ground activity was observed in two well-establishedandrogen-insensitive prostate cancer cell lines. Themechanism for the incomplete repression of promoteractivity by tet likely involves activation of the pro-moter by endogenous factors. It is likely that the highbackground activity will impair the usefulness of thetet-repressible system for expression of growth-inhibitory or apoptosis-inducing genes in PC-3 cells.Screening multiple clones of Tsu-Pr1 cells stably trans-fected with the tTA and inducible vectors may yieldclones with a background that is low enough for suchstudies.

ACKNOWLEDGMENTS

We thank Dr. Warren D.W. Heston for many help-ful discussions. These studies were supported in partby NIH grant DK/CA 47650 and the Edwin Beer Pro-gram at the New York Academy of Medicine. J.E.G.was supported by the Deutsche Forschungsgemein-schaft (Bonn, Germany).

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