[CANCER RESEARCH 59, 1378–1385, March 15, 1999]

Glucocorticoid-induced Cell Death Requires Autoinduction of GlucocorticoidReceptor Expression in Human Leukemic T Cells1

Jyoti Ramdas, Wei Liu,2 and Jeffrey M. Harmon3

Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799

ABSTRACT

In contrast to the negative autoregulation of glucocorticoid receptor(GR) expression seen in most cells and tissues, GR expression is positivelyautoregulated in human leukemic T cells and in other cells sensitive toglucocorticoid-induced cell death. To determine whether positive autoreg-ulation is a necessary component of glucocorticoid-induced cell death, awild-type GR gene under the control of a tetracycline-regulated promoterwas stably transfected into glucocorticoid-resistant cells lacking endoge-nous functional receptor. Transfectants grown in the presence of tetracy-cline contained about 15,000 receptors/cell, a value approximately equal tobasal level GR expression in glucocorticoid-sensitive 6TG1.1 cells beforesteroid treatment. Under these conditions, dexamethasone had a minimaleffect on cell growth, elicited little internucleosomal DNA fragmentation,and induced no cell cycle perturbation. In the absence of tetracycline, GRmRNA and protein expression increased 2–3-fold, and cells expressed48,000 receptors, a level nearly equivalent to that present in 6TG1.1 cellsafter 18 h of autoinduction. Under these conditions, dexamethasone mark-edly inhibited cell growth, caused G1 arrest, and induced significantinternucleosomal DNA fragmentation. These studies therefore suggestthat basal level GR expression is inadequate to mediate glucocorticoid-induced apoptosis in glucocorticoid-sensitive T cells and that positiveautoregulation is a necessary component of this process.

INTRODUCTION

The GR4 is a ubiquitously expressed ligand-dependent transcriptionfactor that regulates the activities of a large number of genes. Ingeneral, studies that have examined the relationship between GRconcentration and biological response have found a direct correlationbetween GR concentration and the magnitude of the response (1). Inparticular, the sensitivity of many lymphoid cell lines to glucocorti-coid-induced growth arrest and apoptosis is directly related to intra-cellular receptor concentration (2–4). Bourgeois and Newby (3)showed that mouse WEHI-7 cells, which contain two functionalcopies of the GR gene, are more sensitive to steroid-induced cell deaththan cells that contain a single copy. Gehringet al.(2) showed a directcorrelation between receptor binding activity and steroid-inducedgrowth inhibition of different clones of S49 cells. More recently,Chapmanet al. (4) demonstrated a correlation between steroid sensi-tivity and the level of GR expression in different clones of receptor-less S49 cells stably transfected with the mouse GR gene. However,the minimum concentration of GR necessary to mediate an apoptoticresponse has not been established. In addition, although there is a

strong correlation between GR concentration and steroid sensitivitywhen clonal cell lines are compared, studies that have attempted tocorrelate GR concentration with therapeutic response in the treatmentof leukemias and lymphomas have proven contradictory (5–11). Thus,additional factors must contribute to determining the sensitivity oflymphoid cells to steroid-induced lymphocytolysis.

We have previously shown that in contrast to the negative autoreg-ulation of GR expression seen in most cells and tissues, the hGR ispositively autoregulated in glucocorticoid-sensitive human leukemic6TG1.1 cells (12). In response to glucocorticoid treatment, hGRmRNA and protein increase several-fold during the first 24 h ofsteroid treatment. These increases appear to be the result of increasedtranscription of the hGR gene and precede the earliest evidence ofglucocorticoid-induced growth arrest and cell death (13). Positiveautoregulation of GR expression has also been confirmed in otherclonal cell lines derived from human leukemic CEM cells (14, 15) aswell as glucocorticoid-sensitive mouse S49 cells (16). In addition, oftwo human myeloma cell lines established from the same patient, thehGR was positively autoregulated in the cell line that was glucocor-ticoid sensitive, but not in the one that was glucocorticoid resistant(17). Thus, positive autoregulation of GR expression may be a nec-essary component of glucocorticoid-induced growth arrest and celldeath.

To investigate the role of positive autoregulation of hGR expressionin steroid-induced growth arrest and cell death, we have introduced afunctional copy of the hGR gene under the control of a tetracycline-regulated promoter into glucocorticoid-resistant ICR27TK.3 cells.ICR27TK.3 cells areDGR/GR753Fand were derived from the glu-cocorticoid-sensitive cell line 6TG1.1, whose genotype isGR1/GR753F(18). The mutant hGR present in these cells is activationdeficient; although it can repress activator protein 1 activity, it cannotinduce transcription from the mouse mammary tumor virus promoteror mediate steroid-induced growth arrest or cell death (19, 20). Usinga stably transfected cell line that expresses a functional hGR genewhose expression is regulated by the concentration of tetracycline inthe growth medium, we show that steroid treatment of transfectedcells that express a concentration of GR equivalent to that present in6TG1.1 cells before steroid treatment elicits only a small decrease incell growth and induces little cell death. In contrast, steroid treatmentof transfected cells that express a concentration of GR more equal tothat seen in 6TG1.1 cells 18–24 h after steroid treatment results inincreased growth arrest and substantial apoptosis, suggesting thatbasal levels of hGR expression are inadequate to mediate an apoptoticresponse and that positive autoregulation of hGR expression mayprovide a mechanism to amplify the hormonal signal in cells pro-grammed to die in response to steroid treatment.

MATERIALS AND METHODS

Plasmids. Plasmid pZeoSV was obtained from Invitrogen (Carlsbad, CA).pZeo was constructed by removing the 622-bpBamHI/ClaI fragment ofpZeoSV containing the SV40 promoter and polyA addition signal sequenceand replacing it with a linker sequence containingXhoI and PvuII sites.Plasmids pUHD10-3 and pUHD15-1 neo were generous gifts of Drs. HermannBujard (Zentrum fur Molekulare Biolgie der Universitat Heidelberg, Heidel-berg, Germany) and Steven Reed (Scripps Research Institue, La Jolla, CA) (21,

Received 11/2/98; accepted 1/21/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by USPHS Grant CA32226 awarded by the National Cancer Institute toJ. M. H. The opinions or assertions contained herein are the private ones of the authors andare not to be construed as official or reflecting the views of the Department of Defense orthe Uniformed Services University of the Health Sciences.

2 Present address: Laboratory of Cellular Biology, National Institute on Deafness andOther Communication Disorders, NIH, Rockville, MD 20850.

3 To whom requests for reprints should be addressed, at Department of Pharmacology,Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Be-thesda, MD 20814-4799. Phone: (301) 295-3248; Fax: (301) 295-3220; E-mail:jharmon@usuhs.mil.

4 The abbreviations used are: GR, glucocorticoid receptor; hGR, human GR; HA,influenza hemagglutinin; EMSA, electrophoretic mobility shift assay; RT-PCR, reversetranscription-PCR.

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22). pRShGRHA17 containing three repeats of the HA epitope was con-structed by subcloning the 1-kbSalI/ClaI fragment of pRShGRa (23)containing the region of the NH2-terminal domain of the hGR (C245 to T272)against which the antipeptide anti-GR antibody 710 was raised (18) intoSalI/ClaI-digested pBluescript SK1 (Stratagene, La Jolla, CA). UniqueXbaI andSacI sites flanking the immunogenic sequence were introduced intothe internal 467-bpStyI fragment by PCR amplification with mutant oligonu-cleotides, and the 1-kbSalI/ClaI fragment of the resulting plasmid was used toreplace the corresponding fragment of pRShGRa to create pGRM. Fouroverlapping, complimentary synthetic oligonucleotides (59-TTCCTT-CTAGAAGGAAACTCGAATGAGGACTACCCATACGACGTCCCAGAC-TACGCT-39, 59-ACTACCCATACGACGTCCCAGACTACGCTTAC-CCATACGACGT CCCAGACTACGCTTACCCATACGACGTCCCA-GACTACGCTACACTGCCCC-39, 59-GACGTCCCAGACTACGCTA-CACTGCCCCAAGTGAAAACAGAAAAAGAAGATTTCATCGAG-CTCTGCACCCC-39, and 59-AGGGGTGCAGAGCTCGATGAAATCTTC-39) encoding three copies of the HA epitope (YPYDVPDYA) were annealed;after filling the gaps with the Klenow fragment of DNAPolI and digestionwith XbaI andSacI, they were used to replace the 149-bpXbaI/SacI fragmentof pGRM to create the hGR expression plasmid pRShGRHA17. The GRprotein containing the HA epitope was fully active in the ligand-dependentinduction of pMSG-CAT and the repression of273 COL-CAT when trans-fected into COS-7 cells as described previously (19). ptetGRa was constructedby amplification of the entire coding region of the GR gene from pRShGRa

with forward primer 59-ACAACACCGCGGATATTCACTGATG-GACTCC-39and reverse primer 59-CGGAATTATTCTTACCAACGGAAA-GATCTACAACA-39, digestion withSacII andXbaI, and insertion intoSacII/XbaI-digested pUHD10-3. The sequence of the GR gene was confirmed bycomplete double-strand sequencing. Transient cotransfection of pUHD15-1neo, ptetGRa, and pMSG-CAT into CV-1 cells confirmed that the GR gene inptetGR was functional and under the control of thetetO sequence; growth inthe presence of 1mg/ml tetracycline repressed chloramphenicol acetyltrans-ferase activity by.50% (data not shown). Finally, pZeotetGRHA was con-structed by replacing the 1-kbSalI/ClaI fragment of ptetGRa with the corre-sponding fragment of pRShGRHA17 and inserting the 3456-bpXhoI/PvuIIfragment of the resulting plasmid intoXhoI/PvuII-digested pZeo.

Cell Culture and Transfection. The glucocorticoid-sensitive T-cell line6TG1.1 and its glucocorticoid-resistant derivative, ICR27TK.3, have beendescribed previously (24). Cells were maintained in RPMI 1640 containing10% fetal bovine serum and grown at 37°C in a humidified atmospherecontaining 5% CO2 as described previously (24). The human B-cell line IM-9was grown under the same conditions. Cell number was determined with aCoulter Counter (model ZM; Coulter Electronics, Inc., Hialeah, FL). For stableand transient transfections, cells were harvested during log-phase growth bycentrifugation at 2003 g for 10 min at 4°C and resuspended in ice-coldelectroporation medium (RPMI 1640) at a concentration of 83 106 cells/ml.Five million cells (0.7 ml) were transferred to a precooled electroporationcuvette (4-cm gap; Bio-Rad Laboratories, Hercules, CA) to which 10mg oflinearized plasmid were added. After incubation for 10 min at 4°C, electropo-ration was performed with a Bio-Rad Genepulser apparatus (with capacitanceextender) at 340 V and 960mF. Electroporated cells were incubated at 4°C for10 min and then transferred to 10 ml of culture medium composed of a 1:1mixture of fresh and conditioned medium containing 10% fetal bovine serum(JRH Biosciences, Lenexa, KS) preequilibrated for 24 h at 37°C in a humid-ified atmosphere containing 5% CO2. The cells were allowed to recover for24 h before the addition of 1 mg/ml Geneticin (Life Technologies, Inc.,Gaithersburg, MD) and the selection of resistant clones by limiting dilution in96-well microtiter plates at 1000 cells/well. Alternatively, when cells wereselected for zeocin resistance, they were plated in 250mg/ml zeocin (Invitro-gen) at a concentration of 0.2 cells/ml.

EMSA. A 42-bp double-stranded oligonucleotide containing the 19-bpinverted repeat sequence of thetetO gene was constructed as describedpreviously (21) and labeled with [g-32P]dATP and T4 polynucleotide kinase.Preparation of nuclear extracts and DNA binding assays were performed asdescribed previously (21).

RT-PCR. Total cell RNA was purified using RNAzol (Tel-Test, Friend-swood, TX) according to the directions of the manufacturer. RT-PCR wasperformed using the Gene-Amp RNA PCR Kit (Perkin-Elmer Corp., FosterCity, CA). First-strand synthesis was performed in 20ml of 10 mM Tris-HCl

buffer (pH 8.3) containing 5 mM MgCl2, 50 mM KCl, 1 mM of each de-oxynucleotide triphosphate, 2.5mM random hexamers, 1 unit of RNase inhib-itor, 2.5 units of murine leukemia virus reverse transcriptase, and 1mg of totalRNA. The reaction was incubated at room temperature for 10 min, at 42°C for15 min, at 99°C for 5 min, and at 5°C for 5 min in a Perkin-Elmer ThermalCycler model 480. Amplification was performed by adding MgCl2 to a finalconcentration of 2 mM and adding sufficient buffer to maintain a concentrationof 50 mM KCl. Primers A (59-TCGAATGAGGACTACCCATACGAC-39), B(59-TCCAGGAAAGCTTGCCTGACAGTA-39), and C (59-ATGGAGATCT-GGTTTTGTCAAGCCC-39) were added to a final concentration of 0.15mM,and AmpliTaq DNA Polymerase (2.5 units; Perkin-Elmer Corp.) was added toinitiate the reaction. After denaturation at 95°C for 3 min and 30 s, amplifi-cation was accomplished by 35 cycles of incubation at 95°C for 30 s, 55°C for30 s, and 72°C for 1 min, followed by a final extension at 72°C for 7 min. PCRproducts were purified using the QIAquick-spin column (Qiagen, Valencia,CA), and 200 ng of each product were electrophoresed at 6 V/cm in a 1.5%agarose gel in 13 Tris-borate EDTA containing 0.5mg/ml ethidium bromide.To quantify the amount of PCR product, DNA was visualized with a UVtransilluminator and photographed using Polaroid type 55 film. Negatives werescanned with a LKB Ultrascan laser densitometer.

Analysis of hGR Protein. The binding of [3H]dexamethasone to intactcells was performed as described previously (24). For immunoblotting, totalcell protein was extracted and solubilized as described previously (13). Protein(50mg) was electrophoresed in an 8% SDS-polyacrylamide gel and transferredto a 0.45mm Protran nitrocellulose membrane (Schleicher and Schuell, Keene,NH), and the filter was incubated overnight at 4°C in TTBS buffer [20 mM

Tris-HCl (pH 7.6) containing 137 mM NaCl and 0.1% Tween-20] containing5% nonfat milk. The filter was washed twice at room temperature for 15 minin 300 ml of TTBS, incubated for 2 h at room temperature in TTBS containinga 1:1000 dilution of anti-hGR antibody 710 (18) or anti-HA epitope antibodyHA.11 (Berkeley Antibody Co., Richmond, CA), washed three times in 300 mlof TTBS, and then incubated in TTBS containing a 1:2000 dilution of donkeyantirabbit IgG conjugated to horseradish peroxidase. Filters were incubatedwith equal portions of luminol and hydrogen peroxide for 1 min, followed byexposure to Hyperfilm (Amersham Pharmacia Biotech, Arlington Heights, IL).For quantification of chemiluminescence, filters were processed using a Bio-Rad Model GS-250 Molecular Imager and Molecular Analyst software.

Flow Cytometry. Cells were harvested by centrifugation at 2003 g for 10min at room temperature and fixed for flow cytometric analysis by a modifi-cation of the procedure of Gorczycaet al.(25). The cell pellet was resuspendedin 1 ml of ice-cold PBS by gentle tapping and transferred to a 1.5-ml microfugetube. After centrifugation at 3203 g for 2 min at 4°C, the pellet wasresuspended in PBS containing 2% paraformaldehyde and incubated at 4°C for15 min. Fixed cells were washed twice with cold PBS, resuspended in 70%ethanol, and stored overnight at220°C. After two washes with cold PBS, thepellet was resuspended in PBS containing 200mg/ml RNase A, 50mg/mlpropidium iodide, and 0.6% NP40 and stored overnight at 4°C. Flow cytom-etry was performed using an Epics Elite Flow Cytometer (Coulter ElectronicsInc.). Data were collected using the Elite program and analyzed using Multi-cycle AV (Phoenix Flow Systems, Inc., San Diego, CA).

RESULTS

Hormone-independent Regulation of hGR Expression.In glu-cocorticoid-sensitive CEM cells, there is a hormone-dependent in-crease in both hGR mRNA and protein before the onset of growtharrest and cell death when cells are exposed to steroid (12, 13). Toexamine the role of autoregulation of hGR expression on glucocorti-coid-induced growth arrest and apoptosis of cultured T cells, a cellline was constructed in which the concentration of the receptor couldbe regulated in a hormone-independent manner. Accordingly, thetetracycline-inducible expression system of Gossen and Bujard (21)was used to express HA-tagged hGR in glucocorticoid-resistantICR27TK.3 cells. The resistance of these cells to steroid-induced celldeath is the consequence of a deletion of one hGR gene and amutation in the other (L753F) that renders it activation-deficient andunable to mediate glucocorticoid-induced growth arrest or cell death

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(Refs. 18 and 26; Fig. 1). Plasmid pUHD15-1 neo, which expressesthe chimeric tTA transactivator protein, was transfected intoICR27TK.3 cells, and neomycin-resistant clones were screened fortTA expression by EMSA using thetetOsequence as a probe and byassayingb-galactosidase activity in cells that were transiently trans-fected with the tetracycline-responsive reporter plasmid pUHC16-3.Cells that expressed the highest level of tTA (as measured by EMSA)and in whichb-galactosidase expression from a transiently transfectedtTA-regulated, reporter plasmid was repressed by growth in mediumcontaining tetracycline (data not shown) were then transfected withplasmid pZeotetGRaHA17 that expresses the HA-tagged GR down-stream of a heptameric repeat of thetetO sequence and a minimaleukaryotic promoter (phCMV9-1) and also contains a gene conferringresistance to zeocin. Cells resistant to both Geneticin and zeocin wereassayed for dexamethasone-induced growth inhibition in the absenceor presence of tetracycline. The majority of drug-resistant clonesshowed greater steroid-induced growth inhibition in the absence oftetracycline. Several of these clones were assayed for hGR expressionin the absence or presence of tetracycline, and clone CEMtetGRHAwas chosen for further study.

The ability of tetracycline to regulate hGR mRNA expression inCEMtetGRHA cells was determined by RT-PCR (Fig. 2). Forwardprimers A and C, which are specific for the transfected HA-taggedhGR gene and the endogenous mutantGR753Fgene, respectively,and reverse primer B, which hybridizes to both genes, were used toamplify mRNA transcribed from the endogenous mutant and trans-fected HA-tagged hGR genes. The 147-bp product from the endoge-nous mutantGR753Fgene is present in samples prepared from bothparental ICR27TK.3 and transfected CEMtetGRHA cells and servesas an internal control for the efficiency of the amplification. Incontrast, the 204-bp product from the transfected gene is present onlyin CEMtetGRHA cells (Fig. 2B). More importantly, growth ofCEMtetGRHA cells in 1mg/ml tetracycline for 4 days resulted in a50% reduction in the amount of mRNA from the transfected gene,demonstrating tetracycline-regulated expression of hGR mRNA inCEMtetGRHA cells.

To examine the regulation of hGR protein, extracts of total cellprotein prepared from parental ICR27TK.3 and transfected CEMtet-

GRHA cells were analyzed by immunoblotting with anti-hGR anti-body 710 or anti-HA antibody HA.11. The results (Fig. 3B) showedthat antibody 710 reacts with the 98-kDa hGR protein present in thehuman B-cell line IM-9 that contains approximately 105 receptors/cell(27) and the glucocorticoid-sensitive T-cell line 6TG1.1, which, in theabsence of steroid, expresses both the mutant L753F hGR protein andapproximately 15,000–20,000 normal receptors/cell (18). Antibody710 also reacted with the mutant hGR (L753F) protein present in bothparental ICR27TK.3 and transfected CEMtetGRHA cells. The amountof immunoreactivity was essentially equal in extracts prepared fromthese two cell lines and was approximately half that present in extractsprepared from glucocorticoid-sensitive 6TG1.1 cells, consistent withthe fact that the wild-type gene is deleted in these cells and indicatingthat there is no reaction of antibody 710 with the HA-tagged GR. Incontrast, antibody HA.11 reacted exclusively with HA-tagged GR in

Fig. 2. Tetracycline represses hGR mRNA expression in CEMtetGRHA cells.A,forward primers A and C (which are specific for the HA-tagged and endogenous mutantGR genes, respectively) were used in combination with reverse primer B for RT-PCR ofhGR mRNA expressed from the transfected wild-type and endogenous mutant hGR genes.B, RT-PCR based quantification of HA-tagged hGR mRNA. Total RNA prepared fromuntransfected ICR27TK.3 cells(Lane 2)or CEMtetGRHA cells grown for 4 days in thepresence(Lane 3)or absence(Lane 4)of 1 mg/ml tetracycline was amplified by RT-PCRas described in “Materials and Methods.”Lanes 1and5 contain a 100-bp ladder and aDNA mass ladder, respectively.

Fig. 1. Inducible expression of HA-tagged hGR in ICR27TK.3 cells. A tetracycline-based inducible expression system was used to express HA-tagged GR in glucocorticoid-resistant ICR27TK.3 cells. Plasmid pUHD15-1 neo expressing the chimeric transactivatortTA (21) was introduced by electroporation, and stable Geneticin-resistant transfectantswere selected as described in “Materials and Methods.” Plasmid pZeotetGRaHA17,which expresses the HA-tagged GR downstream of a heptameric repeat of the tet operator(tetO) sequence and a minimal eukaryotic promoter (phCMV9-1), was then introduced,and stable transfectants were selected in medium containing zeocin. One zeocin-resistanttransfectant (CEMtetGRHA) was chosen for analysis of the role of GR autoinduction inglucocorticoid-induced T-cell apoptosis.

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transfected CEMtetGRHA cells (Fig. 3C); there was no reaction witheither mutant L753F or wild-type protein in ICR27TK.3 cells or in6TG1.1 cells.

The addition of as little as 0.05mg/ml tetracycline to the growthmedium resulted in a 50–75% reduction in the amount of immuno-reactive HA-tagged hGR protein expression (Fig. 4). However, in-creasing the concentration of tetracycline did not elicit a furtherreduction in the amount of hGR protein expressed. Thus, regulation ofhGR expression from the promoter that contains the heptameric repeatof the tetO sequence occurs over a very narrow range of drug con-centration. In addition, even at 1mg/ml tetracycline, it is not possibleto fully repress hGR expression.

To measure the intracellular concentration of receptor, [3H]dexa-methasone binding assays were performed on intact cells. Under theconditions of this assay, there is virtually no binding of [3H]dexam-ethasone to mutant L753F receptors (Ref. 28; data not shown).CEMtetGRHA cells grown in the absence of tetracycline expressedapproximately 48,000 receptors/cell (Fig. 5). In contrast, cells grownin 1 mg/ml tetracycline expressed only 15,000 binding sites/cell (Fig.5). This decrease was comparable to the decrease in immunoreactiveprotein seen by immunoblotting. More importantly, the concentration

of active hGR in CEMtetGRHA cells grown in the presence andabsence of tetracycline corresponds well with the amount of func-tional hGR present in glucocorticoid-sensitive 6TG1.1 cells beforeand 18 h after glucocorticoid treatment. Thus, tetracycline regulationof hGR expression in CEMtetGRHA cells is a suitable system forevaluation of the role of positive hGR autoregulation in glucocorti-coid-induced growth arrest and cell death.

Effect of Receptor Concentration on Glucocorticoid-inducedGrowth Arrest and Cell Death. To ascertain the role of receptorconcentration on glucocorticoid-induced growth arrest, CEMtet-GRHA cells cultured in the absence or presence of 1mg/ml tetracy-cline were grown for 4 days in the absence or presence of 1mM

dexamethasone. Determination of the cell number revealed that cellsgrown in the presence of tetracycline (15,000 receptors/cell) experi-enced a partial growth inhibition in response to steroid. This wasgreater than that seen after the treatment of steroid-resistantICR27TK.3 cells but was substantially less that seen after steroidtreatment of CEMtetGRHA cells grown in the absence of tetracycline(48,000 receptors/cell), which experienced a 75% reduction in growth(Fig. 6). Tetracycline itself had no effect on either hGR expression orcell growth (data not shown). Thus, there is a direct correlationbetween receptor concentration and the steroid-induced inhibition ofcell growth.

The effect of receptor concentration on cell cycle arrest and celldeath was investigated by flow cytometric analysis of cells cultured inthe presence or absence of tetracycline and then grown for 48 h in theabsence or presence of 1mM dexamethasone. Growth of steroid-resistant ICR27TK.3 cells in 1mM dexamethasone had no effect on

Fig. 4. Tetracycline regulation of hGR protein expression. Extracts (50mg of protein)prepared from untransfected ICR27TK.3 cells(Lane 1)or CEMtetGRHA cells grown for4 days in the absence(Lane 2), or presence(Lanes 3–6)of increasing amounts oftetracycline (0.05–1.0mg/ml) were fractionated by SDS-PAGE, and the HA-tagged hGRwas visualized with antibody HA.11 as described in “Materials and Methods.”

Fig. 3. Identification of HA-tagged hGR.A, schematic representation of the replace-ment of the 28-residue portion of the NH2-terminal domain of the hGR against whichantibody 710 was raised with three copies of the HA antigen. Although not indicated, T272

is present in the final construct.B andC, protein (50mg) isolated from IM-9, 6TG1.1,ICR27TK.3, or CEMtetGRHA cells was fractionated by SDS-PAGE, and the hGR wasvisualized using antibody 710(B) or HA.11 (C) as described in “Materials and Methods.”The positions of wild-type GRa and of HA-tagged GR are indicated byarrowsto therightof each panel.

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cell cycle distribution, and no apoptotic cells were detected, as evi-denced by the absence of subdiploid nuclei (Fig. 7). Similarly, steroidtreatment of CEMtetGRHA cells grown in the presence of tetracycline(a condition approximating the initial hGR concentration in steroid-sensitive 6TG1.1 cells) resulted in only a small decrease in theproportion of cells in S phase, with no substantial increase in thenumber of cells with a subdiploid amount of DNA. In contrast, steroidtreatment of CEMtetGRHA cells grown in the absence of tetracycline(a condition approximating the concentration of hGR in 6TG1.1 cellsafter 24 h of steroid treatment) elicited both a decrease in the per-centage of S-phase cells and a substantial number of apoptotic cells(Fig. 7). A comparable result was obtained when apoptosis wasassessed by examining chromatin fragmentation; there was substan-tially more internucleosomal DNA fragmentation in steroid-treatedcells grown in the absence of tetracycline than in cells cultured in thepresence of tetracycline (Fig. 8).

When growth arrest and cell death were examined in steroid-sensitive 6TG1.1 cells in which the hGR is autoinduced (12, 13), theeffects of steroid treatment were similar to those seen in CEMtet-GRHA cells grown in the absence of tetracycline (Figs. 7 and 8); therewas a substantial decrease in the percentage of S-phase cells, a markedincrease in internucleosomal DNA fragmentation, and an increase inthe percentage of dead cells with less than a G1 content of DNA. Thus,glucocorticoid-induced growth arrest and cell death seem to requirehGR concentrations comparable to those seen after receptor autoin-duction.

DISCUSSION

In most cells and tissues, the GR is negatively autoregulated byboth transcriptional and posttranscriptional mechanisms (29, 30).Such negative autoregulation is consistent with the negative feed-back loops characteristic of many endocrine systems. However, wehave previously shown that hGR mRNA and protein are positivelyautoregulated in glucocorticoid-sensitive 6TG1.1 cells at the levelof transcription (13). Positive autoregulation has been observed inother cells programmed to die in response to steroids (16, 17, 31),suggesting that the increase in GR could provide a mechanism foramplifying the hormonal signal in cells destined to undergo apop-tosis. To investigate the role of positive autoregulation in steroid-induced cell death, the hGR gene, under the control of a tetracy-cline-regulated promoter, was stably transfected into cells that lack

functional GR. Glucocorticoid treatment of cells grown in theabsence of tetracycline (induced) resulted in the accumulation ofcells in the G1 phase of the cell cycle, the inhibition of celldivision, and increased chromatin fragmentation. In each case, theresponse was qualitatively similar to the response seen in 6TG1.1cells in which expression of the hGR gene is controlled by its ownpromoter. Because untreated 6TG1.1 cells express approximatelythe same amount of hGR ligand binding activity (15,000 receptors/cell) as CEMtetGRHA cells grown in the presence of tetracycline(24) and because the 2–3-fold increase in hGR protein and ligandbinding activity seen after the removal of tetracycline is compara-ble to the 3– 4-fold increase in hGR protein seen after steroidtreatment of autoinducible 6TG1.1 cells (12, 13), we conclude thatautoinduction is an essential component of steroid-induced celldeath in human leukemic 6TG1.1 cells.

One possible complication to the interpretation of these results isthat in replacing the epitope used to generate anti-hGR antibody 710with a trimer of the HA epitope, the activity of the GR was reduced,thereby requiring increased expression of the HA-tagged receptor toachieve growth arrest and cell death. Two factors suggest that this isnot the case:(a) the region of the GR that was replaced is downstreamfrom the strong transactivating function (t1) present in the NH2-terminal domain of the hGR (32, 33); and(b) more importantly,transient transfection of the HA-tagged GR gene into COS-7 cellsgave the same level of glucocorticoid induction of pMSG-CAT andthe same level of glucocorticoid repression of the activator protein1-responsive reporter plasmid273 COL-CAT as did wild-type GR(data not shown).

Although positive autoregulation of GR expression is seen in sev-eral cell lines sensitive to glucocorticoid-induced cell death, it is notsimply an in vitro phenomenon restricted to such cells. Positiveautoregulation of GR expression is also seen in several regions of thebrain, indicating that it is not a sufficient condition for steroid-inducedcell death (34). There has been only limited characterization of theorganization and regulation of the hGR promoter (35–39). Conse-quently, the mechanism(s) that underlies the differential autoregula-tion of hGR expression is unclear. However, differential autoregula-tion of the closely related androgen and mineralocorticoid receptorshas also been observed (40–42), suggesting that differential tissue-specific autoregulation may be a feature common to other members ofthe nuclear receptor family.

Recently, it has been reported that glucocorticoid-resistantCEM-C1 cells can be rendered sensitive to glucocorticoid-induced

Fig. 5. Tetracycline regulation of [3H]dexamethasone binding activity in CEMtet-GRHA cells. CEMtetGRHA cells were grown for 4 days in medium containing variousconcentrations of tetracycline. Ligand binding activity was determined as described in“Materials and Methods.”

Fig. 6. Effect of hGR concentration on cell growth. ICR27TK.3 or CEMtetGRHA cellsgrown in the absence (2) or presence (1) of 1 mg/ml tetracycline(tet) were treated with1 mM dexamethasone for 4 days. Results are expressed as the number of cells indexamethasone-treated cultures as a percentage of untreated controls.

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cell death by overexpression of rat GR (43). However, we havepreviously shown that CEM-C1 cells expressing approximately thesame basal level of GR as glucocorticoid-sensitive 6TG1.1 cellsexhibit the same level of positive hGR autoregulation as their glu-cocorticoid-sensitive counterparts (13). In addition, it was shown thatboth CEM-C1 cells and murine SAK8 cells could be rendered stably

sensitive to glucocorticoid-induced apoptosis by treatment with 5-aza-cytidine (44, 45), presumably as a result of the demethylation ofglucocorticoid-regulated genes involved in the lytic response. Thus, itis probable that the relative sensitivity of various cells and cell linesto glucocorticoid-induced growth arrest and cell death reflects abalance between the amount of GR present and the sensitivity of the

Fig. 7. Effect of hGR concentration on dexa-methasone-induced cell cycle arrest. Glucocorti-coid-resistant ICR27TK.3 cells (A andB), CEMtet-GRHA cells grown in the presence (C and D) orabsence (Eand F) of 1 mg/ml tetracycline, orglucocorticoid-sensitive 6TG1.1 cells (Gand H)were grown in the absence (A, C, E, and G) orpresence (B, D, F,andH) of 1 mM dexamethasonefor 48 h. Cells were stained with propidium iodideand analyzed by flow cytometry as described in“Materials and Methods.” The percentage of parti-cles with a subdiploid content of DNA is indicatedin each panel.

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hormonally responsive genes involved in the lytic response to regu-lation by GR.

In the absence of tetracycline, relatively high levels of hGR ex-pression were observed. However, the growth of stable transfectantsin the presence of tetracycline did not result in complete repression ofhGR expression. hGR mRNA, protein, and ligand binding activitywere all reduced to approximately 30–50% of the amount seen in theabsence of antibiotic. The reason for this high level of repressedexpression is unclear. However, when CV-1 cells were transientlytransfected with a tTA expression vector, a hGR-responsive reporterplasmid, and atetO-containing hGR expression vector, a similarinability to fully repress hGR activity was observed (data not shown).Thus, the high level of repressed hGR expression in CEMtetGRHAcells grown in the presence of tetracycline is not a T cell-specificphenomenon or a function of the site of integration. Rather, it seemsto be a function of the strong modified cytomegalovirus promoterused to achieve high level expression in the absence of tetracycline.Significant levels of repressed expression have been seen in othersystems as well (46) and probably account for the small amount of cellcycle perturbation and chromatin fragmentation seen after steroidtreatment of CEMtetGRHA cells grown in the presence of tetracy-cline.

Unlike other stimuli that induce programmed cell death, glucocor-ticoid-induced lymphocytolysis requires ongoing protein synthesis(20, 47–49), suggesting that either the synthesis of new protein or thecontinuous presence of a labile protein is required. We have recentlyshown that the labile inhibitory factor IkBa is induced by glucocor-ticoids in 6TG1.1 cells (20). Induction of this factor and the accom-panying repression of nuclear factorkB activity could contribute tosteroid-induced cell death. In addition, the results presented here

suggest that the synthesis of new hGR protein is also required. Thus,the requirement for new protein synthesis could reflect, in part, therequisite autoinduction of hGR expression. If this is the case, thenthere may be more than one component whose induction is requiredfor steroid-induced cell death.

We have demonstrated that autoinduction of hGR expression is anecessary component of glucocorticoid-induced cell death in humanleukemic CEM cells in culture. Studies examining the relationshipbetween hGR concentration and clinical response in human leukemiccells obtained directly from patients have not found the same strongpositive correlation between receptor concentration and response seenfor estrogen receptors in breast cancer (5–11). However, these studiesexamined intracellular hGR concentration in the absence of hormone.Thus, if autoinduction of hGR expression is a necessary component ofthe therapeutic response, it is possible that the absence of a strongcorrelation between receptor concentration and clinical response isdue to the measurement of basal levels rather than induced levels ofhGR expression.

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Fig. 8. Effect of hGR concentration on dexamethasone-induced internucleosomal DNAfragmentation. Glucocorticoid-sensitive 6TG1.1 cells(Lanes 1and 2), glucocorticoid-resistant ICR27TK.3 cells(Lanes 3and4), or CEMtetGRHA cells grown in the absence(Lanes 5and6) or presence(Lanes 7and8) of 1 mg/ml tetracycline were cultured in theabsence(Lanes 1, 3, 5,and7), or presence(Lanes 2, 4, 6,and8) of 1 mM dexamethasonefor 48 h. Internucleosomal chromatin fragmentation was assessed by agarose gel electro-phoresis as described in “Materials and Methods.”Lanes M1andM2 contain 100-bp and1-kb DNA ladders, respectively.

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1999;59:1378-1385. Cancer Res   Jyoti Ramdas, Wei Liu and Jeffrey M. Harmon  CellsGlucocorticoid Receptor Expression in Human Leukemic T Glucocorticoid-induced Cell Death Requires Autoinduction of

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