Molecular and Cellular Endocrinology, 83 ( 1992) 153- 17 1 0 1992 Elsevier Scientific Publishers Ireland, Ltd. 0303.7207/92/$05.00

MOLCEL

153

02689

Methotrexate-induced overexpression of functional glucocorticoid receptors in Chinese hamster ovary cells Deborah

L. Bellingham

‘,I, Madhabananda

Sar ’ and John A. Cidlowski

a,b

” Department of Biochemistry and Biophysics, ” Department of Physiology, and ’ Department of Cell Biology and Anatomy, Lineherger Cancer Cell Biology Program, University of North Carolina, Chapel Hill. NC 27599, USA (Received

Key words: Glucocorticoid

receptor

(human);

22 August

Receptor

1991; accepted

overexpression;

18 October

Chinese

1991)

hamster

ovary cells; Methotrexate

Summary We have used a modified cotransfection and selection strategy to create a series of mammalian cell lines that stably express high levels of intact glucocorticoid receptors. These cell lines were produced by subjecting Chinese hamster ovary (CHO) cells, which had been previously cotransfected with a glucocorticoid-responsive dihydrofolate reductase (DHFR) gene and the human glucocorticoid receptor gene, to growth in increasing concentrations of methotrexate (MTX). By linking the MTX selection process to glucocorticoid receptor function via the DHFR gene, stable cell lines resistant to a range of MTX concentrations (50 nM to 3 PM) were isolated that were strictly dependent upon glucocorticoids for growth. Quantitation of steroid binding capacity in MTX-resistant cells revealed a progressive increase in the number of glucocorticoid receptors as a function of increasing MTX concentration. This increase in receptor content was maximal at the highest level of MTX resistance examined (3 I_LM MTX) and represented a 25fold elevation in glucocorticoid receptor number relative to CHO cells expressing only endogenous hamster receptor. The increases in steroid binding obtained after MTX selection were reflected by similar increases in the level of glucocorticoid receptor protein as determined by immunoblot analysis. Examination of glucocorticoid receptor structure by sucrose density gradient centrifugation revealed that oligomeric (9 S> steroid receptor complexes were formed at all levels of receptor expression. Subcellular localization of the glucocorticoid receptor protein by immunocytochemical staining revealed effective nuclear translocation of the overexpressed receptors in MTX-resistant cells. Functional transfection studies using a glucocorticoid-responsive reporter gene indicated that the additional glucocorticoid receptors in CHO cells were competent to activate transcription. To determine the molecular basis for the MTX-induced increases in functional glucocorticoid receptors, steady-state levels of glucocorticoid receptor mRNA were examined. MTX selection produced a 5 to 7-fold increase in transfected glucocorticoid receptor gene expression relative to untreated cells. MTX-resistant cells also expressed increased levels of a putative hamster glucocorticoid receptor mRNA species. Interest-

Correspondence to: Dr. John A. Cidlowski, CB #7545 460 Medical Science Research Bldg., University of North Carolina, Chapel Hill, NC 27599-7545, USA. Tel. (919) 966-1523; Fax (919) 966-6927. ’ Current address: Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75235-9050, USA. Supported by National Institutes of Health Grant DK 32460.

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ingly, the observed increases in receptor gene expression in these cells could not be accounted amplification of either the human or the hamster glucocorticoid receptor genes.

Introduction Glucocorticoid hormones produce and coordinate a diverse array of physiological responses. These effects are mediated through the actions of a specific intracellular receptor protein, which functions as a highly specialized transcription factor. The glucocorticoid receptor, as well as other members of the steroid hormone receptor family, acquires an increased affinity for nuclear acceptor sites only after binding to cognate ligand (Evans, 1988; reviewed in Carson-Jurica et al., 1990). As a direct consequence of hormone binding, the glucocorticoid receptor dissociates from a large, heteromeric complex that probably contains a dimer of the 90-kDa heat shock protein (Mendel et al., 1986; Denis et al., 1987) and as recently suggested, one or more additional heat shock-related proteins (Sanchez et al., 1990a). Ultimately this structural alteration converts the receptor into a DNA-binding protein, which interacts with specific DNA sequences termed glucocorticoid response elements (GREs) and results in the activation or repression of transcription initiation from nearby promoters (Yamamoto, 1985; Burnstein and Cidlowski, 1989; Beato, 1991). Although significant advances have been made toward achieving a complete understanding of the structure. function and regulation of the glucocorticoid receptor, detailed studies have been complicated by the low intracellular abundance of steroid receptors. A number of approaches have recently been used to overcome this limitation. Procaryotic expression systems have been used to generate large quantities of the glucocorticoid receptor protein (Dahlman et al., 1989; Freedman et al., 1989); however, to date, only truncated forms of the receptor have been successfully overexpressed in Escherichia coli. Receptor derivatives have also been expressed in yeast but efforts to obtain high level expression of full-length glucocorticoid receptors have yielded proteins that were largely insoluble (Schena and

for by

Yamamoto, 1989; Wright et al., 1990). Recently Thompson and coworkers succeeded in obtaining large quantities of full-length glucocorticoid receptor protein using the baculovirus expression system (Srinivasan and Thompson, 1990). Although the recombinant protein was structurally intact and bound to GRE-containing DNA in gel shift assays, transcriptional enhancing activity of baculovirus-expressed glucocorticoid receptors was hormone independent (Tsai et al., 1990). Several mammalian cell systems have been created in an effort to obtain high level expression of otherwise rare, endogenous receptor proteins (Bellingham and Cidlowski, 1989; Israel and Kaufman, 1989; Hirst et al., 1990; Alksnis et al., 1991). These systems, which are based upon the use of amplifiable selection markers such as dihydrofolate reductase (DHFR) or metallothionein, can efficiently direct the overexpression of a heterologous gene following cotransfection of host cells and selection for drug or heavy metal resistance (reviewed in Bebbington and Henschel, 1987). While these strategies have been used to achieve high level expression of steroid receptors, a potential limitation arises from the fact that the selection process is not directly linked to the expression of normal, functional receptors. Several recent reports have indicated that such methods can produce receptors that display aberrant behavior (Kushner et al., 1990; Sanchez et al., 1990b). We have taken an alternative approach to ensure the overproduction of intact, functional glucocorticoid receptors in mammalian cells. By specifically cotransfecting a glucocorticoid-responsive DHFR gene into DHFR-deficient CHO cells together with the gene encoding the human glucocorticoid receptor, cell survival becomes dependent upon the production of functional glucocorticoid receptors. Using this linked cotransfection strategy, we recently obtained a cell line that expressed 5 times more receptor than nontransfected control cells and displayed receptor features that were indistinguishable from endoge-

155

nous hamster glucocorticoid receptors (Bellingham and Cidlowski, 1989). We have now extended this approach to create stable cell lines with even higher levels of glucocorticoid receptors by employing stepwise selection with increasing amounts of methotrexate (MTX). Overexpressed glucocorticoid receptors were structurally and functionally intact as defined by several independent criteria. Interestingly, the MTX-induced increases in receptor levels were not due to amplification of either the human or the hamster glucocorticoid receptor genes but rather to increased expression of transfected human receptor genes as well as apparently silent hamster glucocorticoid receptor genes. Materials

and methods

Materials [6,7-“HlDexamethasone (48.2 Ci/mmol), lz51staphylococcal Protein A (33.2 mCi/mg) and [ “C]chloramphenicol (60 mCi/mmol) were obtained from Du Pont-New England Nuclear (Boston, MA, USA). Dexamethasone was from Steraloids (Wilton, NH, USA). RU38486 was kindly provided by Dr. R. Deraedt, Roussel UCLAF (Romainville, France). Methotrexate was obtained from Lederle Laboratories (Carolina, PR, USA). Acetyl coenzyme A, DEAE-dextran and unlabeled nucleotides were obtained from Pharmacia LKB Biotechnology (Piscataway, NJ, USA). Biotrans nylon membrane was from ICN (Irvine, CA. USA) and nitrocellulose was obtained from Schleicher and Schuell (Keene, NH, USA). [a“*P]UTP (600 Ci/mmol) and [(y-32P]dCTP (3000 Ci/mmol) used to prepare hybridization probes were both obtained from ICN Radiochemicals (Irvine, CA, USA). Riboprobe reaction components (T3 RNA polymerase, RNAse-free DNase, reaction buffers), T4 DNA polymerase, and restriction enzymes were from Bethesda Research Laboratories (BRL). Random primed DNA labeling reactions were performed using a kit that was purchased from Boehringer-Mannheim (Indianapolis, IN, USA). Relative quantitation of bands on autoradiographs was obtained using a GS3000 scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA, USA). Epitope purified rabbit antiserum No. 57, which is an

antipeptide antibody directed against amino acids 346-367 from the amino-terminal portion of the human glucocorticoid receptor, was prepared as previously described (Cidlowski et al., 1990). Acrylamide, bisacrylamide, ammonium persulN, N, N ‘, N ‘-tetramethylethylenediamine fate, (TEMED), sodium dodecyl sulfate (SDS), Tris, sucrose, and agarose were from BRL. Prestained molecular mass standard proteins were also from BRL. Protease inhibitors (phenylmethylsulfonyl fluoride and aprotinin), dimethyl sulfoxide (DMSO) and all other reagent grade chemicals were obtained from Sigma Chemical Co. (St Louis, MO, USA). Cell culture and MTX selection strategy The parental Chinese hamster ovary cell line (MG/hGR) used in these studies was obtained following stable cotransfection with a plasmid containing a modified, glucocorticoid-responsive DHFR gene and a plasmid encoding the human glucocorticoid receptor cDNA. The isolation and characterization of this cell line have been described in detail previously (Bellingham and Cidlowski, 1989). To obtain further overexpression of glucocorticoid receptor, a stepwise methotrexate (MTX) selection scheme was employed. The first round of MTX selection was performed on MG/hGR cells by the inclusion of 50 nM MTX and 5 nM dexamethasone in the culture medium. A resistant colony was obtained (MG/ hGR/ MTXSO) and preliminary characterization of glucocorticoid receptor levels in MTXSO cells indicated that this strategy would be an effective method to overexpress receptor. MTXSO cells were therefore subjected to further stepwise increases in MTX in the continued presence of 5 nM dexamethasone. Following each increase in MTX (250, 750 nM, 1.5, 3 PM), resistant colonies were removed from the culture dish by trypsinization when they reached an average diameter of 2-3 mm. Single colony isolates were then replated in selective medium and allowed to reach confluence several times prior to increasing the concentration of MTX. The MTX-resistant cell lines selected for the most detailed study are designated MTXSO, MTX250, MTX750 and MTX3000, consistent with their level of MTX resistance. All MTX-resistant lines were grown in

monolayer culture in a-minimal essential medium (MEM) lacking nucleosides but supplemented with 8% dialyzed fetal calf serum, 2 mM glutamine, penicillin (100 U/ml> and streptomycin (75 U/ml), 5 nM dexamethasone and the indicated concentration of MTX. Nontransfected CHO cells (denoted DG44) (Urlaub et al., 1983) were included in several experiments as a control for endogenous hamster glucocorticoid receptor. These cells were grown in monolayer culture in complete MEM as previously described (Bellingham and Cidlowski, 1989). A CHO cell line that contains one copy of the DHFR gene was kindly provided by Dr. Thea Tlsty (University of North Carolina, Chapel Hill, NC, USA). These cells (denoted CHO) were maintained in monolayer culture in MEM lacking nucleosides. HeLa S, cells were grown in monolayer culture in Joklik’s minimal essential medium as previously described (Allgood et al., 1990). Cells were counted in a hemocytometer to obtain cell numbers. Quantitation of glucocorticoid receptor numbers MTX-resistant CHO cells were grown for 16 h prior to harvest in MEM lacking exogenous glucocorticoid (but otherwise supplemented as described above). This ‘washout’ procedure was previously employed to displace radioinert dexamethasone and allows efficient radiolabeling of receptor (Bellingham and Cidlowski, 1989). Control CHO cells (MG/hGR) and HeLa cells were grown as described above and were also subjected to a medium change 16 h prior to harvest. Cells were removed from culture plates by incubation in phosphate-buffered saline (PBS) containing 5 mM EDTA for 5 min at 37°C followed by scraping with a rubber policeman. After washing in PBS, cells were collected by centrifugation at 600 xg for 5 min. Equivalent cell numbers were resuspended in 20 mM Tris-HCI pH 7.4, 2 mM EDTA, 20 mM sodium molybdate, 2 mM P-mercaptoethanol, 10% glycerol to a final cell density of 3 x 10’ cells/ml. Cells were sonicated at 4°C with three 10 s bursts and cytosols prepared by centrifuging lysates at 100,000 X g for 1 h at 4°C. Duplicate aliquots (135 ~1) of cytosol (0.1-0.2 mg protein) were incubated for 2.5 h at 0 “C with 50 nM [‘Hldexamethasone. Parallel samples containing ‘H-ligand plus 10 PM unla-

beled dexamethasone were used to determine nonspecific binding, which was never more than 10% of total binding. Free hormone was removed from all samples by adding cytosol to the pellet obtained from centrifugation of an equal volume of a dextran-coated charcoal suspension (1% activated charcoal, 0.1% dextran in 1.5 mM MgCI,). The cytosol/charcoal suspension was vortexed, incubated on ice for 5 min, centrifuged at 10,000 xg for 5 min and 125 pi aliquots were analyzed for radioactivity content by scintillation counting. Saturably bound [ -?H]dexamethasone was determined from the difference between the average total binding ([‘Hldexamethasone) and the nonspecific binding ([ ‘Hldexamethasone plus unlabeled dexamethasone). This value was then used to calculate [ ‘Hldexamethasone binding capacity. Cytosol protein was determined by the method of Bradford (1976). Gel electrophoresis and immunoblot analysis Prior to electrophoretic analysis of receptor protein, control and MTX-resistant cell lines were grown for 12-16 h in fresh MEM lacking exogenous glucocorticoid to displace radioinert dexamethasone. Cells were harvested by scraping, washed in PBS and pelleted as described above. Whole cell lysates were prepared by resuspending cell pellets in lysis buffer (10 mM sodium phosphate pH 7.2, 150 mM NaCl, 1% sodium deoxycholate, 1% Nonidet P-40 (NP-401, 0.1% SDS, 0.5 mM phenylmethylsulfonyl fluoride, and 1 pg/pI aprotinin) at a density of 3 x 10’ cells/ml, essentially as described by Sambrook et al. (1989). Lysates were vortexed briefly and then centrifuged at 15,000 X g for 30 min at 4°C to remove insoluble debris. Supernatants were transferred to fresh microfuge tubes, mixed with an equal volume of 2 x sample buffer (20 mM TrisHCl pH 7.5, 2 mM EDTA, 2% SDS, 10% sucrose, 20 pg pyronin Y tracking dye/ml) (Fairbanks et al., 1971), heated at 100°C for 2 min, and stored at - 70°C. Prior to electrophoresis, protein concentrations were determined by Bradford assay (Bradford, 1976). Samples (200 pg) were resolved by electrophoresis through 7.5% polyacrylamide gels (Fairbanks et al., 1971) and then electroblotted to nitrocellulose according to the method of Towbin et al. (1979). To visualize

glucocorticoid receptor protein, immunoblot analysis was performed. Membranes were incubated with blocking buffer (10 mM Tris-HCl pH 7.4, 10% nonfat dry milk, 0.05% Tween-20, 0.9% NaCl) for 2-3 h at room temperature followed by overnight incubation at 4°C with epitope-purified, anti-glucocorticoid receptor antibody No. 57 at a dilution of 1: 100. This antibody is directed against a 22 amino acid sequence located on the aminoterminal side of the DNA-binding domain of the human glucocortoid receptor and has been described in detail previously (Cidlowski et al., 1990). Following incubation with antibody, filters were briefly washed in blocking buffer at room temperature and then incubated with 5 PCi ‘251conjugated Protein A in 10 ml blocking buffer for 2 h at room temperature. Filters were again washed in blocking buffer followed by several washes in blocking buffer without nonfat milk, air dried and exposed to X-ray film at -70°C. Sucrose gradient ultracentrifugation

MTX-resistant cells were grown in MEM lacking exogenous glucocorticoid for 14 h prior to harvest to displace radioinert dexamethasone as described above. Medium was removed, monolayers were washed with unsupplemented MEM and cells were harvested by scraping in PBS containing 5 mM EDTA. Cells were washed with additional PBS and resuspended in unsupplemented MEM to a final cell density of 3 X 10’ cells/ml. Cells were then incubated with 60 nM [3H]dexamethasone for 2.5 h at 0°C with gentle agitation. Cells were collected by centrifugation at 600 X g for 5 min and resuspended in 20 mM Tris-HCl pH 7.4, 2 mM EDTA, 20 mM sodium molybdate, 2 mM /?-mercaptoethanol, 10% glycerol. Cytosolit extracts were prepared by sonicating cells with 3 x 10 s pulses at 4°C and centrifuging cell lysates at 100,000 X g for 1 h at 4°C to remove insoluble debris. Free hormone was removed from all samples by dextran-coated charcoal treatment as described above. Aliquots (175 ~1) of [3H]dexamethasone-labeled cytosols were layered on 520% (w/v) linear sucrose gradients prepared in 20 mM Tris-HCl pH 7.4, 2 mM EDTA, 20 mM sodium molybdate, 2 mM P-mercaptoethanol and 10% glycerol. Gradients were centrifuged at 190,000 X g for 16 h at 3°C in a Beckman SW50.1

rotor. Ten drop fractions were collected and analyzed for radioactivity content. The sedimentation positions of several protein standards (myoglobin (2 S), transferrin (4.9 S), alcohol dehydrogenase (7.4 S)) were determined in companion gradients. Immunocytocbemical

procedures

Control (MG/hGR) and MTX-resistant CHO cells (MTXSO, MTX750, MTX3000) were plated in two-chamber glass slides in complete MEM and cultured for 3 days at 37°C. The immunocytochemical staining procedure used was essentially as described in Cidlowski et al. (19901. Briefly, after fixation in 2% paraformaldehyde, slides were washed in PBS and permeabilized in 0.2% Triton-X for 20 min. After an additional wash in PBS, cells were treated with 2.0% normal goat serum, washed in PBS, and then incubated with epitope-purified anti-glucocorticoid receptor antibody No. 57 (1: 7500) for 20 h at 4°C. The cells were washed in PBS followed by incubation with biotinylated goat anti-rabbit IgG (1 : 400) for 1 h at room temperature. Immunoreactive proteins were visualized by incubating cells in avidin-biotin-peroxidase (1: 400) for 1 h followed by treatment with a diaminobenzidine-hydrogen peroxide solution for 10 min. Determination of CAT activity

To evaluate directly the ability of glucocorticoid receptors to activate transcription in CHO cells following MTX-induced overexpression, cells were transfected with a reporter plasmid containing the mouse mammary tumor virus (MTV) promoter linked to the chloramphenicol acetyltransferase (CAT) gene. This plasmid (pGMCS) was provided by Dr. Keith Yamamoto (University of California, San Francisco, CA, USA). Transient transfections were performed by the DEAE-dextran method as modified by Lopata et al. (1984). Briefly, cells were plated in duplicate into lo-cm dishes at a density of 1-3 x 10h cells/dish in complete MEM. The following day, medium was replaced with MEM lacking exogenous glucocorticoid for an additional 14 h. 10 pg pGMCS reporter plasmid and 5 pg of RSV P-galactosidase control plasmid (pRSV-P-gal, kindly provided by Dr. William Rutter, University of California, San Francisco, CA, USA) were mixed with

15X

DEAE-dextran and incubated with cells for 4 h at 37°C. After incubation with the DNA/DEAEdextran mixture, cells were washed and exposed to 10% DMSO for 90 s. Complete MEM with or without 100 nM dexamethasone was then added to each group and cells were incubated for 18 h at 37°C. Cells were harvested in PBS and assayed for CAT activity essentially as described by Gorman et al. (1982). Typically 30 pg of cell extract was used per reaction, which was incubated for 3 h at 37°C. [‘“ClChloramphenicol and its acetylated forms were separated by thin layer chromatography in a solvent of CHClJmethanol (95 :5). After autoradiography, the amount of CAT activity in each sample was assayed by excising radioactive spots from silica gel plates and counting in Scintiverse (Fischer Scientific Co.). Relative levels of conversion were calculated after the transfection efficiencies of all cell lines had been determined. These values were determined by calorimetric assay of p-galactosidase activity (An et al., 1982) produced by the control plasmid pRSV-P-gal and were essentially identical for all lines tested. Northern blot analysis Total RNA was isolated from control and MTX-resistant cell lines by lysis in 4 M guanidinium thiocyanate and centrifugation through a cushion of cesium chloride (Chirgwin et al., 1979). 20-40 pg samples of RNA were denatured in glyoxal and DMSO and size fractionated on 1% agarose gels essentially as described by Sambrook et al. (1989). After electrophoresis, RNA was transferred to Biotrans nylon membrane for 1824 h and immobilized by ultraviolet light KJV> crosslinking (UV Stratalinker 1800, Stratagene). Filters were then hybridized by standard methods with the following ‘*P-labeled cRNA probes: human glucocorticoid receptor antisense RNA or chick p-actin antisense RNA. These cRNA probes were generated from the dual promoter vector pT7/T3-18 (Bethesda Research Laboratories) containing either the human glucocorticoid receptor cDNA (Giguere et al., 1986) or the chick p-actin cDNA, according to the procedure recommended by the supplier. After hybridization, membranes were extensively washed at 70°C and exposed to X-ray film.

Glucocorticoid receptor and DHFR gene copy number analysis Analysis of DHFR and glucocorticoid receptor gene copy numbers was performed on genomic DNA samples according to the method of Brown et al. (1983). Genomic DNA was extracted from all cell lines by lysis in SDS as described by Sambrook et al. (1989). DNA samples were then denatured in sodium hydroxide, neutralized and applied to nitrocellulose (0.5 pg/well) using a slot-blot manifold. Prior to hybridization with either DHFR or glucocorticoid receptor-specific probes, all DNA samples were normalized to the intensity of the hybridization signal obtained from an actin gene probe. A genomic titration curve was generated with increasing amounts of plasmid DNA containing either DHFR or human glucocorticoid receptor genes, which were denatured and applied to nitrocellulose as described above. Identically loaded membranes were then hybridized with either a randomly primed, ‘*Plabeled DHFR DNA fragment (3000 bp EcoRI/ fst I fragment) or a human glucocorticoid receptor cDNA fragment (2500 bp SalI/XhoI fragment). Following autoradiography, the intensities of the hybridizing bands were quantified by scanning densitometry. Results MTX-resistant CHO cells require glucocorticoids for growth We have previously shown that cotransfection of CHO cells with a plasmid containing a glucocorticoid-responsive DHFR gene and a plasmid encoding the human glucocorticoid receptor cDNA results in stable expression of intact human glucocorticoid receptors in mammalian cells (Bellingham and Cidlowski, 1989). Using this linked strategy, a stable cell line was obtained that expresses 5 times more glucocorticoid receptors than nontransfected CHO cells. When this cell line, denoted MG/hGR, was selected for resistance to a low concentration of methotrexate (MTX), preliminary observations indicated that resistance was associated with approximately a lo-fold higher level of glucocorticoid receptors relative to CHO cells that had not been transfected with receptor.

159

Based on these findings, we extended the MTX selection strategy in a stepwise fashion to generate cell lines that express progressively higher levels of glucocorticoid receptors. More specifically, MTX selection was performed on MG/ hGR cells by the addition of the following doses of MTX to the culture medium: 50, 250, 750 nM, 1.5. 3 FM MTX. Clonal lines were isolated at each step and allowed to achieve stable growth prior to increasing the concentration of MTX. The MTX-resistant cell lines selected for the most detailed study are designated MTXSO, MTX250, MTX750 and MTX3000. Since glucocorticoid receptor-driven expression of DHFR was designed to be a critical determinant of cell survival, selections for MTX resistance were performed in the presence of 5 nM dexamethasone. To examine directly whether MTX-resistant cells depend on glucocorticoid for survival, these lines were subjected to growth in MTX in either the absence or presence of 5 nM dexamethasone. Cell densities were evaluated after 7 days of growth by staining with crystal violet. As seen in Fig. 1 (Control), there is a substantial amount of growth when MTXSO, MTX250, MTX750, and MTX3000 cell lines were propagated under control conditions (i.e. in medium containing the indicated concentration of MTX and 5 nM dexamethasone). However, when these cells were withdrawn from 5 nM dexamethasone, cell growth was completely inhibited (Fig. 1, DEX Withdrawn). This growth arrest was observed in all lines tested, suggesting that cotransfected cells retain a strict dependence on glucocorticoids for survival with each increase in MTX. To evaluate whether this dependence is the result of a glucocorticoid receptor-mediated event, MTX-resistant cells were plated into control medium containing the indicated concentration of MTX and 5 nM dexamethasone and were then treated with the glucocorticoid antagonist RU486 (Fig. 1, 5 nM DEX + RU486). When 1 PM RU486 was present in the medium, cell growth was markedly reduced in all MTX-resistant lines. This result supports the conclusion that cell survival in MTX is mediated via a glucocorticoid receptor-dependent process. There does appear to be slightly more cell growth in the groups that were treated with RU486 relative to those that were simply

5 nM (Control)

DEX Withdrawn

5 nM DEX + RU486 (1 pM)

MTX750

Fig. 1. MTX-resistant cell lines are dependent on exogenous glucocorticoids for growth. Stepwise selections for MTX resistance were performed on stably cotransfected CHO cells as described in Materials and methods. After expansion of single colony isolates, a series of MTX-resistant cell lines (MTX.50, MTX250, MTX750, MTX3000) were plated into 6-well cluster plates at a density of 2 X lo4 cells/well and allowed to attach overnight in control medium. Control medium is defined as medium that contains the indicated concentration of MTX and 5 nM dexamethasone. After cell attachment, control medium was removed from all wells and was replaced with medium containing either the indicated concentration of MTX and 5 nM dexamethasone (Control), medium containing the indicated concentration of MTX but lacking dexamethasone (DEX Withdrawn), or medium containing the indicated concentration of MTX, 5 nM dexamethasone and 1 PM RU486 (5 nM DEX + RU486). Media were changed in all wells every 2-3 days. After 7-9 days of treatment, cell densities were assessed by fixation in 10% phosphate-buffered formalin and staining with 0.2% crystal violet as previously described (Bellingham and Cidlowski, 1989). The staining patterns shown are representative of the results obtained in three independent experiments.

withdrawn from dexamethasone (Fig. 1, DEX Withdrawn vs. 5 nM DEX + RU486). This effect may be due to partial agonist activity of RU486, which has been reported by others (SchweizerGroyer et al., 1988). Together, these growth inhibition studies suggest that all MTX-resistant cells display an absolute dependence on functional glucocorticoid receptors.

160

Quantitation of glucocorticoid receptor number in MTX-resistant cell lines Based on this physiological linkage between survival in MTX and glucocorticoid receptor function, is increased MTX resistance related to

TABLE

I

QUANTITATION OF [‘HIDEXAMETHASONE BINDING CAPACITY IN CONTROL AND MTX-RESISTANT CHO CELLS Cotransfected CHO cells (MG/hGR). MTX-resistant CHO cells (MTX50, MTX250, MTX750, MTX3000) and control HeLa S, cells were grown in various media as described in Materials and methods. Media were changed in all flasks 16 h prior to harvest and were replaced with either control medium (MG/hGR, HeLa S,) or medium lacking exogenous glucocorticoids (MTX-resistant cells) in order to displace radioinert dexamethasone. Single-point hormone binding assays were performed by incubating duplicate aliquots (I35 ~1) of cytosol for 2.5 h at 0°C with 50 nM [‘Hldexamethasone. Parallel samples containing ‘H-ligand plus 10 PM unlabeled dexamethasone were used to determine nonspecific binding, which was never more than 10% of total binding. After removal of unbound hormone, 125 ~1 aliquots were analyzed for radioactivity content by scintillation counting. Saturably bound [‘Hldexamethasone was determined from the difference between the average total binding ([3H]dexamethasone) and the nonspecific binding ([‘Hldexamethasone plus unlabeled dexamethasone). This value was then used to calculate the number of [“Hldexamethasone binding sites in each cell line. Cell line

DG44 ’ MG/hGR MTX50 MTX250 MTX750 MTX3000 HeLa S,

[3H]Dexamethasone

binding

sites

fmol receptor/ mg protein a

Receptors/ cell h

100 351- 364 530- 586 1,135 931- 941 1,090-1,165 132- 169

6,600 30,100 80,900 141,200 125,000 174,700 22,000

” Range of values obtained from two independent experiments performed in an identical manner. Each binding reaction typically contained 0.1-0.2 mg protein as determined by Bradford assay (Bradford, 1976). h The number of glucocorticoid receptors per cell in each of the CHO lines was calculated based upon the relative number of [‘Hldexamethasone binding sites per cell obtained for control HeLa S, cells, which were found to contain 20,000-22,000 receptors per cell by Scatchard analysis of binding data (Cidlowski and Cidlowski, 1981). ’ [‘HlDexamethasone binding capacity was determined by Scatchard analysis as previously reported (Bellingham and Cidlowski, 1989).

the actual number of glucocorticoid receptors? There are several cases where it has been demonstrated that the relative magnitude of a particular glucocorticoid response is directly correlated with the level of glucocorticoid receptors (Gehring et al., 1984; Vanderbilt et al., 1987). To determine if there was a corresponding increase in glucocorticoid receptor number after MTX selection, receptors were quantified by single saturation dose binding assays using a concentration of 50 nM [ “Hldexamethasone. Since single-point binding assays yield underestimates of receptor numbers relative to values obtained by Scatchard analysis of multiple-point binding data, HeLa S, cells were used as an internal standard in this assay. Scatchard analysis has previously demonstrated that HeLa S, cells contain approximately 2 X 104 glucocorticoid receptors per cell (Cidlowski and Cidlowski, 1981). The number of glucocorticoid receptors in each of the cell lines examined in the present study was therefore calculated based upon the relative number of [“Hldexamethasone binding sites per cell obtained for HeLa S, cells under identical conditions. Table 1 shows that progressive increases in the level of MTX resistance are associated with increases in the number of [“Hldexamethasone binding sites per cell. The only exception to this trend was observed with MTX7.50 cells, which do not appear to be substantially different in receptor content from MTX2.50 cells. The greatest increase in glucocorticoid receptor number (2.7fold) occurred when MG/hGR cells were subjected to an initial dose of 50 nM MTX. Subsequent increases in MTX resistance were associated with additional 1.4- to 1.7-fold increases in glucocorticoid receptor number. When nontransfected CHO cells (Table 1, DG44) are compared with MTX3000 cells, it is clear that the MTX selection process has produced a 25fold increase in glucocorticoid receptor content over endogenous hamster receptor levels. Immunochemical analysis of glucocorticoid receptor protein in CHO cells after MTX selection In view of the increase in the relative number of [ 3H]dexamethasone binding sites following MTX selection, were these changes reflected by similar increases in the amount of intact gluco-

161

corticoid receptor protein? To address this issue, glucocorticoid receptors were examined by immunoblot analysis. Whole-cell extracts were prepared from MTX-resistant cell lines as well as from control CHO cells and probed with an antipeptide antibody (No. 57) to the human glucocorticoid receptor (Cidlowski et al., 1990). As seen in Fig. 2, there is a highly immunoreactive protein species visible at approximate M, 90,000-94,000 in each of the MTX-resistant lines examined (lanes MTXSO, MTX750 and MTX 3000). For comparison, extracts were also prepared from non-MTX-treated cotransfectants (lane MG/hGR), which contain 2-3 X lo4 glucocorticoid receptors per cell, and from CHO cells (lane DG44*), which express only endogenous hamster receptors. The immunoreactive protein observed at M, 94,000 in cotransfected MG/hGR cells comigrates with a protein species that is saturably bound by [ ‘Hldexamethasone mesylate, which is a covalent affinity label of glucocorticoid receptors (Simons and Thompson, 1981). Moreover, this same immunoreactive species is visible as a faint band in nontransfected CHO cells that contain a low level of endogenous hamster receptors (Fig. 2, DG44* vs. MG/hGR). Based on these results, it appears that the prominent immunoreactive species detected by antibody No. 57 in the MTX-resistant cell lines represents intact glucocorticoid receptor protein.

TABLE

MG/hGR MTX50 MTX250 MTX750 MTX3000

Sucrose gradient analysis of oligomeric glucocorticoid receptor structure

Recent studies have provided evidence to indicate that the macromolecular structure of the

2

SUMMARY Cell line

Comparison of the relative levels of immunoreactive receptor protein in each of the cell lines shown in Fig. 2 indicates that, in general, resistance to higher concentrations of MTX is correlated with increased levels of glucocorticoid receptor protein. There is a marked increase in receptor protein in MTXSO cells but only a modest change in MTX750 cells (Fig. 2 and Table 2). These observations are consistent with the steroid-binding data presented in Table 1. When the concentration of MTX was increased from 750 nM to 1.5 PM (not shown), and then increased to 3 PM, receptor levels continued to increase (Fig. 2 and Table 2). Based on these results as well as those in Table 1, the ability of cotransfected CHO cells to survive in higher doses of MTX seems to correlate reasonably well with increases in the level of glucocorticoid receptor protein. There are two minor immunoreactive bands visible in the MTX-resistant samples (Fig. 2, MTXSO, MTX750, MTX3000), which migrated below the intact glucocorticoid receptor at about M, 68,000 and 42,000. It is likely that these proteins are proteolytic degradation products of the receptor.

OF THE PROPERTIES Receptors/ cell a

30,000 81,000 141,000 125,000 175,000

OF GLUCOCORTICOID

RECEPTORS

IN MTX-RESISTANT

Relative level of receptor protein ’

Sedimentation coefficient (nonactivated)

1.0 5.5 ND 4.0 9.2

-9s -9s -9s ND -9s

CELL LINES Nuclear localization

’

’ _ ++ ND + +++

a Receptor numbers were determined by saturation dose binding assays as described in the legend to Table 1. by scanning densitometry. The amount of receptor is normalized ’ ‘251-labeled bands at _ 94 kDa in Fig. 2 were quantitated relative amount of immunoreactive receptor protein assayed for non-MTX-treated MG/hGR cells. ’ Values obtained under nonactivating conditions as described in Fig. 3. d Immunocytochemical localization of receptors performed under normal growth conditions as described in Fig. 4. ND, not determined in experiment presented.

to the

162

PB-

BSA-

ov-

Fig. 2. Immunoblot analysis of glucocorticoid receptor protein levels in MTX-resistant cell lines. Nontransfected CHO cells (DG44” 1, control cotransfectants (MG/hCR) and MTX-resistant CHO cells (MTXSO, MTX750, MTX3000) were cultured as described in Materials and methods. Whole cell extracts were prepared from all lines, resolved by SDS-gel electrophoresis and transferred to nitrocellulose. Samples were normalized to equivalent protein concentrations (200 pg) prior to loading. To detect immunoreactive glucocorticoid receptor protein, filters were incubated with epitope-purified, anti-human glucocorticoid receptor antibody No. 57 tcidlowski et al., 1990). Antibody signals were detected with ‘z”I-conjugated Protein A and autoradiograph~. Positions of molecular mass standards are indicated to the left of the figure and are as follows: phosphorylase b (PB), 109 kDa; bovine serum albumin (BSA), 71.8 kDa: and ovalbumin (OVt, 45.8 kDa.

nonactivated (non-DNA-binding) glucocorticoid receptor is much more complex than was initially proposed. Unliganded receptors (aporeceptors) appear to contain a variety of associated proteins in addition to the 90-kDa heat shock protein (hsp 90) (Sanchez et al., 1990a), and may be derived from very large heteromeric complexes (Bresnick et al., 1990). We have previously reported that stably transfected human glucocorticoid receptors expressed in MG/hGR cells sediment as _ 9 S complexes on sucrose gradients under nonactivat-

ing conditions in the presence of sodium molybdate (Bellingham and Cidlowski, 1989). This sedimentation profile was identical to that of endogenous hamster glucocorticoid receptors examined under the same conditions. These findings suggest that at least a 5-fold increase in receptor expression can be tolerated by host CHO cells without depleting any receptor-associated factors that are involved in forming nonactivated glucocorticoid receptor complexes. Using transient transfection strategies to achieve high level expression of glucocorticoid receptors, Pratt et al. (1988) have demonstrated that COS-7 monkey cells have the capacity to form a large number of 9 S complexes with the human glucocorticoid receptor. However, since only a small proportion of cells are actually transfected in transient assays it is possible that potentially limiting, receptor-associated factors are furnished by nontransfected cells during cytoso1 preparation. As a result of the unavoidabIe mixing that occurs within heterogenously transfected cell populations, intact oligomeric receptor complexes would be correctly formed under these conditions without knowing whether any receptor-associated factors were truly limiting. To examine the oligomeric structure of stably transfected receptors, we performed sucrose gradient analysis on overexpressing cell lines. Control (MG/hGR) and MTX-resistant CHO cells were incubated with [‘Hldexamethasone under nonactivating conditions and glucocorticoid receptor complexes were analyzed on 520% sucrose density gradients containing sodium molybdate. As seen in Fig. 3, a single peak of radioactivity was detected in all cell lines. Based on the relative positions of several protein standards, the sedimentation coefficient of this peak was calculated to be N 9-9.4 S. This value is in close agreement with the results obtained by several investigators for endogenoug nonactivated glucocorticoid receptor complexes, which are known to be composed of a hormone binding monomer and various heat shock-related proteins (Mendel et al., 1986; Denis et al., 1987). The overall symmetry and the relatively uniform position of each of the peak shown in Fig. 3 suggest that nonactivated glucocorticoid receptor complexes in MTX-resistant cells are structurally identical to

163

the complex present in control MG/hGR cells. These findings imply that the receptor-associated factors involved in forming oligomeric receptor complexes are not limiting in CHO cells overexpressing human glucocorticoid receptors.

0

10

20

30

T

B

Fraction

Number

Fig. 3. Sucrose gradient analysis of glucocorticoid receptors in MTX-resistant cell lines. Control (MG/hGR) and MTX-resistant CHO cells (MTXSO, MTX250, MTX3000) were cultured as described in Materials and methods. Media were changed in all flasks 14 h prior to harvest and were replaced with media lacking exogenous glucocorticoid. This washout procedure displaces radioinert dexamethasone from hormone-treated cells and allows efficient radiolabeling of glucocorticoid receptors. Cells were incubated with 60 nM [7H]dexamethasone for 2.5 h at 0°C and cytosols prepared. After removal of unbound steroid, samples were layered on 5-20% (w/v) linear sucrose gradients containing 20 mM sodium molybdate and centrifuged at 190,000 X g for 16 h at 3°C. Ten drop fractions were collected and analyzed for radioactivity content. Samples are indicated by the following symbols: MG/hGR co), MTXSO (W). MTX250 (A ), MTX3000 (0). Sedimentation markers were analyzed in a companion gradient and are as follows: myoglobin (2 S), transferrin (4.9 S), and alcohol dehydrogenase (7.4 S). Only the position of alcohol dehydrogenase is indicated in the figure. B, bottom of gradient; T, top of gradient.

Subcellular localization of glucocorticoid receptors in MTX-resistant cells In the absence of hormone, glucocorticoid receptors are typically localized to the cytoplasm of target cells. This has been demonstrated in a number of different cell lines and tissues by immunochemical studies using several different anti-glucocorticoid receptor antibodies (Wikstrom et al., 1987; Cidlowski et al., 1990). However, upon exposure to hormone, glucocorticoid receptors are found associated with the nucleus of target cells. Since MTX-resistant CHO cells were selected and grown in the continuous presence of dexamethasone, we wished to examine the subcellular distribution of glucocorticoid receptors in these cells under normal growth conditions. NonMTX-treated MG/hGR cells and various MTXresistant cell lines were therefore subjected to immunocytochemical analysis using the anti-glucocorticoid receptor antibody (No. 57) described above. As seen in Fig. 4 (left panel), incubation of MG/hGR cells with anti-receptor antibody revealed that immunoreactive glucocorticoid receptors are localized to the cytoplasm of CHO cells under normal growth conditions (i.e. in the absence of exogenous hormone). In contrast, MTX selection in the presence of dexamethasone yields additional glucocorticoid receptors that are largely associated with the nucleus (Fig. 4, panels MTXSO, MTX750, MTX3000). Indeed, MTX3000 cells exhibit a highly lobular pattern of glucocorticoid receptor immunoreactivity localized within the nucleus and virtually undetectable staining in the cytoplasm. These findings are consistent with efficient nuclear translocation of overexpressed glucocorticoid receptors in the presence of ligand. Functional activity of glucocorticoid receptors after MTX-induced overexpression Based on phenotypic evidence (Fig. 11, CHO cells selected for increased resistance to MTX are dependent upon functional glucocorticoid receptors for growth. This dependence is presumably linked to a demand for receptor-driven expression of the DHFR gene. However, it is possible that MTX-treatment could select for cells having mutations in the structure and/or function of the overexpressed receptor protein. These

164

cells would be viable simply because DHFR is constitutively expressed. Indeed, the potential difficulty of maintaining the fidelity of heterologous proteins produced after MTX-induced overexpression has been reported (Kaufman and Sharp, 1982). In view of this possibility, we tested the ability of overexpressed glucocorticoid receptors in CHO cells to activate a glucocorti~oid-responsive reporter gene. A plasmid that contains the mouse mammary tumor virus (MTV) promoter linked to the chloramphenicol acetyltransferase gene (CAT), was transfected into control and MTX-resistant ceils. Table 3 shows that DG44 cells displayed only a minimal response (2- to 3-fold) to dexamethasone, which is presumably due to the functional activity of the small number of endogenous hamster glucocorticoid receptors expressed in CHO cells. In contrast, a 2% to 30-fold induction of CAT activity was observed in MG/hGR cells following treatment with dexamethasone. Since these cell lines were transfected with equal efficiencies, the difference in CAT activity between DG44 and MG/hGR cells is likely to be due to the expression of additional glucoc(~rticoid receptors. Moreover, a 37- to 45-fold induction in CAT activity was obtained in MTX3000 cells in response to dexamethasone. This result suggests that MTX-induced overexpression of glucocorti-

MGlhGR

MTX50

TABLE

3

GLUCOCORTICOID-INDUCED CONTROL AND MTX-RESISTANT

CAT ACTIVITY CHO CELLS

IN

Cells were cotransfected with pGMCS reporter plasmid and pRSV P-gal as described in Materials and methods. After removal of DNA, cells were grown for I8 h in the absence or presence of IO0 nM dexamethasone. Cells were then harvested and processed for analysis of CAT activity (Corman et al.. 19821. As a control for transfecti(~l~ efficiencies, lysates were also analyzed for fl-galactosidase activity by calorimetric assay (An et al., 1982) Cell line

CAT activity (induction factor i dexamethasone) Expt.

DG44 MG/hGR MTX3000

3.2 28.5 45.3

1

”

Expt. 2 3.4 30.3 37.7

” Fold induction of CAT activity by dexamethasone was calculated by determining the extent of acetylation of [ ‘~C]chloramphenicol in control and dexamethas[~ne-treated samples. Data are expressed as the ratio of CAT activity in dexamethasone-treated samples to untreated samples obtained in two independent experiments.

coid receptors in CHO cells does not preclude the receptor from functioning as a transcriptional activator. These functional studies are consistent with the apparent structural integrity of the overexpressed receptors as revealed by immunoblot

MTX750

MTX3000

Fig. 4. Immunocytochemical localization of glucocorticoid receptors expressed in MTX-resistant CHO cells. Control (MG/hGR) and MTX-resistant CHO cells (MTXSO, MTX750, MTX3000) were plated in Z-well chamber slides in MEM as described in Materials and methods. h?TX-resistant cells were supplemented with the appropriate concentration of MTX and 5 nM dexamethasone. tmmunoreacti~~ gluc~orticoid receptor protein was detected by incubating cells with anti-glu~ocorticoid receptor antibody and staining with avidin-biotin-peroxidase as previously described (Cidlowski et al., 1990). Magnification: X650.

165

:, A”

,,

_:_

*hamster

*human

GR

GR

Fig. 5. Northern blot analysis of glucocorticoid receptor mRNAs in MTX-resistant cell lines. Total RNA was isolated from nontransfected CHO cells (DG44* ), control cotransfectants (MG/hGR) and MTX-resistant CHO cells (MTXSO, MTX750, MTX3000). Samples (20 pg, with the exception of DG44*, 40 kg) were denatured, separated on a 1% agarose gel. and transferred to a nylon membrane. The filter was hybridized with the following “P-labeled cRNA probes: human glucocorticoid receptor antisense RNA (GR, upper panel) or chick p-actin antisense RNA (Actin, lower panel). The positions of the transfected human glucocorticoid receptor transcript (Human GR) and the endogenous hamster glucocorticoid receptor transcript (Hamster GR) are indicated to the right of the figure. The positions of radiolabeled molecular weight markers are indicated to the left of the figure.

analysis 3).

(Fig. 2) and sedimentation

profiles

(Fig.

Analysis of glucocorticoid receptor gene expression in MTX-resistant cells To determine the molecular basis for the increased production of functional glucocorticoid receptors in MG/hGR-derived cells after selection for MTX resistance, the steady-state levels of glucocorticoid receptor mRNAs in control and MTX-resistant cell lines were analyzed. Total RNA was isolated from MTX.50, MTX750 and MTX3000 cell lines as well as from parent an d control (DG44) CHO cells, and (MG/hGR) subjected to Northern blot analysis (Fig. 5). We have previously observed that an abundant

cells mRNA of - 3.5 kb is detected in MG/GR after hybridization with a human glucocorticoid receptor cDNA probe (Bellingham and Cidlowski, 19891, which corresponds to the predicted size of the transfected human glucocorticoid receptor transcript. As shown in Fig. 5 (lanes MTXSO, 750, 30001, this transcript is also detected in all MTX-resistant cell lines. After selection of MG/hGR cells with an initial dose of 50 nM MTX, there is a marked 4- to 5-fold increase in human glucocorticoid receptor mRNA (Fig. 5, lane MTXSO). Additional increases in MTX resistance beyond 50 nM were not reflected by relative changes in the levels of human glucocorticoid receptor mRNA that were as large as this initial increase. The amount of human glucocorticoid receptor mRNA in MTX750 cells (Fig. 5, lane MTX750) appears to be largely unchanged compared to MTXSO cells. This result was not surprising in view of the data obtained on the levels of receptor protein in these cell lines (Table 1 and Fig. 2). Nonetheless, following selection for resistance to 3 PM MTX, the steady-state level of human glucocorticoid receptor mRNA in CHO cells underwent an additional 1.5- to 2-fold increase (Fig. 5, lane MTX750 vs. lane MTX3000). Hybridization with an actin probe reveals that, with the exception of the DG44 sample (see below), equivalent amounts of RNA were loaded in each sample lane (Fig. 5, actin). In addition to the prominent human receptor transcript observed at 3.5 kb in MTX-resistant cells, an additional cross-hybridizing species was detected at approximately 7 kb. This transcript size is consistent with the transcript size that has been reported for other rodent receptors (Danielson et al., 19861, suggesting that it corresponds to endogenous hamster receptor mRNA. Since the hamster glucocorticoid receptor gene has not been cloned, it is not possible to make an unequivocal identification. However, this 7 kb transcript is the only hybridizing species detected in CHO cells that have not been transfected with the human glucocorticoid receptor gene (Fig. 5, lane DG44*), providing an additional line of evidence that it is endogenous hamster receptor mRNA. The intensity of this hybridization signal is very weak in nontransfected CHO cells; 40 pg total RNA was required to produce a detectable band

in the experiment shown in Fig 5. In contrast, when one-half the amount of RNA (20 pg) from each of the MTX-resistant cell lines was hybridized with a glucocorticoid receptor probe, the relative level of this 7 kb transcript appears to be at least 2- to 3-fold higher compared to DG44 cells (Fig. 5, lanes MTXSO, MTX750, MTX3000 vs. lane DG44”). This putative hamster receptor mRNA was not visible in the MG/hGR sample containing roughly the same amount of total RNA. These findings raise the interesting possibility that the linked selection strategy employed in this study has selected for MTX-resistant CHO cells that overexpress endogenous hamster glucocorticoid receptors as well as transfected human glucocorticoid receptors.

GR DG44

Cl-i0 I-Ma Genomic DNA 0.5 2.5

Characterization of glucocorticoid receptor and DHFR gene copy numbers in MTX-resistant cells

The rationale for using the DHFR gene as a selectable marker to obtain stable overexpression of glucocorticoid receptors in CHO cells was based upon the known coamplification properties of the DHFR gene following selection with the folate antagonist MTX (Kaufman and Sharp, 1982). In view of the increase in transfected human receptor gene expression observed in Fig. 5 following MTX selection, was this result due to a DHFR-linked, gene coamplification mechanism? To address this question, the relative copy numbers of the gtucocorticoid receptor and the DHFR genes in each of the MTX-resistant ceil lines was determined (Fig. 6). Prior to hybridizing control and MTX-resistant DNAs with DHFR and glucocorticoid receptor-specific gene probes, all sample concentrations were normalized to the intensity of the hybridization signal obtained from an actin gene probe. Normalized quantities of various DNAs were then applied in duplicate to nitrocellulose using a slot-blot manifold and hybridized with either a DHFR or a glucocorticoid receptor gene probe. Densitometric analysis of the DHFR hybridization signal from MG/hGR cells (Fig. 6, MG/hGR) indicated that approximately 40 copies of the DHFR gene are present in the DNA sample analyzed. Similar analysis of the number of glucocorticoid receptor genes indicated that about 30 copies of the glucocorticoid

12.5

-

50

-

250

Gene Copy Number Standaids Fig. 6. Gene copy number analysis of DHFR and glucocorticoid receptor genes. Genomic DNA samples (Genomic DNA) were denatured and applied to nitrocellulose filters using a slot-blot manifold. Haploid genome equivalents (Gene Copy Number Standards) corresponding to 0.5, 2.5, 12.5, 50 and 250 copies of either the DHFR or the glucocorticoid receptor gene were similarly treated and used to generate standard curves based on hybridization signal intensities. Duplicate filters were hybridized with a randomly primed “‘P-labeled DHFR fragment (DHFR) or a randomly primed “P-labeled glucocorticoid receptor fragment (GR). Gene copy numbers were calculated as previously described (Bellingham and Cidlowski, 1989).

receptor gene are present in MG/hGR DNA. The high copy number of the glucocorticoid receptor gene in MG/hGR cells is presumably due to transfected human glucocorticoid receptor sequences since the endogenous hamster gene was not even detected in nontransfected DG44 cells by this assay (Fig. 6, DG44). The large number of DHFR genes present in MG/hGR cells is due exclusively to transfected gene sequences since both copies of the endogenous DHFR gene have been deleted in DG44 cells Wrlaub et al., 1983). A line of CHO cells known to contain one copy (per haploid genome) of the endogenous hamster

167

DHFR gene was included for signal comparison (Fig. 6, CHO); however, neither the low copy number DHFR gene nor the endogenous hamster glucocorticoid receptor gene was detected in these cells. A similar result was obtained when HeLa DNA was examined (Fig. 6, HeLa). The number of DHFR genes in HeLa cells is apparently below the level of detection of this assay, whereas a faint hybridization signal corresponding to approximately one copy of the glucocorticoid receptor gene was observed. Densitometric analysis of the number of DHFR genes in MTXSO cells reveals that there was a small (1.2-fold) increase in the number of DHFR genes following initial MTX selection, from 40 to about 60 copies (Fig. 6, MTXSO vs. MG/hGR). A similar increase (1.4-fold) in the number of glucocorticoid receptor genes was also observed in MTXSO cells following MTX selection. This coordinate increase in the number of DHFR genes and glucocorticoid receptor genes in MG/hGR cells in response to MTX treatment may be contributing to the 3- to 5-fold increase in glucocorticoid receptor protein (Fig. 2, Table 1) and mRNA levels (Fig. 5) in MTXSO cells. However, this increase is not sufficient to account completely for the observed increase in glucocorticoid receptor levels. Moreover, when the number of glucocorticoid receptor and DHFR genes in CHO cells resistant to higher levels of MTX was examined (Fig. 6, MTX750, MTX3000), no further changes in the copy number of either gene were detected. These findings suggest that the additional increases in glucocorticoid receptor levels that were obtained after selection of CHO cells in concentrations of MTX higher than 50 nM occurred by mechanism(s) other than gene amplification. Discussion

A number of bacterial and eucaryotic expression systems have been developed in an effort to obtain the high levels of protein that are necessary for in vitro analysis of glucocorticoid receptor structure and function (Schena and Yamamoto, 1988; Dahlman et al., 1989; Freedman et al., 1989; Srinivasan and Thompson, 1990). While these approaches have proven informative, there are many aspects of glucocorticoid receptor

biology that will ultimately require analysis within a whole-cell system. Unfortunately, in vivo studies of steroid hormone receptor structure and function have been complicated by the fact that receptor proteins are normally expressed in very low levels in mammalian cells. Several different approaches have been taken to address this problem. For example, cotransfection strategies based upon amplifiable selection markers such as DHFR or metallothionein have recently been used to generate stable, high level expression of steroid receptors in mammalian cell lines (Israel and Kaufman, 1988; Alksnis et al., 1990; Hirst et al., 1990; Kushner et al., 1990). However, in the absence of a direct selection for receptor function, the possibility of selecting for cell lines that are unable to emulate all the features of native receptors becomes a real concern. In an effort to circumvent this problem, we have taken a different approach and modified the usual DHFR-based co-expression strategy to generate CHO cells that stably express human glucocorticoid receptors, By rendering the DHFR gene responsive to glucocorticoids and then applying MTX pressure, we reasoned that the acquisition of resistance to MTX should be physiologically coupled to glucocorticoid receptor-driven expression of DHFR. Moreover, this linkage should demand that the additional glucocorticoid receptors induced by MTX selection are produced in a functional form. Several lines of evidence independently support the conclusion that we have accomplished these objectives. When stably cotransfected MG/hGR cells were chronically subjected to the selective pressure of MTX, the resistant cells that arose displayed a strict, receptor-mediated dependence on exogenous glucocorticoid for survival. This glucocorticoid-dependent phenotype was retained with each successive increase in MTX up to the highest level of MTX selection examined (Fig. l), thus demonstrating that there is a physiological linkage between glucocorticoid receptor function and cell survival in MTX-resistant cells. The successful establishment of such a linkage between MTX resistance and glucocorticoid receptor function is reflected by progressive increases in the actual number of glucocorticoid receptors with higher doses of MTX. It therefore

appears that modification of the standard DHFR-based selection in MTX can still produce the desired result of directing overexpression of a heterologous gene. Quantitation of steroid-binding capacity in MTX-resistant cells (Table 1) revealed a progressive increase in receptor content that was maximal at the highest level of MTX examined (3 PM) and represented a 25fold increase over CHO cells that normally express a low level of endogenous receptors. One exception to this trend was observed with the MTX750 cell line, which did not appear to have acquired any additional receptors in response to higher MTX pressure. The molecular basis for this effect is unclear; however, similar observations have been reported by other investigators using MTX selection schemes (Murray et al., 1983; Hirst et al., 1990). Even with the transient plateau in receptor levels in MTX750 cells, increased expression was restored following selection in higher concentrations of MTX (MTX3000). The relative increases in steroid-binding capacity induced by MTX are reflected by comparable changes in the level of immunoreactive glucocorticoid receptor at M, 94,000 (Fig. 2). It is particularly significant that MTX does not appear to have produced any changes in the structure of the receptor protein. Furthermore, sucrose gradient analysis of nonactivated oligomeric glucocorticoid receptors overexpressed in the MTX-resistant lines (Fig. 3) indicates that CHO cells retain the ability to form oligomeric, 9 S complexes when receptors are expressed up to 25 times higher than normal. At the subcellular level, overexpressed glucocorticoid receptors are associated with the nucleus in the presence of hormone (Fig. 4), suggesting that normal, nuclear translocation mechanisms also remain operative in CHO cells following MTX selection. These properties are summarized in Table 2. Overexpressed receptors in MTX-resistant CHO cells were competent to activate transcription of a glucocorticoid-responsive CAT reporter plasmid (Table 3). In view of the much larger number of glucocorticoid receptors expressed in MTX3000 cells (1.7 x lo5 receptors per cell) compared to MG/hGR cells (3 X lo4 receptors per cell), it was somewhat surprising that the level of inducible CAT activity was not corre-

spondingly higher. Given the number of glucocorticoid-responsive DHFR genes that are already present in MTX-resistant CHO cells, it is conceivable that the transiently transfected CAT genes are competing with the resident DHFR genes for necessary transcription factors and thus preventing full inducibility of CAT. Indeed, the limiting nature of transcription factor(s) required for glucocorticoid inducibility has been reported by others (Bocquel et al., 1989; Bruggemeier et al., 1990). Whether a similar situation exists in CHO cells is currently unknown. Nonetheless, the lack of any impairment of CAT reporter gene activity in MTX3000 cells relative to MG/hGR cells supports the conclusion that modification of the typical MTX-induced overexpression scheme does not have any detrimental effects on glucocorticoid receptor function. It is interesting to note that in the present studies overexpression of glucocorticoid receptors was obtained in the continued presence of 5 nM dexamethasone, which is a requirement for cell growth in MTX. Glucocorticoids are known to induce down-regulation of endogenous receptors (Cidlowski and Cidlowski, 1981; Dong et al., 1988) as well as transfected glucocorticoid receptors (Burnstein et al., 19901. Similarly, we have observed that MG /hGR cells, which are not grown in dexamethasone, also undergo ligand-induced down-regulation of glucocorticoid receptor expression (Bellingham, D.L., Sar, M. and Cidlowski, J.A., submitted for publication). Thus it appears that the MTX-resistant cell lines that were derived from MG/hGR cells have developed an altered capacity to undergo ligand-induced down-regulation of glucocorticoid receptors. We may have selected for cells having a down-regulation-defective phenotype; however, this possibility will require further study. A particularly unexpected result of this work concerns the apparent molecular basis for the overexpression of glucocorticoid receptors obtained in CHO cells in response to MTX treatment. Northern blot analysis (Fig. 5) of steadystate glucocorticoid receptor mRNA levels revealed relative increases in transfected human glucocorticoid receptor gene expression that were generally consistent with the MTX-induced increases in glucocorticoid receptor protein (Fig. 2).

169

However, the level of a cross-hybridizing _ 7 kb transcript, which is thought to correspond to endogenous hamster glucocorticoid receptor, was also increased as a function of MTX concentration. Based on these findings, we speculate that the modified MTX selection strategy employed in this study may have selected for CHO cells that overexpress endogenous hamster glucocorticoid receptors as well as transfected human glucocorticoid receptors. To date, the hamster glucocorticoid receptor gene has not been cloned to allow investigation of this hypothesis at the gene level. Species-specific anti-glucocorticoid receptor antibodies might be able to provide some insight by discriminating between hamster and human receptor proteins. Based on Southern blot analysis, it does not appear that any rearrangements or alterations in either the endogenous hamster receptor genes or the transfected human glucocorticoid receptor genes occurred following selection with MTX (data not shown). Quantitative analysis of the number of DHFR and glucocorticoid receptor genes (Fig. 6) indicates that no detectable gene amplification events occurred in MTX-resistant CHO cells after the initial selection of MG/hGR cells in 50 nM MTX. This was surprising in light of the fact that glucocorticoid receptor mRNA levels still continued to rise in CHO cells with additional MTX treatment up to the highest concentration of MTX examined (Fig. 5). Based on these results, it appears that the MTX-induced increases in glucocorticoid receptor expression in CHO cells occurred by mechanism(s) other than gene amplification. MTX can affect genome stability (Pallavicini et al., 19901, which could give rise to variations in glucocorticoid receptor gene expression at the transcriptional and/or the post-transcriptional level. For instance, previously silent glucocorticoid receptor genes might be recruited following MTX selection. Alternatively, MTX selection may select for cells that have elevated rates of receptor gene transcription and/or more stable glucocorticoid receptor mRNAs. These issues will require further study. The absolute levels of glucocorticoid receptor expression achieved in MTX3000 cells (175,000 receptors/cell) are about 2-fold higher than the levels that are normally found in certain tumor

cell lines, which express only endogenous glucocorticoid receptors. For example rat HTC cells are among the highest known sources of endogenous glucocorticoid receptor but they express only - 100,000 glucocorticoid receptors/ cell (Hirst et al., 1990). Admittedly, the levels of glucocorticoid receptor expression that we have obtained in CHO cells thus far are modest relative to other overexpression systems that employ MTX selection strategies which are not dependent on glucocorticoid receptor function (Hirst et al., 1990; Alksnis et al., 1991). There are several possible reasons for this, of which the most obvious is likely to be related to the lack of significant amplification of the glucocorticoid receptor gene in our MTX-resistant cell lines. We speculate that the reason for obtaining only minimal amplification of the glucocorticoid receptor gene may be a consequence of DHFR gene expression being under glucocorticoid control. The DHFR gene used in this study has been modified such that the majority of the endogenous promoter has been deleted and replaced with a GRE-containing fragment from the long terminal repeat of the mouse mammary tumor virus (Bellingham and Cidlowski, 1989). If the level of DHFR transcription from this plasmid is adequate when MTX-resistant CHO cells are grown in the continued presence of dexamethasone, then there is no selective pressure to amplify the DHFR gene. Indeed, a lack of gene amplification has been reported in CHO cells when the expression of a transfected DHFR gene was placed under the control of a promoter from the long terminal repeat of Harvey sarcoma virus (Murray et al., 1983). Although glucocorticoid receptor-dependent expression of a modified DHFR gene may not be compatible with gene amplification mechanisms in CHO cells, the MTX-induced pressure to maintain high levels of DHFR expression nonetheless creates a selective advantage for cells that express high levels of functional glucocorticoid receptors. Extending this reasoning, a unique situation is created in which it is possible to select for cells that express higher levels of endogenous glucocorticoid receptors as well as transfected receptors. The identification of a putative hamster glucocorticoid receptor mRNA species that

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is increased in CHO cells resistant to higher doses of MTX (Fig. 5) supports this hypothesis. Based on the data presented, we have designed an overexpression system that effectively selects for structurally and functionally intact glucocorticoid receptors in mammalian cells. To our knowledge this is the first report in which a selection strategy that links receptor function with cell survival has been used to achieve stable overexpression of intact glucocorticoid receptors in mammalian cells. The ability to select for functional receptors represents a useful and potentially general strategy to obtain homogenous populations of mammalian cells that are well suited for the in vivo analysis of steroid receptor structure and function. Acknowledgments We thank Dr. Kerry Burnstein for providing invaluable assistance with cell culture. We also thank Drs. Victoria Allgood and Yoshie ItohLindstrom for their helpful suggestions and assistance with CAT assays. This work was supported by National Institute of Health Grant DK 32460. References Alksnis, M., Barkhem, T., Ahola, H., Wright, T., Gustafsson, J.-A. and Nilsson, S. (1991) J. Biol. Chem. 266, 1007% 10085. Allgood, V.E., Powell-Oliver, F.E. and Cidlowski, J.A. (1990) J. Biol. Chem. 265, 12424-12433. An, G., Hidaka, K. and Siminovitch, L. (1982) Mol. Cell. Biol. 2, 1628-1632. Beato, M. (1991) FASEB J. 5, 2044-2051. Bebbington, C.R. and Hentschel, C.C.G. (1987) in DNA Cloning: A Practical Approach, Vol. 3 (Clover, D.M., ed.), pp. 163-188, IRL Press, Oxford. Bellingham, D.L. and Cidlowski, J.A. (1989) Mol. Endocrinol. 3, 1733-1747. Bocquel, M.T., Kumar, V., Stricker, C., Chambon, P. and Gronemeyer, H. (1989) Nucleic Acids Res. 17, 2581-2595. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Bresnick, E.H., Dalman, F.C. and Pratt, W.B. (1990) Biochemistry 29, 520-527. Brown, P.C., Tlsty, T.D. and Schimke, R.T. (1983) Mol. Cell. Biol. 3, 1097-1107. Bruggemeier, U., Rogge, L., Winnacker, E.-L. and Beato, M. (1990) EMBO J. 9, 2233-2239. Burnstein, K.L. and Cidlowski, J.A. (1989) Annu. Rev. Physiol. 51, 683-699.

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