Functional Activities of the A and B Forms of the Human Androgen Receptor in Response to Androgen Receptor Agonists and Antagonists

Tianshu Gao and Michael J. McPhaul

Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas 75235


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The androgen receptor (AR) is present in many cells in two forms. The B form migrates with an apparent mass of 110 kDa and constitutes more than 80% of the immunoreactive receptor in most cell types. The A form of the AR migrates with an apparent mass of 87 kDa, appears to derive from internal translation initiation at methionine-188 in the AR open-reading frame, and usually constitutes 20% or less of the immunoreactive AR present. Previous experiments designed to examine the functional capacity of the A and B forms of the AR have been hampered by marked differences in the expression levels of the two isoforms, as the nucleotide sequence surrounding the codon encoding methionine-188 causes it to be used inefficiently as a translation initiation site. To circumvent this, we altered the nucleotide sequence surrounding methionine-188 to render it more similar to that surrounding the codon encoding methionine-1. Transfection of a cDNA containing these changes resulted in similar levels of expression of A and B forms of the AR as assessed by immunoblot assays using antibodies directed at an epitope preserved in both. Functional activities of these cDNAs were assessed using cotransfection assays that employed two model androgen-responsive genes (MMTV-luciferase and PRE2-tk-luciferase) in response to mibolerone, a potent androgen agonist, in three different cell lines. These studies demonstrated subtle differences in the activities of the A and B isoforms, which depended on the promoter and cell context. Additional studies failed to reveal any major differences in the responses of the AR-A and AR-B isoforms to a variety of androgen agonists and antagonists, suggesting that the previously reported functional defect of the AR-A is due principally to its level of expression. When assays of AR function are performed under conditions in which levels of expression of the two isoforms are equivalent, the AR-A and AR-B possess similar functional activities.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The androgen receptor (AR) is a member of the steroid hormone-thyroid hormone-retinoic acid family of nuclear receptors (1, 2) and mediates the responses of tissues to the male hormones, testosterone and 5{alpha}-dihydrotestosterone. The cDNA nucleotide sequence predicts a protein approximately 917 amino acids long comprised of a highly conserved DNA-binding domain, a carboxy-terminal hormone-binding domain, and a large amino terminus (3, 4, 5, 6). The form of the AR predicted by the translation of the complete open reading frame (termed AR-B) migrates with an apparent molecular mass of 110 kDa on SDS-polyacrylamide gels.

During our studies of affected members of a family with complete testicular feminization, we detected a novel shortened form of the human AR (7). Subsequent experiments revealed that this AR isoform [termed AR-A because of its similarity in structure to the A form of the progesterone receptor (PR)] differed from the structure predicted for the B form of the AR in that it lacked an intact amino terminus, yet contained intact DNA- and hormone- binding domains. Further analysis revealed that this form of the AR is expressed in a variety of normal tissues, albeit at low levels, and is derived from initiation of translation at methionine-188 instead of methionine-1 of the AR open reading frame (8, 9).

The detection of two distinct forms of the AR raised a number of issues. First, since the original family in which the AR-A form of the receptor was detected exhibited a phenotype of complete testicular feminization, it was not clear whether this defect of androgen action was caused by the reduced levels of receptor expressed or to the impaired function of this form of the AR (7). Second, as noted above, the structures of the A and B forms of the AR bear a striking resemblance to the more fully characterized A and B forms of the PR. A number of studies have suggested that the A and B forms of the PR exhibit distinct differences of function, with respect to the target genes that they regulate, the ligands to which they respond, and the way in which they interact with target DNA sequences (10, 11, 12, 13, 14, 15). The similarities evident between the isoforms of the AR and PR suggested that specific roles might exist for the AR-A and AR-B isoforms in the regulation of androgen-responsive genes as well.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Alteration of Nucleotide Sequence Surrounding Methionine-188 Permits the Expression of the AR-A and AR-B Isoforms at Equivalent Levels in the Absence of Hormone in Transfection Assay
Previous studies suggested that the defect of AR function in patient 776 was caused, at least in part, by the reduced levels of AR-A expressed in fibroblasts established from the affected subject (7). This low level of expression is likely caused by the nucleotide sequence environment surrounding methionine-188, which is a poor fit to Kozak consensus sequence rules for translation initiation and predicts poor translation efficiency (16). To circumvent this limitation, sequences 5' to methionine-188 were replaced by those that are normally 5' to methionine-1 by site-directed mutagenesis (Fig. 1Go). The effect of this change was assessed by measuring the level of expression in transfected cells using antibodies that recognize both receptor isoforms (amino acid residues 200–220 in AR-B). As shown in Fig. 2Go, transfection of the resulting expression plasmid (PS367) into COS cells resulted in the synthesis of levels of immunoreactive AR equivalent to those observed after transfection with a cDNA encoding the full-length AR (CMV 3.1).



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Figure 1. Schematic Structures of the AR-A and AR-B Isoforms

The AR-A and AR-B isoforms differ in the length of their amino termini. The cDNA used in the initial studies of the functional activity of the AR-A isoform (7) contained a premature termination codon at amino acid residue 60, and the surrounding nucleotide sequence predicted a poor efficiency of translation initiation at methionine-188. To permit the expression of the isoforms in a fashion where equivalent amounts of each are expressed, the cDNA segment 5' to methionine-188 in plasmid 776 (CMV776) was mutated to correspond to that 5' to methionine-1 in CMV3.1 (Ref. 6). The resulting plasmid (in which the codon encoding methionine-188 is located immediately 3' to the segment normally 5' to the ATG encoding methionine-1) was designated PS367. The black boxes shown in the plasmids CMV3.1 and CMV776 indicate the positions of the glutamine repeats. Underlined sequences shown in PS367 indicate a BglII restriction endonuclease cleavage site by which the segment amplified by PCR was ligated into CMV5 vector.

 


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Figure 2. Alteration of the Segment 5' to Methionine-188 Results in High Level Expression of the AR-A Isoform

Monolayers of COS cells were transfected in parallel with expression plasmids encoding either the hAR (CMV3.1; AR 1–917), CMV776, or PS367. Forty hours after transfection, the cells were harvested, and extracts were prepared and analyzed by Western analysis. The antibody used (U407) recognizes an epitope (amino acids 200–220) that is common to both the AR-A and AR-B isoforms. The numbers in parentheses indicate the quantity of protein loaded in each lane.

 
AR-A and AR-B Demonstrate Differences of Function When Assayed in the CV-1 Cell Line Using Mouse Mammary Tumor Virus (MMTV) and PRE2-tk Promoter Fusions
Having demonstrated that the levels of immunoreactive AR expressed were similar when equivalent levels of cDNA were transfected, we then performed experiments to compare their functional activities. To do so, we transfected increasing amounts of the human (h) AR-A and hAR-B expression vectors and constant amounts of the MMTV-luciferase reporter gene into CV1 cell monolayers and incubated the transfected cultures with 2 nM dihydrotestosterone (DHT) in each experiment. The results (shown in Fig. 3AGo) revealed substantial differences in the dose-response curves of the two isoforms. After transfection with low concentrations of cDNAs encoding either isoform, increasing activation of the reporter gene was observed. Although in both instances the maximum stimulation of the reporter gene was achieved between 0.1 µg and 0.4 µg of AR cDNA, at the region of peak activity AR-A exhibits approximately half the level of reporter gene activity measured for cells transfected with the AR-B cDNA (Fig. 3AGo). This result suggests that the amino-terminal segment (amino acid residues 1–188) that is not present in the AR-A isoform is important for the formation of receptor complexes capable of stimulating maximal activation of the MMTV promoter in CV1 cells.



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Figure 3. Dose Response of AR-A and AR-B Function Analyzed in CV1 Cells Using the MMTV Luciferase and PRE2-tk Luciferase Reporter Gene

Monolayers of CV1 cells were transfected with different quantities of cDNA encoding AR-A (PS367) and AR-B (CMV3.1) and the androgen-responsive reporter gene MMTV luciferase (A) and PRE2-tk luciferase (B). Transfection efficacy was assessed by measurement of the ß-galactosidase activity encoded by the control plasmid, CMV-ß-galactosidase, which was included in each transfection. Each point on this graph is derived from one of six separate cell samples (triplicate points each from cells stimulated in parallel with no hormone or 2 nM mibolerone). This experiment is representative of three independent experiments.

 
Different results were obtained when a different promoter, PRE2-tk, was used to assay the functions of the AR isoforms in the same cells (CV1 cells). When assayed using this reporter gene, AR-A functioned approximately twice as well as AR-B (Fig. 3BGo). This result suggests that the two AR isoforms interact with components of the transcription apparatus in a fashion that is promoter-specific. Experiments conducted transfecting cDNAs encoding both isoforms demonstrated no evidence that the AR-A isoform acted in a dominant negative fashion (data not shown).

Cell- and Promoter-Specific Behavior of AR Isoforms in Stimulating the Activity of Androgen-Responsive Reporter Genes
To determine how the A and B isoforms would function in different cell types to activate these model androgen-responsive reporter genes, we transfected 200 ng of AR-A and AR-B cDNA in parallel with either the MMTV or PRE2-tk reporter plasmids into the CV1, DU145, and PPC1 cell lines and assayed reporter gene activity after incubation with 2 nM DHT in each experiment. This protocol was chosen as this amount of AR cDNA induced the activity of the androgen-responsive genes by both isoforms to maximal levels (Fig. 3Go).

When CV1 cells were transfected with cDNAs encoding the AR isoforms and the MMTV promoter reporter plasmids, AR-A exhibited approximately half of the level of activity observed in transfections using the AR-B cDNA. These results correspond to those obtained in the dose response curves presented in Fig. 3AGo. Although the measurements of AR function in the different cell lines cannot be compared directly, it is clear that similar patterns in the relative activities of the A and B isoforms are seen when AR function is measured in the DU145 and PPC1 cell lines using the MMTV reporter, as when performed in the CV1 cell line. In each instance, the B form of the AR is approximately twice as active in stimulating reporter gene activity as the A form assayed in parallel (Fig. 4AGo).



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Figure 4. The Effects of Cell Types and Reporter Gene Promoters on the Activities of AR-A and AR-B

Cells were transfected with a constant amount of expression plasmid (200 ng) encoding the AR-A or AR-B isoforms, reporter plasmid [MMTV luciferase (A) or PRE2-tk luciferase (B), 10 µg], and transfection control plasmid CMV-ß-galactosidase (1 µg). After stimulation with no hormone or 2 nM mibolerone for 48 h, luciferase and ß-galactosidase activities were measured. Each value is derived from six separate points (three transfections treated with no hormone and three treated with 2 nM mibolerone). Owing to the different cell lines and reporter genes used, only the values for the AR-A and AR-B isoforms in a given cell line and promoter can be directly compared.

 
The results obtained when the capacities of these AR isoforms to activate the PRE2-tk promoter fusion were assayed in the same panel of cell lines were less constant. When assayed in the CV1 cell line, AR-A isoform was found to be more potent than the AR-B isoform (Fig. 4BGo, results consistent with those depicted in Fig. 3BGo). When assayed in the DU145 cell line, the two isoforms exhibited similar capacities to activate the reporter gene. In the PPC1 cell line, the level of reporter gene activation achieved by AR-A was lower than that of AR-B. These differences suggest that the AR-A and AR-B hormone-receptor complexes interact with the PRE2-tk promoter in a fashion that varies among cell types. Despite these differences, each reporter gene used displayed similar levels of activation in response to saturating concentrations of DHT and testosterone (data not shown).

AR-A and AR-B Cannot Be Distinguished by Antiandrogens in Cotransfection Assay
The A and B forms of the PR have functions that can be distinguished pharmacologically. To examine whether this was true for the A and B AR isoforms, we examined the response of these isoforms to different AR agonists and antagonists. These assays were performed by transfecting the AR-A and AR-B cDNAs in parallel into CV1 cells in combination with the MMTV-luciferase reporter gene and treating the transfected cells with varying concentrations of antiandrogens alone or in combination with saturating concentrations of 5{alpha}-DHT. The results that we obtained are shown in Fig. 5Go and are intriguing in several respects.



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Figure 5. Effects of Antiandrogens on AR-A and AR-B Function

CV1 cells were transiently transfected by the addition of a calcium phosphate precipitate containing the cDNAs (200 ng) encoding either AR-A (PS367) or AR-B (CMV3.1), the reporter plasmid MMTV-luciferase (10 µg), and a control plasmid, CMV-ß-galactosidase (1 µg), and incubated with the indicated combinations of ligands for 48 h. Panels A, B, and C display the activities observed in experiments using hydroxyflutamide and flutamide, while panel D indicates the results of an experiment using bicalutamide (Casodex).

 
As expected, hydroxyflutamide functioned as a potent antagonist to the activation of the AR isoforms by DHT (Fig. 5AGo). In these experiments, the inclusion of 0.5 µM hydroxyflutamide inhibited the reporter gene activity induced by saturating concentrations of 5{alpha}-DHT by more than 90%. Additional inhibition of AR function was not evident when the concentration of hydroxyflutamide was increased further. Instead, as the concentration of hydroxyflutamide was increased, increasing levels of reporter gene activation were observed. When these experiments were performed using hydroxyflutamide alone, it was possible to demonstrate that the agonism observed at the higher hydroxyflutamide concentrations was a function of the properties of hydroxyflutamide itself.

When this same type of experiment was repeated using flutamide, a different result was observed. Flutamide showed less potent capacity to antagonize DHT action than hydroxyflutamide, requiring concentrations of 2 µM to achieve a 90% inhibition of DHT-stimulated AR function. Of interest, as the concentrations of flutamide were increased (from 0.5 to 2 µM), only a progressive inhibition of AR function was observed. In keeping with this observation, flutamide was devoid of any intrinsic agonism in functional assays when added alone to the transfected cells (Fig. 5BGo). Experiments in which side-by-side comparisons were performed confirmed this finding (Fig. 5CGo).

The findings presented above are not confined to hydroxyflutamide, and other high-affinity AR antagonists—even those that have been suggested to be pure antiandrogens—exhibit a mixture of agonist and antagonist properties. A representative experiment is shown in Fig. 5DGo for Casodex (bicalutamide). At low concentrations, Casodex exhibits a potent capacity to antagonize the activation of the AR-B by 2 nM 5{alpha}-DHT. As was observed with hydroxyflutamide, however, as the concentration of Casodex is increased, increasing agonism becomes evident. As shown in Table 1Go, the AR antagonists demonstrate similar behaviors when assayed in the PPC-1 and DU145 cell lines.


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Table 1. AR Antagonists Display Similar Activities in the PPC-1, DU145, and CV1 Cell Lines When Assayed Using the MMTV Luciferase Reporter Gene

 
In the experiments depicted in Fig. 5Go, A, B, and D, the activity of AR-A isoform was measured in parallel with those of the AR-B isoform. In each instance, the patterns of response observed for the AR-B and AR-A isoforms were similar.

Activation of the AR Isoforms Is Not Always Accompanied by Increased Levels of Immunoreactive AR
Androgen causes the levels of AR to increase when assayed by ligand binding (17) or by immunoblot assays (18), and this increase appears to result from a change in the half-life of the receptor protein. To determine whether the agonism observed for the AR antagonists (such as hydroxyflutamide and Casodex) also caused changes in the levels of AR, we performed immunoblot assays of cells transfected with cDNAs encoding the different isoforms after treatment with either no hormone or with the different antagonists. The results of these experiments are shown in Fig. 6Go and are interesting in two respects. The first is that the level of AR does not change in cells transfected with AR-B and treated with AR antagonists, even under circumstances in which substantial agonism is observed (e.g. at 5 µM hydroxyflutamide). These experiments also show, unexpectedly, that the increase in the level of AR-B that is observed after treatment with androgen agonists, such as mibolerone, is not observed in cells transfected with the AR-A isoform. This finding suggests that the amino- terminal segment of AR-B is critical for the increase in receptor levels after treatment with ligand.



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Figure 6. Increase in the Levels of Immunoreactive AR Do Not Parallel the Agonistic Effects Observed in Response to Selected Antiandrogens

Monolayers of CV1 cells were transfected in parallel with expression plasmids encoding either the hAR-B (CMV3.1 AR 1–917) and AR-A (PS367 AR 188–917). Twenty four hours after transfection, cells were treated with DHT and different antiandrogens at different concentrations in MEM containing 5% charcoal- treated serum. Forty hours later, the cells were harvested, and extracts were prepared and analyzed by Western analysis. The antibody used (U407) recognizes an epitope (amino acids 200–220) that is common to both the AR-A and AR-B isoforms. The quantity of protein loaded in each lane is the same (~100 µg).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Different receptor forms derived from a single structural gene have been described for a number of different members of the steroid-thyroid-retinoic acid family of nuclear receptors (1, 19, 20, 21). Of these, the two different forms of the hAR bear the most striking similarity to the A and B forms of the PR. As in the case of the PR, the expression of two different forms of the hAR has been detected in a number of different tissues. Unlike the PR-A isoform, the hAR-A has been detected at relatively low and constant levels and no cell type or tissues have been identified to date in which only the AR-A is expressed as the predominant isoform (8, 9). Despite this, the similar structures of the AR and PR isoforms suggested that the AR-A isoform might display distinctive properties in the regulation of androgen-responsive genes.

In our prior studies, a patient with complete testicular feminization expressed only a shortened form of AR (AR-A), which appeared to be derived from initiation of translation at methionine-188 (7). The low and relatively constant level of AR-A that was detected in the fibroblasts of this patient was also found to be present in the fibroblasts of normal subjects and in most target tissues (8). The unfavorable context surrounding the AUG triplet encoding methionine-188 appeared to account for the low level of expression in vivo and in transfection studies and complicated studies to analyze the functional capacity of the two isoforms. For this reason, we constructed an AR-A expression vector (PS367) in which the sequence upstream of methionine-1 was introduced 5' to methionine-188. As the result of these changes, the expression levels between AR-A and AR-B were similar and permitted a more thorough analysis of their functional activities.

When assayed in CV1 cells, AR-A and AR-B showed different dose-response curves in transfection assays using an MMTV-luciferase reporter gene fusion to measure receptor function. The activities of both isoforms were maximal at low concentrations of transfected cDNA, and decreasing levels of reporter gene activity were observed as increasing amount of cDNA are added. Of note, at the level where maximal activation was achieved, the AR-B isoform was approximately twice as active as the AR-A isoform. When such assays are repeated using a reporter gene controlled by the PRE2-tk promoter in CV1 cells, the level of induction by AR-B was half that stimulated by AR-A. This finding suggests that the amino-terminal segment of the AR (amino acid residues 1–187) interacts differently with the proteins that regulate transcription from these two promoters. These inferences are further supported by the results of assays using these same reporter genes in different cell lines. In these experiments, while the activity of the AR-B was higher using the MMTV promoter in all of the cell lines, the PRE2-tk promoter gene fusion was activated to varying levels by the AR-A in the DU145, PPC1, and CV1 cell lines. The demonstration that the amino terminus confers upon the AR-B isoform activities not exhibited by the AR-A isoform is consistent with studies that have localized a distinct transactivation domain to the corresponding segment of the human PR (22).

In some instances, isoforms of nuclear receptors have demonstrated differential responses to pharmacological manipulations (10, 11, 12). For this reason, we explored the effects of a variety of AR antagonists on AR function. In these experiments, using a reporter gene controlled by the MMTV promoter in CV1 cells, a variety of antiandrogens failed to display any major qualitative differences in the activities of AR isoforms. In each instance the results of assays using AR-A were similar to those obtained using AR-B.

While the results of these functional studies did not reveal unique activities of the AR isoforms, the properties exhibited by the different antagonists in these experiments were intriguing. First, it is evident that of the compounds tested, only flutamide failed to exhibit agonism in our assays. While low concentrations of each of the higher affinity antagonists (Casodex, hydroxyflutamide) effectively blocked the stimulation of the AR by saturating concentrations of agonist (DHT), increasing agonism was evident as the concentrations of these AR antagonists were further increased. Of note, our observations differ somewhat from the findings of other groups who have examined the activities of hydroxyflutamide and Casodex in reporter gene assays. In two studies, these compounds exhibited little agonistic activity and appeared to function as pure antagonists (23, 24). By contrast, Kemppainen and Wilson (25) noted the agonism inherent in the behaviors of hydroxyflutamide and Casodex. The results reported in the present study are in best agreement with these latter investigations. The reasons for the discordances are not clear, but may be explained in part by the reporter genes used, by the concentrations of antagonist assayed, or by the specific cell types employed.

The mechanisms by which ligands for nuclear receptors exhibit both agonism and antagonism are not clear. Molecules exhibiting such a mixture of activities have been identified that act to antagonize the actions of ligands for many members of the nuclear receptor family, including androgens, estrogens, and progestins (12, 24, 25, 26, 27, 28). Detailed studies of the activities of such compounds have suggested models in which the receptor assumes distinct conformations that permit the liganded receptor to interact with the transcription apparatus in a productive or nonproductive fashion (29, 30). The degree of agonism observed has been found to vary substantially, depending on cell type and promoter context. In the case of the ER, the agonistic properties of some antiestrogens have been correlated with the activity of a specific transactivation domain (TAF-1) located within the amino terminus of the receptor (27, 28, 31). Recent work suggests that the levels and capacity of the liganded receptor to recruit specific coactivators and repressors to the transcription complex are important determinants of the degree of agonism or antagonism that is observed (32, 33).

While such models provide a framework with which to explain the tissue- and cell type-specific behaviors of some antagonists, they do not provide a ready explanation for one aspect of the behaviors of the AR antagonists studied in the current work. Namely, these models do not offer a rationale for the emergence of agonistic behavior of some AR antagonists at concentrations well above that required to inhibit the activation of the AR by saturating concentrations of DHT. Such observations could be reconciled by a contrasting view of the actions of steroid hormone receptor antagonists, such as that afforded by Jensen and co-workers (34, 35). In this view, a two-site model has been proposed to assist in accounting for the mixed agonist-antagonist properties displayed by some receptor antagonists. In such a model, in addition to competing for the cognate hormone-binding site, antihormones react with a second domain in the receptor, which is not recognized by cognate ligand and which plays a role in antagonist action (34, 35). Such a model would consider the difference between antihormones of different classes to result from their different relative affinities for the two binding regions. While such models have not been experimentally tested extensively, differences in the number of binding sites have been detected using [3H]tamoxifen and estradiol as ligands in binding assays. In a similar vein, the demonstration that a mutant PR deleted for the receptor carboxy terminus is able to bind and respond to RU486 but not to progesterone is also consistent with the existence of separable sites for the binding of progesterone antagonists and agonists within the PR hormone-binding domain (36). Finally, with respect to the mechanisms by which AR antagonists function, the existence of additional binding sites would offer a ready explanation for the appearance of agonism at concentrations of antagonist well above that needed to interrupt the action of AR agonists.

Flutamide is rapidly and extensively metabolized in vivo to hydroxyflutamide by hydroxylation. While both molecules have been shown to block the effects of androgens in target tissues (37, 38, 39, 40), flutamide has been estimated to do so with a 25-fold lower affinity relative to hydroxyflutamide for AR in vitro (41). For this reason, subsequent attempts to develop higher affinity AR antagonists have used hydroxyflutamide as a starting point. Our results suggest that antagonists with such structures (particularly with larger side chain substituents) may possess, by their very nature, a certain degree of inherent agonism that is unmasked at higher concentrations. It is conceivable that increased formation or accumulation of metabolites (such as hydroxyflutamide) within cells might contribute to the apparent resistance of prostate cancers that occurs in patients treated with such compounds. Such a mechanism would offer an alternative explanation for the flutamide withdrawal syndrome, particularly in those cases in which an AR mutation is not identified.

Finally, the addition of ligand to cultured cells has been shown to result in an increase in the level of AR, measured either using ligand-binding assays (17) or Western blotting (18). Previous studies have demonstrated that this effect is posttranslational and requires both an intact ligand-binding domain and the amino-terminal segment of the receptor molecule. The studies of Zhou et al. concluded that an interaction of the amino and carboxy termini of the protein were required to observe a ligand-induced stabilization of the receptor (42). These inferences were further supported by the results of immunoblot assays that measured the levels of expression of the intact AR and a number of mutant ARs containing deletions of varying sizes within the amino terminus (43). The present work suggests that amino acids within the most amino-terminal segments of the receptor participate directly in these interactions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals
Restriction enzyme were purchased from New England Biolabs, Inc. (Beverly, MA). PCR reagents were obtained from Perkin Elmer (Branchburg, NJ). 125 I-labeled goat F-(AB) antirabbit IgG was purchased from DuPont NEN research products (Boston, MA). Samples of the antiandrogens flutamide (batch no. IRQ-BTA-7-D-76D) and hydroxyflutamide (batch no. 26492–79) were generously provided by R. Neri (Schering Co., Bloomfield, NJ). Sample of the Casodex (ICI 176334 batch no PP-0195) was provided by G. Kolvenbag (Zeneca Pharmaceuticals, Macclesfield, Cheshire, U.K.). The solutions used were freshly prepared before use. 5{alpha}-Dihydrotestosterone was purchased from Steraloids, Inc. (Wilton, NH).

Site-Directed Mutagenesis
The segment 5' to methionine-188 in the original plasmid encoding the A-form of the AR [CMV776 (Ref.7)] was modified by replacing it with the sequence that is 5' to methionine-1 in the AR expression plasmid CMV3.1. This mutagenesis was accomplished by using two oligonucleotides (AR-As: 5'-ACACAGATCTAGGTGGAAGATTCAGCCAAGCTCAAGGATGCAACTCCTTCAGCAACAGCAGCAGGAA-3' and AR-Aas: 5'-GGCTGAGGGTGACCCAGAACCGGGT-3') as primers and the plasmid CMV3.1 (6) as template in a PCR reaction. The resulting PCR fragment, encoding the methionine-188 as the initiator methionine [i.e. deleting nucleotides 163–723 of the AR cDNA sequence (6)], was digested with the restriction enzymes NcoI and BglII and ligated into the 0.6-kb fragment purified after digestion of a sample of the CMV 3.1 hAR expression plasmid digested with the same restriction endonucleases. Vector fragments derived from the expression vector (CMV3.1) cleaved with the same enzymes. As shown in Fig. 1Go, the resulting plasmid (designated PS367) encodes a protein that is identical to hAR-B, except it lacked the segment encoding amino acids 1–187 of the hAR open reading frame. In this environment, the initiator methionine of the hAR-B (the codon encoding methionine-188 in CMV 3.1) is predicted to be a much better fit to the Kozak consensus sequence for translation initiation (16).

Cell Culture and Transient Transfection Assays
Stock cultures of the CV1 (monkey kidney fibroblast-like cell line) and DU145 cell lines (a metastatic human prostate carcinoma cell line) were obtained from the American Type Culture Collection and were maintained in MEM (GIBCO/BRL, Gaithersburg, MD) containing 10% (vol/vol) FCS and 1% penicillin and streptomycin. PPC1 cell line (considered to be a variant of PC-3 cell line) was obtained from Arthur Brothman (Salt Lake City, UT) and maintained in RPMI 1640 containing 10% (vol/vol) FCS and 1% penicillin and streptomycin.

Transfection assays were performed as described (43). The day before transfection, cells were trypsinized and plated at a density of 2 x 105 cells per well in six-well plates (each well 35 mM diameter) for reporter gene assays of AR function and a density of 1 x 106 cells per 10-cm dish for immunoblot analyses. Each six-well plate or 10-cm dish was transiently transfected by the addition of calcium phosphate precipitate containing the indicated concentrations of hAR isoform expression plasmid, the androgen-responsive reporter plasmid (10 µg), and 1 µg of a control plasmid (CMV-ß-galactosidase) in 12 ml (for six-well plates) or 10 ml (for each 10-cm dish) of culture medium for 24 h. After these incubations, the medium was removed and replaced with fresh MEM containing 5% charcoal-stripped serum alone or containing the various ligands. Forty-eight hours later, the cell cultures were harvested and assayed for luciferase activity and ß-galactosidase activity or harvested for immunoblot assays. In each experiment, measurements of the function of individual ARs were assessed in at least three separate transfections (wells) for each in the absence or presence of hormone. The results of these individual measurements were averaged and compared with the results obtained using the AR-B included in each experiment. In the text and legends, the functional assay results are presented either as stimulated luciferase values or as fold induction. This latter value is calculated by dividing the stimulated luciferase values by the basal luciferase values.

Immunoblots
Immunoblots were prepared as previously described. After transfer, the filters were incubated with affinity-purified antibody (anti-Internal A antibody) from rabbit U407 (8) that recognizes amino acids 200–220 of the hAR protein, which is preserved in both the AR-A and AR-B isoforms.


    FOOTNOTES
 
Address requests for reprints to: Michael J. McPhaul, M.D., Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-8857.

This work was supported by NIH Grants DK-03892 and 47657, a grant from the Robert A. Welch foundation (I-1090), and a grant from the Perot Family Foundation.

Received for publication July 14, 1997. Revision received January 2, 1998. Accepted for publication January 29, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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