A C619Y Mutation in the Human Androgen Receptor Causes Inactivation and Mislocalization of the Receptor with Concomitant Sequestration of SRC-1 (Steroid Receptor Coactivator 1)

Lynne V. Nazareth, David L. Stenoien, William E. Bingman, III, Alaina J. James, Carol Wu, Yixian Zhang, Dean P. Edwards, Michael Mancini, Marco Marcelli, Dolores J. Lamb and Nancy L. Weigel

Department of Molecular and Cellular Biology (L.V.N., D.L.S., W.E.B., A.J.J., C.W., Y.Z., M.M., M.M., D.J.L., N.L.W.), Scott Department of Urology (D.J.L.), and Department of Medicine (M.M.) Baylor College of Medicine Houston, Texas 77030
Department of Pathology (D.P.E.) University of Colorado Health Science Center Denver, Colorado 80262


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Androgen ablation therapy is a primary treatment for advanced prostate cancer, but tumors become refractive to therapy. Consequently, the role of the androgen receptors (ARs) and of mutations in the AR in prostate cancer has been a subject of much concern. In the course of analyzing tumors for mutations, we identified a somatic mutation that substitutes tyrosine for a cysteine at amino acid 619 (C619Y), which is near the cysteines that coordinate zinc in the DNA binding domain in the AR. The mutation was re-created in a wild-type expression vector and functional analyses carried out using transfection assays with androgen-responsive reporters. The mutant is transcriptionally inactive and unable to bind DNA. In response to ligand treatment, AR619Y localizes abnormally in numerous, well circumscribed predominantly nuclear aggregates in the nucleus and cytoplasm. Interestingly, these aggregates also contain the bulk of the coexpressed steroid receptor coactivator SRC-1, suggesting, in analogy to AR in spinal bulbar muscular atrophy, that this mutant may alter cellular physiology through sequestration of critical proteins. Although many inactivating mutations have been identified in androgen insensitivity syndrome patients, to our knowledge, this is the first characterization of an inactivating mutation identified in human prostate cancer.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Because of the importance of androgens in prostate growth and development and the initial success of androgen ablation in treatment of advanced prostate cancer (1, 2), much attention has been devoted to the role of androgen receptors (ARs) (3, 4) in prostate cancer. In normal prostate, androgens induce production of growth factors in the stromal cells that cause growth of the epithelial cells (5). The ARs in the epithelial cells induce the expression of various proteins characteristic of the differentiated state including prostate-specific antigen (6). Tumors develop from the epithelial cells and ultimately become independent of the prostate stromal cells. After an initial response to androgen ablation, the tumors in the prostate and their metastases eventually become unresponsive to endocrine therapy (7). Whether AR continues to play a role in androgen-independent disease is an open question. In many cases, it is likely that the cells have developed independent means of growth. AR usually continues to be expressed after androgen ablation therapy (4, 8) and the gene is sometimes amplified (9). Moreover, under some circumstances, activation of cell-signaling pathways induces AR activity in the absence of hormone (10, 11). Because the AR is encoded on the X chromosome and therefore is present as a single copy in males, attention has also been focused on whether the AR is mutated in prostate cancer tumors and the potential consequences of these mutations.

There have been several investigations of AR gene alterations/mutations in prostate cancer (3, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). It appears that the incidence of mutations increases dramatically in patients with more advanced disease (3, 12, 16, 17, 18) as there are fewer reports of mutations in early stage disease (3, 12, 17). AR mutants that respond to the AR antagonist, flutamide, as an agonist have been detected in patients who have failed flutamide therapy (20), suggesting a role for these mutations in the response of the tumors. In the course of searching for AR mutations in prostate cancer samples, we identified a mutation at cysteine 619, a non-zinc-coordinating cysteine immediately carboxyl terminal of the second zinc finger that is conserved in all steroid receptors. The consequences of amino acid substitutions at this position in the glucocorticoid receptor vary from no effect to partial inactivation, depending upon the substitution and the species of glucocorticoid receptor (25, 26). We find that this mutation in the AR not only inactivates the receptor, but also causes hormone-dependent aggregation of the AR. Protein aggregates have been detected in a number of diseases caused by expansion of CAG repeats, resulting in extended polyglutamine tracts (27, 28, 29, 30, 31). Included among these is spinal bulbar muscular dystrophy (SBMA), which is caused by an expansion of the polyglutamine tract in the amino terminus of the AR (30). We and others (32, 33) have found that this expansion induces hormone-dependent aggregates, although the morphology differs from those of the C619Y mutant. Our finding that the C619Y mutant binds and causes colocalization of SRC-1 (steroid receptor coactivator 1), suggests that transcriptionally inactive AR may nonetheless alter cellular physiology.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The AR gene has been cloned and mapped to the X chromosome (34, 35, 36, 37, 38, 39). It is comprised of eight exons generally containing between 910 and 919 amino acids, depending on the variable length of the polyglutamine repeat in the amino terminus of the receptor. In the course of screening primary prostate cancer tumors and associated lymph node metastases for AR mutations using single-strand conformational polymorphism, we found a substitution of a tyrosine for the conserved cysteine carboxyl terminal of the second zinc finger (M. Marcelli, M. Ittmann, S. Mariani, R. Sutherland, R. Nigam, L. Murthy, Y. Zhou, D. DiConcini, E. Puxeddu, A. Esen, J. Eastham, N. L. Weigel, and D. J. Lamb, submitted). The location of the mutation in the AR DNA binding domain is depicted in Fig. 1AGo. Because a length of 919 amino acids is used for Web-based collections of AR mutations (http://www.mcgill.ca/androgendb), we have termed this mutation C619Y to conform to this nomenclature.



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Figure 1. Functional Analysis of the Mutant AR

The location of the mutation in the AR DNA binding domain is indicated by the arrow in panel A. CV-1 cells (panel B) and PC-3 cells (panel C) were transfected with 0.5 µg of the GRE2E1bCAT reporter alone (first set of bars) or in conjunction with 62.5 ng (CV1 cells) or 5 ng (PC-3 cells) of the WT or mutant AR (C619Y) indicated by the second and third sets of bars, respectively. The cells were treated with ethanol vehicle, 10 nM, or 0.1 nM R1881. Error bars are as indicated (SD of the mean for triplicates).

 
Mutation of Cysteine 619 to Tyrosine Results in a Loss of Transcriptional Activity
To determine whether this mutant is transcriptionally active, the androgen-inducible GRE2E1bCAT reporter was transfected into CV1 cells (Fig. 1BGo) or PC-3 prostate cancer cells (panel C) in the absence or presence of the wild type (WT) or mutant (C619Y) AR expression vectors. Cells were treated with vehicle (ethanol), or the synthetic androgen, R1881 (10 nM or 0.1 nM). As expected, the reporter alone is not activated by R1881 in either cell line (shown in the first set of bars). The WT receptor is strongly activated by either concentration of R1881 (the second set of bars). However, the C619Y receptor is not activated at either level of androgen (the third group). In data not shown, the C619Y receptor, cotransfected with an estrogen-responsive ERE2E1bCAT reporter, was also found to be inactive, indicating that the mutation did not alter the specificity of interaction with this DNA response element.

C619Y Does Not Undergo the Hormone-Dependent Change in Mobility on SDS-PAGE Analysis That Is Characteristic of WT AR
To be certain that mutation did not alter receptor expression, high-salt extracts were prepared from COS-1 cells transfected with WT or C619Y receptors and analyzed by SDS-PAGE followed by immunoblotting. One of the characteristics of the WT AR is hormone-dependent phosphorylation resulting in reduced mobility on SDS gels. The AR is detected as a doublet of 112–110 kDa corresponding to different phosphorylation isoforms (40, 41), as indicated by the arrow (Fig. 2Go). Lanes 1–4 represent WT receptor and lanes 5–8 represent C619Y receptor (duplicates). Lanes 1 and 2 and lanes 5 and 6 were from control (ethanol-treated cells) extracts, and lanes 3 and 4 and lanes 7 and 8 were from cells treated with 10 nM R1881. Expression levels for the receptors are similar. The WT receptor shows an increase in the 112-kDa form in the presence of R1881 (the upper band becomes more pronounced compared with control) as previously reported (41). In contrast, the C619Y receptor does not show a corresponding increase in the 112-kDa form on steroid treatment, suggesting a failure to undergo a hormone-dependent posttranslational modification.



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Figure 2. Expression of WT and C619Y AR

An immunoblot of COS-1 cells transfected with WT or C619Y receptors and treated with ethanol vehicle or 10 nM R1881 is shown. Thirty micrograms of total protein were electrophoresed (in duplicate) on a 6.5% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with monoclonal antibody AR441 to the AR. Lanes 1–4 correspond to WT AR and lanes 5–8 to C619Y AR-transfected extracts. Lanes 1, 2, 5, and 6 correspond to vehicle treatment, and lanes 3, 4, 7, and 8 represent R1881-treated extracts. The AR protein is visualized as a doublet of 112–110 kDa as shown by the arrow.

 
The Mutant Receptor Has A Slightly Lower Steroid Binding Affinity Than WT Receptor
Since the mutation is carboxyl terminal of the second zinc finger of the DNA binding domain, and we did not detect a hormone-dependent change in mobility on SDS-PAGE gels, we next tested the hormone binding affinity of the receptors. COS-1 cells were mock transfected (GRE2E1bCAT DNA) or transfected with WT or C619Y expression vectors, and a whole-cell binding assay was performed using [3H]R1881. Scatchard analysis (Fig. 3AGo) showed that the dissociation constants (Kds) for the WT and C619Y receptors are 0.84 nM and 1.90 nM, respectively. This difference of a factor of 2 has been detected consistently in five separate experiments. Because transactivation was tested at concentrations of hormone that would essentially saturate the hormone binding site (10 nM), this difference is unlikely to explain the lack of transcriptional activity.



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Figure 3. Effect of the C619Y Mutation on Steroid Binding

A, Hormone binding affinity. COS-1 cells were mock transfected or transfected with 10 ng of the WT and C619Y AR expression vectors and treated with doses of [3H]R1881 ranging from 0.1 nM to 4 nM for 24 h. Cells were washed with PBS and bound receptor was extracted with 100% ethanol. Nonspecific counts were subtracted from total counts using the mock-transfected values to yield specific counts. A graph of bound/free vs. bound (nM) was plotted. The WT receptor is indicated by the solid circles, whereas the C619Y receptor is shown by the open circles. B, R1881 dissociation rate of the mutant AR is not altered. COS-1 cells were transfected with WT and C619Y receptors and were incubated with [3H]R1881 or radioinert R1881 for varying lengths of time as described in Materials and Methods. A graph of exponential counts bound vs. time (hours) was plotted to compare the R1881 dissociation rates for the two receptors. The WT receptor is depicted by the solid circles and the C619Y receptor by the open circles.

 
Dissociation Rates of R1881 for the C619Y and WT Receptors Are Similar
Zhou et al. (42) have described AR mutations with near-normal hormone binding affinity, but dramatically increased dissociation rates that reduce activity of the mutant. We next measured the dissociation rate of R1881 from C619Y. COS-1 cells were transfected with WT or C619Y expression vectors, and a dissociation rate assay was performed using [3H]R1881. From the plot of exponential counts bound over time (Fig. 3BGo), it can be seen that the two receptors have nearly parallel slopes, implying that their off rates are similar. In fact, when a plot of percent counts bound over time was done, the t1/2 values for the WT and C619Y receptors were very similar (217 and 204 min, respectively; data not shown).

In Vitro Analysis of DNA Binding
To test for DNA binding, extracts from R1881-treated COS cells containing WT AR, C619Y, or no receptor were incubated with labeled ARE (androgen response element) +/- AR441 antibody and analyzed by electrophoretic mobility shift assay (EMSA) as described in Materials and Methods (Fig. 4AGo). Although there are a number of DNA complexes formed by COS cell extracts alone (lane 5), there is an additional complex in cells transfected with AR (lane 1) that is supershifted by addition of AR441 (lane 2). In contrast, AR619Y fails to form a DNA complex either in the absence (lane 3) or presence (lane 4) of AR441. That this was not due to differences in expression levels is shown in the Western blot in Fig. 4BGo.



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Figure 4. Electrophoretic Mobility Shift Assay

COS cell extracts containing WT receptor, C619Y, or no receptor were incubated with radiolabeled ARE -/+ monoclonal antibody AR441 and analyzed by EMSA as described in Materials and methods. Lane 1, WT receptor; lane 2, WT + AR441; lane 3, C619Y; lane 4, C619Y + AR441; lane 5, mock; lane 6, probe alone. The position of the AR-ARE complex is as indicated by the arrow on the gel.

 
The Mutant Receptor Does Not Bind to AREs in Whole Cells
Because EMSA conditions may be more stringent than in vivo DNA binding, a promoter interference assay was used as described previously to directly assess DNA binding in vivo (43, 44). The promoter interference reporter plasmid contains three consensus progesterone response element (PRE)/AREs positioned between the TATA box and the transcription start site of the chloramphenicol acetyltransferase (CAT) gene, all driven by the constitutive cytomegalovirus (CMV) enhancer/promoter. In theory, binding of AR to the AREs will sterically hinder assembly of the transcription complex and reduce CAT gene expression. As shown in Fig. 5Go, as increasing amounts of AR WT expression vector are transfected into cells, the transcription of the CMV-PRE3-CAT reporter is inhibited in a dose-dependent manner. However, the C619Y receptor does not significantly inhibit expression of the reporter gene, indicating that it does not bind to the AREs in vivo.



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Figure 5. Promoter Interference Assay

COS-1 cells were transfected with increasing amounts of WT or C619Y receptor expression vectors (ranging from 0 ng to 2 ng) and with the 0.1 µg of CMV-PRE3-CAT reporter. The cells were treated with 0.1 nM R1881 and harvested to assay for potential inhibition of CMV-driven CAT activity by the receptors binding to the AREs in the reporter construct. The WT receptor is represented by the solid circles and the C619Y receptor by the open circles.

 
Subcellular Localization of the AR and the Steroid Receptor Coactivator SRC-1
To determine whether the subcellular localization of AR was altered in the C619Y mutant, we transfected HeLa cells and looked at receptor distribution using the AR441 antibody. As shown in Fig. 6Go, in the absence of hormone, both WT and C619Y receptors exhibited a diffuse and predominantly cytoplasmic distribution [panels A (WT) and D (C619Y)]. The addition of 5 nM R1881 for 16 h resulted in the nuclear localization of both receptors (panels B and E). The WT AR localizes in a hyperspeckled distribution in the nucleoplasm (panel B). Remarkably, after 16 h of hormone treatment, the C619Y receptor is found localized to ligand-dependent aggregates in the nucleus and sometimes in the cytoplasm (panel E). Nuclear aggregates are uniform in shape and can range from barely detectable to 1–2 µm in diameter. That this pattern is not a fixation artifact is shown in panels C and F in which GFPAR [green fluorescent protein (GFP)-linked AR] displays normal nuclear distribution whereas the GFPAR619Y mutant forms aggregates. The differences in appearance of these aggregates in panels E vs. F is at least partially due to the inability of the antibody to penetrate the aggregates, resulting in nonuniform staining, whereas all ARs in panel F contain GFP. Although aggregate formation has been reported previously for artificially generated AR mutations in the zinc-coordinating cysteines (45) and for mutants with deletions that eliminate one or more of these cysteines, not all DNA binding mutants form aggregates (D. L. Stenoien and M. Mancini, unpublished results). To our knowledge, this is the first observation of hormone-dependent aggregate formation by an AR containing a single amino acid substitution. This suggests that substitution of the cysteine by the tyrosine affects the protein folding of the AR molecule, such that it forms aggregates with other AR molecules and possibly with other factors.



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Figure 6. The C619Y Mutation Alters the Hormone-Dependent Subnuclear Localization of the AR

Immunofluorescence was performed on HeLa cells transfected with WT or C619Y receptors. Panels A (WT) and D (619Y) show untreated cells immunostained with anti-AR and a FITC-conjugated secondary antibody. Both WT and C619Y have a diffuse and predominantly cytoplasmic distribution in the absence of hormone. Panels B (WT) and E (C619Y) show cells treated 16 h with 5 nM R1881. WT receptor translocates to the nucleus and adopts a hyperspeckled distribution in response to hormone. C619Y also translocates to the nucleus in response to hormone but forms intranuclear aggregates of varying size that are substantially different from foci containing WT receptor. Nuclear aggregates containing C619Y range in size and can be up to 2 µm in diameter. C619Y aggregates are found in almost all cells with detectable levels of immunofluorescence, suggesting that aggregates do not result from overexpression. In some cells cytoplasmic C619Y aggregates can be seen. To demonstrate that C619Y aggregates are not a staining or fixation artifact, GFP fusions of WT and C619Y were transfected into HeLa cells and microscopy was performed on live, unfixed cells. Panels C (GFP-WT) and F (GFP-C619Y) show live cells after hormone treatment. Both GFP-WT and GFP-C619Y localize similarly to their immunostained, untagged counterparts. Bar = 10 µm.

 
Because the C619Y retains hormone binding activity, we next asked whether the mutant would cause SRC-1 to associate with the aggregates in vivo. To test this, cotransfection experiments were performed with a green fluorescent SRC-1 (GFP-SRC-1) construct (32). This construct behaves similarly as WT SRC-1 in terms of its coactivation and localization properties (D. L. Stenoien, S. Onate, and M. Mancini, unpublished observations). When cotransfected with C619Y in the absence of hormone, C619Y is predominantly cytoplasmic whereas GFP-SRC-1 exhibits diffuse nuclear staining (Fig. 7DGo) indistinguishable from its distribution in the absence of AR (Fig. 7CGo). In the presence of R1881, GFP-SRC-1 has the same hyperspeckled distribution as AR and overlaps considerably with AR staining (Fig. 7AGo). In parallel experiments, cotransfection with the C619Y construct in the presence of R1881 leads to GFP-SRC-1 localizing with C619Y aggregates (Fig. 7BGo). After cell division, these aggregates can also be seen in the cytoplasm (data not shown). Thus, this transcriptionally inactive form of AR appears to retain the ability to associate with and, in this case, sequester SRC-1.



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Figure 7. GFP-SRC-1 Colocalizes with Both WT and C619Y Receptors

HeLa cells were transfected with WT+GFP-SRC-1 (A), C619Y+GFP-SRC-1 (B and D), or GFP-SRC-1 alone (C). Cells were treated 16 h with 5 nM R1881 (A and B), and immunofluorescence was performed on fixed cells using anti-AR antibody and Texas Red-conjugated secondary antibody. In cells treated with hormone (A and B), there is substantial overlap of both WT and C619Y signal (top row) with GFP-SRC-1 fluorescence (middle row); merged images (bottom row) demonstrate the high degree of overlap. In the absence of cotransfected receptor (C), GFP-SRC-1 has a nuclear distribution. To demonstrate that C619Y and GFP-SRC-1 colocalization is dependent upon hormone, immunofluorescence was performed on cells in the absence of hormone (D). No overlap of staining is observed with the predominantly cytoplasmic C619Y and the nuclear GFP-SRC-1. Bars = 10 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mutation described in this paper, in contrast to most of the previously described mutations in prostate cancer, is a somatic mutation causing loss of transcriptional function (an inactivating mutation). Germline inactivating mutations are often found in androgen insensitivity syndrome (AIS), and Takahashi et al. (46) reported the presence of inactivating mutations (frame-shift, nonsense, and missense) in latent carcinomas in the Japanese but not American populations. In contrast, no inactivating mutations were identified in clinical carcinoma cases from either country. Fenton et al. (20) have identified several AR mutations in patients that failed flutamide treatment which allow the AR to utilize flutamide as an agonist. In contrast, the C619Y mutation was identified in a patient who was initially classified as having organ-confined cancer until lymph node metastases were identified during surgery. This naturally occurring mutation involved substitution of a highly conserved cysteine in the DNA binding domain, which obliterates AR function. Although this residue is not one of the cysteines involved in binding zinc, it is conserved across members of the steroid receptor superfamily. Replacement of the corresponding cysteine with a glycine residue in the human glucocorticoid receptor results in a receptor that is unable to bind DNA and unable to stimulate transcription of an inducible MMTVCAT reporter, but represses the {alpha}-glycoprotein hormone promoter up to 70% of WT function (26). In contrast, replacement of the same residue by alanine or serine in the rat glucocorticoid receptor did not compromise receptor function (25). Hence, it was not immediately clear what effect the tyrosine substitution at this position in AR would have on receptor function. The AR mutant is transcriptionally inactive and does not bind DNA in vitro or in vivo. The hormone binding affinity is slightly lower, but this difference is insufficient to account for loss of activity.

The immunolabeling studies performed shed some light on how this mutation may alter AR function. The C619Y receptor, like the WT receptor, translocates from the cytoplasm to the nucleus during 16 h of androgen treatment, indicating that the mutation does not completely block the hormone-induced localization of AR. However, once inside the nucleus, it adopts an altered distribution and forms discrete foci that develop into large clusters of mutant receptor. These aggregates are observed in more than 90% of the cells expressing detectable levels of AR. In the absence of hormone, the apparently normal cytoplasmic appearance of the mutant suggests that the heat shock proteins bind to the mutant and either correct or mask the misfolded area. Hormone treatment results in dissociation from heat shock proteins, thus allowing potentially abnormal interactions of the AR containing the misfolded region with other AR molecules and possibly with other proteins. Whether the change in folding induced by the mutation produces a hydrophobic surface that induces aberrant interaction with additional AR molecules leading to aggregration or whether the misfolded protein is recognized by the chaperone machinery inducing aggregration remains to be determined. Abnormal protein aggregation through expanded polyglutamine tracts has been reported in a number of diseases (27, 28, 29, 30). Among these is SBMA or Kennedy’s disease, in which AR aggregates have been detected in the cytoplasm and/or nucleus of affected motor neurons (31), upon immunocytochemical staining (30). The AR aggregates in the patients and in transfected cells differ from the C619Y aggregates both in their size (much larger) and cellular localization (30, 32). Interestingly, many SBMA patients also exhibit symptoms of AIS as they get older, suggesting that the activity of the receptors with expanded polyglutamine repeats is compromised. SBMA is not due to a simple lack of sufficient AR as demonstrated by the fact that most AIS patients do not have SBMA. Hence the presence of the abnormal protein must have a deleterious effect on cell physiology.

The C619Y mutant also fails to undergo the characteristic hormone-dependent increase in phosphorylation detected by altered mobility on SDS-PAGE analysis of the WT AR. This implies that the phosphorylation either requires specific subcellular localization to a region in the nuclear matrix containing the appropriate kinases (47, 48, 49) or DNA binding. Alternatively, misfolding within the aggregates (or elsewhere) blocks access of the kinase to the phosphorylation sites. Nevertheless, the C619Y receptor colocalizes with the coexpressed SRC-1 in the aggregates, demonstrating that interactions between steroid receptors and coactivators may occur in the absence of DNA binding and transcription. This interaction presumably occurs through interactions of SRC-1 with the hormone-binding domain as in the WT, since, as judged by hormone binding affinity, the mutation has little effect on the hormone-binding domain. The ability of C619Y to sequester SRC-1, and potentially other factors that interact with AR, suggests that mutants that are transcriptionally inactive may still have profound effects on cell function through reduction in available levels of other factors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
All cell culture media and reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD). R1881 (radiolabeled and radio inert forms) was obtained from NEN Life Science Products (Boston, MA), [{alpha}-32P]dGTP was from ICN Pharmaceuticals (Costa Mesa, CA) and poly-L-lysine was from Sigma. All other chemicals were reagent grade.

Case Report
The patient was a 62-yr-old white male found to have metastatic prostate cancer (stage D1, Gleason grade 3+3) during exploratory surgery. Metastatic deposits were found in the right iliac and hypogastric lymph nodes. The mutation was identified in this tissue (M. Marcelli, M. Ittmann, S. Mariani, R. Sutherland, R. Nigam, L. Murthy, Y. Zhou, D. DiConcini, E. Puxeddu, A. Esen, J. Eastham, N. L. Weigel, and D. J. Lamb, in preparation).

Transfection
Monkey kidney CV-1 and COS-1 cells and human prostate PC-3 cells were transfected by the nonrecombinant adenoviral-mediated DNA transfer technique (11, 50). Plasmid DNA for transfection (62.5 ng of AR expression vector for CV1 cells, 5 ng for PC-3 cells, and 10 ng for steroid binding assays along with 0.5 µg of the androgen-inducible GRE2E1bCAT reporter) (51) were incubated with the coupled virus (at a multiplicity of infection of 750:1 for CV1 cells, and 500:1 for PC-3 and COS-1 cells) for 30 min. For EMSAs, 100 ng AR expression vector and 0.5 µg CMV-ß-galactosidase carrier DNA were used. Subsequently, additional poly-L-lysine (1.3 µg/µg of DNA) was added to shrink the DNA onto the viral surface. The virus-DNA complex was added to the cells and allowed to infect the cells for 2 h in serum-free medium, after which time the medium was supplemented with charcoal-stripped serum to a final concentration of 5%. Each experiment was performed a minimum of three times.

Cell Treatment
Twenty-four hours after transfection, the transfected CV-1, COS-1, and PC-3 cells were treated with 10 nM or 0.1 nM R1881 (methyltrienolone from NEN Life Science Products or 0.2% ethanol vehicle (control) or as described in the individual procedures.

Chloramphenicol Acetyltransferase (CAT) Assay
After 24 h of treatment, the cells were harvested by scraping in TEN buffer (40 mM Tris, 1 mM EDTA, 150 mM NaCl, pH 8.0) and pelleted by centrifugation. Cells were resuspended in 0.25 M Tris, pH 7.5, and lysed by freeze thawing. Protein concentrations were determined by a microtiter plate assay (Bio-Rad Laboratories, Inc. Hercules, CA) that is a modification of the Bradford assay (52). The CAT activity was determined by incubation of 2.5–20 µg of total protein with [3H] chloramphenicol (20 µCi/µmol)(NEN Life Science Products and butyryl-CoA as previously described (53). Acylated chloramphenicol was extracted using a 2:1 mixture of tetramethyl pentadecane:xylene and counted in a scintillation counter.

AR Antibody Generation and Characterization
The peptide CSTEDTAEYSPFKGGYTK, corresponding to amino acids 301–317 of the human AR, with an added cysteine residue at the amino terminus for cross-linking, was synthesized and coupled to Keyhole Limpet Hemocyanin by Macromolecular Resources, Colorado State University (Ft. Collins, CO). Immunizations, cell fusions, and construction of hybridomas were done as previously described (54). The antibody was purified from a cell culture supernatant by 50% ammonium sulfate precipitation followed by purification on a protein G Sepharose column. The antibody specifically detects AR by Western blot analysis, immunoprecipitation, and immunocytochemistry (D. P. Edwards and N. Weigel, unpublished observations).

Western Blot Analysis
Cells were harvested in 0.4 M NaCl homogenization buffer (50 mM potassium phosphate, pH 7.4, 50 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, and 12 mM {alpha}-monothioglycerol) with protease inhibitors (leupeptin, antipain, aprotinin, benzamidine HCl, chymostatin, pepstatin, phenylmethylsulfonyl fluoride). Thirty micrograms of protein were electrophoresed on a 6.5% SDS-PAGE gel, transferred to nitrocellulose using a liquid transfer apparatus (Bio-Rad Laboratories, Inc.), and the filter was processed as follows. The membrane was blocked with a solution of Tris-buffered saline (TBS, 10 mM Tris, 150 mM NaCl, pH 7.5 solution) containing 1% milk for 10 min, incubated with the anti-AR antibody described above (at a concentration of 0.05 µg/ml) for 1 h, washed with TBS, incubated with a secondary rabbit antimouse antibody (2:10,000 dilution of a 1 mg/ml stock) for 1 h, washed with TBS, followed by TBS + 0.1% Tween. The membrane was then incubated with antirabbit horseradish peroxidase (1:10,000 dilution) for 1 h and washed with TBS + 0.3% Tween solution, followed by TBS + 0.1% Tween. The signal was detected using the ECL kit from Amersham Pharmacia Biotech (Arlington Heights, IL).

Scatchard Analysis
A whole-cell binding assay was performed in transfected COS-1 cells as described in Ref. 55 . Cells were transfected with WT, C619Y, or GRE2E1bCAT DNA as a control. After 24 h, the cells were treated with [3H]R1881 (specific activity = 86 Ci/mmol) at doses ranging from 0.1 to 4 nM for 24 h. Cells were washed five times with ice-cold PBS and [3H]R1881 extracted with ethanol. Samples were counted in scintillation cocktail in a LS6500 counter (Beckman Coulter, Inc., Fullerton, CA). Nonspecific counts (from mock-transfected cells) were subtracted from total counts to yield specific counts bound. The Kd values for the two receptors were determined by Scatchard analysis.

R1881 Dissociation Rate Analysis
The off-rate for steroid dissociation from the receptor was calculated as described by Zhou et al. (42). Briefly, COS-1 cells in six-well plates were transfected with 10 ng of AR WT or C619Y receptors. The next day, the medium was replaced with serum-free medium. Five nanomolar [3H]R1881 was added to all of the wells, and then cells were incubated in the absence (total) or presence of a 100-fold molar excess of radioinert R1881 (nonspecific) for 2 h at 37 C. Cells were washed twice with ice-cold PBS, the medium was replaced, and once again the cells were incubated in the absence or presence of a 1000-fold molar excess radioinert R1881 at 37 C. At the designated time points (0, 1, 2, 3, 4, and 5 h), cells were washed twice with PBS, and the hormone was extracted in ethanol and counted in scintillation cocktail. Specific binding was obtained by subtracting nonspecific counts from the total counts. A plot of bound counts vs. time was used to determine whether the two receptors have similar t1/2 values (time required for dissociation of half of the counts bound to the receptor). The t1/2 for each receptor was calculated from a plot of percentage counts bound vs. time.

Electrophoretic Mobility Shift Assay
The ARE oligo DNA used for band shift analysis is a single steroid response element from the tyrosine amino transferase gene as described by Tsai et al. (56). The probe was labeled with {alpha}-[32P]dGTP (3000 Ci/mmol) using DNA polymerase (Klenow fragment) and purified using a Nensorb column (NEN Life Science Products). COS-1 cells that were transfected with WT AR or C619Y DNA and treated with 10 nM R1881 were freeze thawed and harvested in 0.4 M NaCl TEDG (10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, and 10% glycerol), and cell extracts were prepared as before. Additional R1881 was added to the cell extract to a final concentration of 10 nM. A reaction mix was prepared containing 4 µl labeled ARE (~300,000 cpm), 14 µl 10% Ficoll, 15 µl 1 mg/ml nuclease-free BSA, 15 µl 0.1 M dithiothreitol, 45 µl 35 µg/ml pBR322 cut with HinF, and 15 µl of TEDG as described previously (57). Incubations included two µg of cell extracts, 5 µl of reaction mix +/- 1 µl AR441 anti-AR antibody in a final volume of 17 µl. Samples were incubated on ice for 40 min and run on a 5% gel in TAE (40 mM Tris acetate, 1 mM EDTA, pH 8.3). The gel was prerun at 200 V for 30 min in the cold room and, after loading, was run at 200 V until the bromophenol blue dye moved to one inch from the bottom of the gel. Labeled complexes were detected by autoradiography after the gel was dried.

Promoter Interference Assay
A promoter interference reporter plasmid (CMV-PRE3-CAT) described previously (43) was used to determine the ability of the AR to bind to DNA in whole cells. The plasmid has the constitutive CMV promoter followed by three synthetic PRE/ARE oligonucleotides inserted into the unique SacI site of CMV-TATA-CAT (kindly provided by Dr. Benita Katzenellenbogen) located between the TATA box and the start of transcription of the CAT gene. COS-1 cells were cotransfected as before with 0–2 ng of AR WT or C619Y expression vectors and 0.1 µg of CMV-PRE3-CAT plasmid and treated with 0.1 nM R1881 for 24 h before harvesting. Two micrograms of total protein were assayed for CAT activity.

Immunolabeling and Image Acquisition
Hela cells were grown in OptiMEM medium (Life Technologies, Inc.) supplemented with 4% FBS. For the transfections, cells were plated onto poly-D-lysine coated coverslips in 35-mm wells at a density of 1 x 105 cells per well. The GFP-WTAR and GFP-SRC-1 plasmids were made as described previously (32). The GFP-C619Y plasmid was generated using the same protocol as for GFP-WT. The C619Y sequence was cut out of the original vector using an internal XbaI and a 3'-BglII site. This segment was subcloned into a pEGFP-C1-AR(aa1–108) vector using the XbaI and BamHI sites to generate the GFP-C619Y plasmid. Cells were transfected with 0.1–0.5 µg of DNA (AR expression vectors and/or the GFP-SRC-1 construct) using the calcium phosphate mammalian cell transfection kit (5 Prime-> 3 Prime, Inc, Boulder, CO). Cells were shocked with 10% dimethylsulfoxide for 3 min and then transferred to DMEM with 5% stripped FBS. After 4 h, the cells were treated with ethanol or 5 nM R1881 for 16 h. Cells were then fixed with a 4% paraformaldehyde solution in PEM buffer (80 mM piperazine-N,N,'-bis(2-ethanesulfonic acid), 1 mM EDTA, 1 mM MgCl2, pH 7.4), extracted with 0.5% Triton X-100 in PEM, and blocked with 5% milk in TBST (0.1 M Tris, 0.15 M NaCl, 0.1% Tween-20, pH 7.4). Cells were incubated with the monoclonal antibody to the AR (1 µg/ml in TBST with 5% milk) for 2 h, followed by incubation with fluorescein- or Texas Red-conjugated goat antimouse secondary antibody (Southern Biotechnology Associates, Birmingham, AL) diluted 1:600 in TBST with 5% milk for 1 h. A Z-series (0.1 µm steps) of optical sections was digitally imaged on a Delta Vision Deconvolution Microscopy System (Applied Precision, Inc., Issaquah, WA) and deconvolved using a constrained iterative algorithm to generate high-resolution images. All image files were digitally processed for presentation in Adobe Photoshop (Adobe Systems, Inc., San Jose, CA). Shown in each image is a single Z-section from typical cells. For live microscopy, HeLa cells were plated onto 40-mm coverslips in 6-cm dishes and transfected with 1 µg of either GFP-WTAR or GFP-C619Y. After hormone treatment, cells were transferred to a live cell chamber (Bioptechs, Inc., Butler, PA) and maintained in DMEM with 5% stripped FBS and 5 nM R1881 at 37 C. Image acquisition and processing were performed as above.


    ACKNOWLEDGMENTS
 
We wish to thank Dr. John Cidlowski (NIEHS, Research Triangle Park, NC) for providing us with the GRE2E1bCAT reporter, as well as Kurt Christensen and Teri Ladtkow, University of Colorado Cancer Center Tissue Culture/Monoclonal Antibody Core Facility (Boulder, CO), for assistance with production of the AR antibody. Imaging was performed in the Department of Cell Biology’s Integrated Microscopy Core; we thank Frank Herbert for technical support.


    FOOTNOTES
 
Address requests for reprints to: Dr. Nancy L. Weigel, Department of Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030.

This work was supported in part by NIH Training Grant HD-07165 (L.V.N.), a Cancer Research Foundation of America fellowship to L.V.N., a Merck United Negro College Fund fellowship (A.J.J.), NIH Training Grant T32 DK-07696 (A.J.J.), and NIH Grant CA-68615 as well as a generous gift from the Association for the Cure of Cancer of the Prostate to D.J.L., M.M., and N.L.W.

Received for publication September 18, 1998. Revision received August 10, 1999. Accepted for publication August 16, 1999.


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