Dehydroepiandrosterone Activates Mutant Androgen Receptors Expressed in the Androgen-Dependent Human Prostate Cancer Xenograft CWR22 and LNCaP Cells

Jiann-an Tan, Yousuf Sharief, Katherine G. Hamil, Christopher W. Gregory, De-Ying Zang, Madhabananda Sar, Paul H. Gumerlock1, Ralph W. deVere White1, Thomas G. Pretlow1, Stephen E. Harris, Elizabeth M. Wilson, James L. Mohler1 and Frank S. French1

Laboratories for Reproductive Biology Departments of Pediatrics (J.-A.T., K.G.H., C.W.G., D.-Y.Z., M.S., E.M.W., F.S.F.), Biochemistry and Biophysics (E.M.W.), Surgery (Y.S., J.L.M.), and Pathology (J.L.M.) and The Lineberger Comprehensive Cancer Center (E.M.W., F.S.F., J.L.M.) School of Medicine The University of North Carolina Chapel Hill, North Carolina 27599 Department of Urology and Medicine (P.G., R.dW.) University of California Davis Medical Center Sacramento, California 95817 Department of Medicine (S.E.H.) The University of Texas Health Science Center San Antonio, Texas 78284-7756 Department of Pathology (T.G.P.) School of Medicine Case Western Reserve University Cleveland, Ohio 44160


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
An androgen receptor (AR) gene mutation identified in the androgen-dependent human prostate cancer xenograft, CWR22, changed codon 874 in the ligand-binding domain (exon H) from CAT for histidine to TAT for tyrosine and abolished a restriction site for the endonuclease SfaNI. SfaNI digestion of AR exon H DNA from normal but not from prostate cancer tissue indicated H874Y is a somatic mutation that occurred before the initial tumor transplant. CWR22, an epithelial cell tumor, expresses a 9.6-kb AR mRNA similar in size to the AR mRNA in human benign prostatic hyperplasia. AR protein is present in cell nuclei by immunostaining as in other androgen-responsive tissues. Transcriptional activity of recombinant H874Y transiently expressed in CV1 cells in the presence of testosterone or dihydrotestosterone was similar to that of wild type AR. With dihydrotestosterone at a near physiological concentration (0.01 nM), H874Y and wild type AR induced 2-fold greater luciferase activity than did the LNCaP mutant AR T877A. The adrenal androgen, dehydroepiandrosterone (10 and 100 nM) with H874Y stimulated a 3- to 8-fold greater response than with wild type AR and at 100 nM the response was similar with the LNCaP mutant. H874Y, like the LNCaP cell mutant, was more responsive to estradiol and progesterone than was wild type AR. The antiandrogen hydroxyflutamide (10 nM) had greater agonist activity (4- to 7-fold) with both mutant ARs than with wild type AR. AR mutations that alter ligand specificity may influence tumor progression subsequent to androgen withdrawal by making the AR more responsive to adrenal androgens or antiandrogens.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human prostate cancer is androgen dependent initially, but after androgen withdrawal therapy it acquires the ability to grow in the absence of testicular androgens. Androgen receptor (AR) is expressed in the majority of untreated prostate cancers and in most that recur during androgen withdrawal therapy. This continued presence of AR suggests that androgen responsiveness may persist in tumors that recur during therapy. Major goals of current research on prostate cancer include identifying mechanisms that stimulate recurrent growth. One mechanism could involve mutations that alter the normal AR ligand-binding specificity, thereby enabling AR activation by adrenal androgens or antiandrogens used in cancer treatment. Other possible mechanisms include ligand-independent activation of AR and adaptation to alternate growth regulators.

The first AR missense mutation identified in human prostate cancer was in the LNCaP cell line (1, 2, 3, 4). Subsequently, amino acid substitutions were reported in organ-confined as well as metastatic tumors (5, 6, 7, 8, 9, 10, 11). Of the several AR mutants in prostate cancer that have been characterized thus far, the majority are functional and can mediate androgen-induced transactivation, in contrast to loss of function germ-line AR mutations that cause androgen insensitivity.

CWR22 is an androgen-dependent human prostate cancer xenograft propagated in male nude mice (12, 13). Upon androgen withdrawal, CWR22 prostate-specific antigen mRNA and protein decrease rapidly, cells undergo apoptosis (13, 14), and tumors regress in size; but after several months they recur in the absence of testicular androgens in a manner characteristic of human prostate cancers (13, 14). In this report a mutant AR with altered ligand specificity is identified and shown to be expressed in CWR22 epithelial cells. Ligand-dependent activation of the mutant AR in transient cotransfection assays is compared with that of the mutant AR in LNCaP cells. The CWR22 AR mutant is transcriptionally active in response to testicular androgen like wild type AR but differs from wild type in that it is also activated by the adrenal androgen, dehydroepiandrosterone, the antiandrogen, hydroxyflutamide, and by estradiol and progesterone.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Localization of AR in the CWR22 Human Prostate Cancer Xenograft
The majority of nuclei in CWR-22 stained positively with the AR antibody AR-52 (15), some more intensely than others (Fig. 1BGo). A preimmune control panel (Fig. 1CGo) indicated that staining in Fig. 1BGo is AR specific. Hematoxylin-eosin staining (Fig. 1AGo) illustrates the predominance of epithelial cells with lack of surrounding stroma. Northern blot analysis of CWR22 total RNA revealed a 9.6-kb AR mRNA (Fig. 2AGo) similar in size to the AR mRNA expressed in benign prostatic hyperplasia tissue (15). In an RT-PCR assay, expression of AR and prostate-specific antigen (PSA) mRNAs in CWR22 was similar to that in the androgen-responsive LNCaP cell line (Fig. 2BGo) and contrasted with the undetectable or very low levels in the androgen- unresponsive cell lines, DU145, PC3, and TSU-PR1. These results are consistent with the androgen-responsive and relatively differentiated state of CWR22. In mice bearing CWR22 xenografts, Wainstein et al. (13) observed that serum PSA concentrations correlate with tumor size, and blood levels of PSA decrease after androgen withdrawal. More recently, Gregory et al. (14) demonstrated a decrease in CWR22 PSA mRNA levels within 48 h after androgen withdrawal.



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Figure 1. Immunohistochemical Analysis of AR Protein Expressed in Androgen- Stimulated Epithelial Cells of CWR22 Tumor from an Intact Nude Mouse

Frozen tissue sections were fixed and immunostained with protein A-purified AR antibody, AR-52. A, Hematoxylin-eosin; B, AR-52; C, peptide-absorbed AR-52 control.

 


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Figure 2. Expression of AR mRNA in the Androgen-Dependent Human Prostate Cancer Xenograft, CWR22, and the Androgen-Responsive LNCaP Cell Line

A, Northern hybridization of CWR22 total RNA from an androgen-stimulated nude mouse using a human AR cDNA probe. The major AR mRNA species is 9.6 kb. B, Southern hybridization of AR and PSA, RT-PCR products from LNCaP, DU145, PC-3, and TSU-PR1 cells and CWR22. DU145, PC-3, and TSU-PR1 are androgen-unresponsive human prostate cancer cell lines. Although not apparent in this figure, PC3 cells contained a very low level of AR mRNA.

 
Identification of the AR Gene Mutation in CWR22
To search for the presence of an AR gene mutation, DNA from CWR22 was PCR amplified using sets of intron primers that bracket each exon (16, 17). Exon A was amplified using four overlapping sets of primers. All exons amplified to the correct size, indicating no major deletions. Analysis of AR exon H by denaturing gradient gel electrophoresis revealed extra doublet bands (Fig. 3BGo), suggesting a mismatch in the heteroduplex of wild type and CWR22 DNA. No similar indication of an AR gene mutation was detected in exons A through G. Automated sequencing of exon H DNA detected a C to T mutation at position 4046, numbered according to Lubahn et al. (16), that changed codon 874 from CAT coding for histidine to TAT coding for tyrosine (Fig. 3Go, A and C). The mutation was detected also by RT-PCR from CWR22 total RNA and manual sequencing of the cDNA region encoded by exon H. Substitution of T for C 4048 altered a SfaNI recognition sequence (GCATC to GTATC). SfaNI did not cut exon H DNA from the original CWR22 xenograft, indicating that the AR mutation was likely present in the tumor before transplantation. In contrast, SfaNI cut AR exon H DNA amplified from paraffin-embedded normal testicular tissue of the same patient, indicating absence of the C to T mutation (data not shown). Thus, H874Y was a somatic mutation in the prostate tumor not present in germline DNA.



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Figure 3. A Single Base Substitution C to T Changed His 874 to Tyr (H874Y) in the CWR22 AR

A, Schematic showing location of the missense mutation in exon H of the steroid-building domain. B, Denaturing gradient gel electrophoresis of exon H PCR-amplified DNA from wild type and CWR22 AR genes. C, Nucleotide sequence of the portion of CWR22 AR exon H containing a T substituted for the wild type C. The four nucleotides represented by solid, broken, and dashed lines are color coded in the original tracing.

 
Steroid-Dependent Transactivation of the CWR22 AR Mutant in Comparison with the LNCaP Cell Mutant AR
Since AR mutation H874Y in CWR22 tumor cells is located near AR mutation T877A of the LNCaP cell line, we compared the transcriptional activities of these mutants in response to a variety of ligands including adrenal and sex steroids and the antiandrogen, hydroxyflutamide. The mutant AR in LNCaP cells was identified earlier by cloning the AR cDNA from an LNCaP cell cDNA library in the {lambda}-ZAPII cloning vector (2, 3). AR mutations were constructed in the expression vector, pCMVhAR (18, 19), and cotransfected into CV1 cells together with a mouse mammary tumor virus long terminal repeat-luciferase reporter vector.

CWR22 AR mutant H874Y induced higher luciferase activity than wild type AR (P < 0.01) at 0.01 nM testosterone (T), a concentration close to the physiological range of free T in human blood (20) (Fig. 4Go). The response of H874Y to dihydrotestosterone (DHT) at 0.01 nM was similar to that of wild type AR and about 2-fold more active (P < 0.001) than LNCaP mutant T877A. Activity induced by 1 nM androstenedione was similar with H874Y or wild type AR and slightly higher (P = 0.001) than T877A (Fig. 4Go). Little transactivation was observed at lower concentrations of androstenedione. Because androstenedione has low affinity for the wild type AR (6), its induction of transcriptional activity with both mutant and wild type ARs likely resulted from conversion to T and DHT in CV1 cells. Relative responses of wild type and mutant ARs to T and DHT and to androstenedione were similar.



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Figure 4. Ligand-Dependent Transactivation by CWR22 (H874Y) and LNCaP (T877A) Mutants with T, DHT, and Androstenedione (ASD)

Transient cotransfection assays were performed in CV1 cells using a mouse mammary tumor virus long terminal repeat-luciferase reporter gene (2.5 µg) together with the full-length wild type human AR, pCMVhAR, or mutant AR expression vector (0.1 µg). Cells were incubated 40 h in the presence and absence of steroid at the concentrations indicated. Cells were lysed and luciferase activity measured in a luminometer as described in Materials and Methods. Data are plotted as means ± SEM.

 
The adrenal androgen dehydroepiandrosterone (DHEA) at 1 nM stimulated luciferase activity 2-fold with mutant and wild type ARs. At 10 nM DHEA, H874Y transcriptional activity was 3-fold greater (P < 0.001) than that of wild type AR and increased to about 8-fold greater (P < 0.001) at 100 nM DHEA (Fig. 5Go). At 100 nM DHEA, transcriptional activity with H874Y was similar to that of T877A. DHEA had little agonist activity with wild type AR up to 100 nM, indicating there was minimal conversion of DHEA to T or DHT in CV1 cells. This concentration of DHEA is similar to that reported in human prostate tissue as discussed below.



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Figure 5. Transactivation by CWR22 (H874Y) and LNCaP (T877A) Mutant and Wild Type AR with the Adrenal Androgen DHEA

Transient cotransfection assays were performed in CV1 cells as described in Fig. 4Go and Materials and Methods with concentrations of DHEA as indicated.

 
Estradiol at 0.1 nM stimulated 2-fold greater (P < 0.001) luciferase activity with H874Y than wild type AR, and this difference increased to several fold (P < 0.001) at 1 nM estradiol (Fig. 6Go). Induction of transcription with T877A was also greater (P < 0.001) than with wild type AR at 1 nM estradiol but was only about half that of H874Y. The physiological level of total serum estradiol in the adult male is approximately 0.1 nM whereas free estradiol is 1–2% of the total (20). There was little increase in transcription at 0.01 nM estradiol suggesting H874Y would not be activated in the adult male; however, aromatase activity in human prostate may cause tissue levels to be higher than in serum. The CWR22 mutant AR transcriptional response to 1 nM progesterone was greater (P < 0.01) than that of the wild type AR but less (P < 0.01) than the LNCaP mutant (Fig. 6Go). Total serum progesterone concentration in adult males, ~1 nM, is similar to that in females during the follicular phase of the menstrual cycle (20) and may be sufficient to activate a mutant AR in prostate.



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Figure 6. Transactivation by CWR22 (H874Y) and LNCaP (T877A) Mutant and Wild Type AR in the Presence and Absence of Estradiol (E2) or Progesterone at the Concentrations Indicated

Transient cotransfection assays were performed as described in Fig. 4Go and Materials and Methods.

 
The antiandrogen hydroxyflutamide had low agonist activity with mutant and wild type ARs at concentrations of 1 nM or less (Fig. 7Go). However, at 10 nM hydroxyflutamide, H874Y and T877A transcriptional activities were 4- and 6-fold greater (P < 0.001) than that of wild type AR (Fig. 6Go). Maximal luciferase activities stimulated by DHEA and hydroxyflutamide with the mutant ARs were 15–20% of activities induced by T and DHT.



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Figure 7. Transactivation by CWR22 (H874Y) and LNCaP (T877A) Mutant and Wild Type AR in the Presence and Absence of the Antiandrogen Hydroxyflutamide (OH-FL) at the Concentrations Indicated

Transient cotransfection assays were performed in CV1 cells as described in Fig. 4Go and Materials and Methods.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CWR22 is an androgen-dependent human prostate cancer epithelial cell xenograft lacking in stroma cells. We have shown that CWR22 expresses a mutant AR, H874Y, with altered ligand responsiveness. Absence of SfaNI digestion of PCR-amplified exon H DNA indicated CWR22 tumor cells express only the mutant AR. The CWR22 mutant AR mediated a transcriptional response to T and DHT similar to that of wild type AR. DHEA, estradiol, progesterone, and hydroxyflutamide induced greater transcriptional responses with the H874Y mutant than with wild type AR. H874Y-mediated transcriptional responses to DHEA and estradiol were greater whereas responses to progesterone and hydroxyflutamide were less than those of the LNCaP mutant T877A.

DHEA-induced transactivation by H874Y and T877A was highest with 100 nM DHEA, a concentration likely maintained in human prostate tissue by adrenal secretion of DHEA and DHEA-sulfate. Total serum DHEA in adult males (mean, ~25 nM) is within the range of total serum testosterone. However, relatively more serum DHEA should be available to prostate cells because it circulates loosely bound to albumin (20). Serum DHEA-sulfate can be 300–500 times the concentration of DHEA, and the sulfatase present in human prostate converts DHEA-sulfate to DHEA (21). Mean concentrations of DHEA-sulfate and DHEA in nonhyperplastic tissue specimens obtained by total prostatectomy were ~300 and 90 pmol/mg DNA, respectively (equivalent to 300 and 90 nM, assuming ~1 mg DNA/g tissue) (21). In a patient undergoing androgen withdrawal therapy by castration or LH suppression, DHEA could be in a concentration range sufficient to activate mutant ARs such as H874Y, T877A, or the AR mutant V715M identified in a prostate cancer bone marrow metastasis by Culig et al. (6). In human prostate, low levels of {Delta}4,5 isomerase activity limit DHEA conversion to {Delta}4 androgens (21). However, small amounts of DHT were formed from DHEA and DHEA-sulfate in human benign prostatic hyperplasia tissue (22).

Estradiol derived from peripheral conversion of adrenal androstenedione is a potential agonist for mutant ARs in prostate cancer. Estradiol induced an increase in luciferase activity with both the CWR22 and LNCaP mutants at a concentration of 10-9 M, less than the amount required for half-maximal stimulation of LNCaP cell growth (10-8 M). In our transcription assays, estradiol was a greater agonist with the CWR22 mutant AR than with the LNCaP mutant. However, CWR22 does not grow in female nude mice, indicating growth stimulation by H874Y is not activated by female mouse levels of estradiol. It was assumed the LNCaP mutant AR mediates the growth-stimulating effect of estradiol on LNCaP cells (2, 3, 4). However, a recent report demonstrated that LNCaP cells also contain estrogen receptors based on ligand binding and immunocytochemical assays (23). The antiestrogen ICI-182,780 inhibited estradiol stimulation of growth but did not inhibit growth induced by DHT, suggesting the growth effect of estradiol was mediated by estrogen receptors rather than by the T877A mutant AR. It remains to be determined whether higher concentrations of estradiol stimulate CWR22 tumor growth by way of the mutant AR. Estrogen receptors were not detected by immunostaining CWR22 tumor cells with three different antibodies to the human estrogen receptor, a finding in agreement with other immunohistochemical studies showing weak or undetectable staining of estrogen receptors in benign prostatic hyperplasia or prostate cancer epithelial cells (24). It is not yet established whether the human homolog of rat prostate estrogen receptor ß (25) is expressed in CWR22.

Hydroxyflutamide is the active metabolite of flutamide, an androgen antagonist used in the treatment of prostate cancer. Both H874Y and T877A mediated increased transcriptional activity at 10 nM hydroxyflutamide whereas there was no effect with wild type AR. Another AR mutant in human prostate cancer, V715M, was unresponsive to 100 nM hydroxyflutamide (6) but responded 2-fold greater than wild type AR to micromolar concentrations as did the prostate cancer AR mutant V730M (26). In the absence of androgen, hydroxyflutamide is an agonist with wild type AR at concentrations of 1 µM or higher (27, 28). As noted by Wong et al. (27) and by Kemppainen and Wilson (29), this could be of clinical importance in the regression of prostate cancer observed in some patients whose flutamide treatment has been withdrawn (30, 31). Prostate cancer patients treated with high-dose flutamide can have plasma levels of hydroxyflutamide greater than 1 µM. During androgen withdrawal therapy, hydroxyflutamide could function as an agonist when concentrations greatly exceed the concentration of androgen, although its affinity for wild type AR is at least 1 order of magnitude less than that of DHT (6, 27). AR mutants such as H874Y and T877A with enhanced responsiveness to hydroxyflutamide would be more active than wild type AR in mediating growth effects of hydroxyflutamide on prostate cancer cells. Half-maximal stimulation of LNCaP cell growth in culture was achieved with 10-8 M hydroxyflutamide (data not shown), in agreement with other studies (32, 33, 34). In general, the relative growth-stimulating activity of an AR agonist correlates with its binding affinity for AR and induction of AR-mediated transactivation in transient cotransfection assays (2, 3, 4, 32, 33, 34, 35, 36, 37).

Of the different AR missense mutations reported in clinical prostate cancer (reviewed in Refs. 11 and 38 and B. Gottlieb, Androgen Receptor Gene Mutations Database), most that were characterized functionally retain androgen-dependent transcriptional activity. V730M AR from a stage B, organ-confined prostate cancer had no loss of androgen binding or transcriptional activity (5, 39). The LNCaP cell line containing AR mutant T877A was derived from a lymph node metastasis. Three additional mutations, V715M, T877S, and H874Y, were identified in bone metastases (6, 7). Each of the mutants exhibited essentially normal transactivation in response to DHT. The V715M mutant showed no steroid binding abnormality but had increased transcriptional activity relative to wild type AR in response to the adrenal androgens, DHEA and androstenedione (6). Mutants H874Y and T877S were reported to have enhanced transcriptional activity with progesterone and estradiol at concentrations of 10 and 100 nM (7). Mutant Q919R at the carboxyl terminus of AR remained functional in response to DHT, with only partial loss of activity (40). The only loss of function mutations reported in association with prostate cancer are a single base change that introduced a stop codon (Trp 794 to stop) in the steroid-binding domain (9) and C619Y in exon C close to the DNA-binding domain (40). By analogy with the crystal structures of the thyroid hormone receptor and retinoic acid receptor {gamma} ligand-binding domains (41, 42), AR ligands are buried in a hydrophobic pocket formed by several {alpha}-helices folded in a way such that widely separated amino acids are brought together to form the pocket. AR amino acids 874 and 877 are located within a conserved heptad repeat of hydrophobic residues (38, 43) in {alpha}-helix 11 (41).

Since AR is required for androgen-dependent growth of prostate cancer cells, tumors may select for AR mutants that retain activity. Most prostate cancers that relapse during androgen withdrawal therapy express AR at levels similar to androgen-dependent prostate cancer (Refs. 44 and 45 and J. L. Mohler, unpublished). Functional AR mutants with enhanced responsiveness to adrenal androgens and/or antiandrogens could have an active role in the survival and recurrent growth of prostate cancer cells. Moreover, the altered spectrum of ligand responsiveness may potentiate ligand-independent activation of AR in relapsed prostate cancers with increased kinase activities (46, 47, 48).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Monkey kidney CV1 cells were obtained from the American Type Culture Collection. DMEM/high glucose and DMEM:F12 were from GIBCO (Grand Island, NY), and FCS was purchased from Hyclone Laboratories, Inc. (Logan, UT). CV1 cell lysis buffer was supplied by Ligand Pharmaceuticals Inc. (San Diego, CA); D-luciferin was from Analytical Luminescence (Westlake Village, CA). Hydroxyflutamide was provided by R. O. Neri Schering Corp. (Bloomfield, NJ); other steroids, buffers, and chemicals were from Sigma (St. Louis, MO), E.M. Science (Fort Washington, PA) and Fisher (Pittsburgh, PA). Taq polymerase and AMV reverse transcription were from Promega (Madison, WI), and Deep Vent DNA polymerase and SFaNI were from New England Biolabs (Beverly, MA).

Human Prostate Cancer Tissue (CWR22)
CWR22 is an androgen-dependent, human prostatic carcinoma xenograft established from a transurethral resection and grown subcutaneously in male athymic nude mice, each containing a 12.5-mg testosterone pellet (Innovative Research, Sarasota, FL) implanted subcutaneously (12). The tumor was stage D with osseous metastases, Gleason grade 9. After removal of CWR22 xenografts from nude mice, tumor tissue was frozen immediately in liquid nitrogen and stored at -80 C.

Immunocytochemistry
Small pieces of fresh tumor tissue (~1 cm2 x 0.3 cm thick) were frozen in isopentane precooled in liquid nitrogen on brass mounts using minced rat liver cushions as an adhesive. Tissue was stored in liquid nitrogen until sectioned at 6 µm thickness using a cryostat at -31 C. Preparation of antibody AR-52 and the method for immunostaining were as described previously (15, 49, 50).

Nucleic Acid Extraction and Northern Hybridization of Human AR
Frozen CWR22 tumor tissue was thawed, finely minced with scalpels, digested with proteinase K at 37 C overnight, and DNA extracted with phenol-chloroform. RNA was removed by RNase digestion and phenol-chloroform extraction. For extraction of normal testicular tissue DNA from the same patient, 6 µm thick sections were cut from paraffin blocks and deparaffinized in xylene. Tissue was digested with proteinase K, and DNA was isolated by NaCl deproteinization (10). Total RNA was extracted by a single-step method using acid-guanidinium thiocyanate-phenol-chloroform (51). Northern hybridization of AR mRNA was as described by Quarmby et al. (52).

Cell Lines
The human prostate tumor cell lines LNCaP, DU145, and PC-3 were obtained from the American Type Culture Collection (Rockville, MD) and maintained according to their instructions. The TSU-Pr1 cell line was generously provided by Dr. Edward Gelmann (Georgetown University School of Medicine, Washington DC) and maintained in DMEM supplemented with 10% FBS. Cultures were harvested for RNA analysis at approximately 80% confluence.

RT-PCR Analysis of Human AR and PSA mRNA
RT-PCR analysis of AR mRNA was done essentially as previously described (45). Briefly, total RNA was extracted from the cell lines and xenografts using guanidinium isothiocyanate and CsCl density gradient centrifugation. Total RNA was transcribed to cDNA using RT primed with random hexamers. Complementary DNA equivalent to 25 ng of the initial total RNA was subjected to RT-PCR using the following oligonucleotides as primers for the AR transcripts: PG45 (upstream, sense) 5'-CCTGATCTGTGGAGATGAAGCTTC-3' and PG46 (downstream, antisense) 5'-TGTCGTGTCCAGCACACACTACAC-3'. These primers are located in exons B and D, respectively, and create a 495-bp PCR product from cDNA. The oligonucleotide PG42 (5'-TGGGAGCCCGGAAGCTGAAGAAAC-3') (spanning the junction of exons C and D) is internally positioned to the two primers and served as the probe for detecting the products in Southern blotting. PCR was run for 36 cycles under the conditions of 95 C (30 sec), 60 C (30 sec), and 72 C (30 sec). The 36 cycles were followed by a 10-min extension phase at 72 C. Southern blotting of the PCR products was done as previously reported using a nonisotopic enzyme chemiluminescent detection system (53). For analysis of expression of PSA transcripts, the primers PSA-494 (upstream, sense) 5'-TACCCACTGCATCAGGAACA-3' and PSA-894 (downstream, antisense) 5'-GTCCAGCGTCCAGCACACAG-3' were used to create a 421-bp product (54). PCR reaction parameters were 94 C for 15 sec, 60 C for 15 sec, and 72 C for 45 sec for 35 cycles followed by a 10-min extension at 72 C. The oligonucleotide used as the probe for Southern blotting of PSA RT-PCR products was PSA-559 5'-ACACAGGCCAGGTATTTCAG-3'. Sample loadings were adjusted to equivalent levels of c-N-ras as previously described and validated by Fishman et al. (55)

Analysis of the AR Gene in CWR22
Amplification of AR gene DNA.
Exons B through H were amplified individually using GC-clamped intron primers. Due to its large size, exon A was amplified in four fragments using exon primers (16, 17). PCR was performed for 30 cycles of denaturation (95 C for 1 min), annealing (60 C for 1 min), and extension (72 C for 2 min). In each PCR a negative control containing no DNA was included. PCR product size was verified by electrophoresis on 1% agarose gels in Tris-borate buffer containing 2 mM EDTA, 50 mM Tris base, 50 mM boric acid, pH 8.0 (TBE), and ethidium bromide staining.

Denaturing Gradient Gel Electrophoresis.
Electrophore-sis of amplified AR was performed on denaturing gradient gels as described (17). Heteroduplexes for each exon were formed by mixing equal amounts of test sample and wild type PCR products. DNA was denatured at 95 C and reannealed by slow cooling to room temperature.

RT-PCR for Splicing Errors.
Total RNA (1 µg) was reverse transcribed into cDNA using AMV RT and oligo (dT)20 primer. Primers for PCR were chosen to amplify all cDNA splice sites from exons A through H, a region of 1296 bp. Amplified AR cDNA fragments were electrophoresed in 1% agarose gels in TBE and stained with ethidium bromide.

Restriction Site Analysis.
Approximately 200 ng of PCR product of exon H amplification were incubated with 2 U of the restriction enzyme SfaNI in a total volume of 20 µl of NE Buffer 3 for 16 h as recommended by New England Biolabs (Beverley, MA). After incubation, the enzyme was heat inactivated and the DNA analyzed by electrophoresis in a 1% agarose gel in TBE buffer.

Subcloning and Sequencing the CWR22 Mutation
An aliquot of the PCR reaction used in denaturing gradient gel electrophoresis was chloroform extracted and ethanol precipitated. The DNA ends were made blunt with Klenow fragment (Promega, Madison WI) and phosphorylated with T4 kinase (Promega). These fragments were cloned into pBluescript II SK- (Stratagene, La Jolla, CA) cut with EcoRV (Promega), and dephosphorylated with calf intestinal phosphatase (Boehringer Mannheim, Indianapolis, IN).

DNA sequence was obtained from four individual clones using double-stranded sequencing with Taq Dideoxy Terminator and an Applied Biosystems 373A automated DNA sequencer (Foster City, CA). Standard M13 vector forward and reverse primers were used. Sequence for both strands of each clone was analyzed using a Genetics Computer Group (GCG) program (Madison, WI).

Construction of Mutant AR Expression Vectors
The CWR22 mutant AR H874Y vector was constructed in pCMVhAR using the two-step PCR method as described (17, 18). LNCaP AR was cloned from an LNCaP cDNA library in {lambda} Zap-II (2, 3). The LNCaP mutant AR T877A cDNA (2) HindIII/BamHI fragment replaced the wild type HindIII/BamHI fragment in pCMVhAR. Expression vector constructs were analyzed by DNA sequencing.

Transcription Assay
Transcriptional activities of wild type and mutant ARs were analyzed by transient cotransfection of African green monkey CV1 cells using a mouse mammary tumor virus long terminal repeat-luciferase reporter vector as described (27). CV1 cells were maintained at 37 C under 5% CO2 in high glucose DMEM with 4.5 g/liter glucose supplemented with 5% FBS. On the day before transfection, 4.5 x 105 cells per 6-cm culture dish were grown in the same medium for about 20 h until 70–80% confluent. Expression vector DNA (100 ng) and reporter vector DNA (2.5 µg) were transfected into CV1 cells using CaPO4 DNA precipitation (56). Cells were incubated 40 h in serum-free medium with and without steroids with a change in medium at 24 h and harvested on the plate using lysis buffer (Ligand Pharmaceuticals Inc., San Diego, CA). The luciferase assay was performed as described (27). Background activity was defined as light units determined in the presence of the AR expression vector and the absence of ligand. Stimulation of luciferase activity is expressed as fold increase over background based on five or more independent experiments. Background activities of the AR expression vectors in light units were: wild type hAR 1817 ± 521, H874Y 2904 ± 1039, and T877A 3718 ± 2153. Student’s t test and the computer program Sigmastat were used for statistical analysis. Data are expressed as the means ± SEM of three to 11 experiments.


    ACKNOWLEDGMENTS
 
We thank K. Michelle Cobb and Yeqing Chen for technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Frank S. French, M.D., Laboratories for Reproductive Biology, University of North Carolina, School of Medicine/CB 7500, Chapel Hill, NC 27599-7500.

This work was supported by NIH Grants AG-11343, HD-04466, P30-HD-18968 (DNA, Cell Culture and Histochemistry Cores), T32-HD-07315 (J-A.T. and C.W.G.), CA-57179 (T.G.P.) and CA-55792, CA-55792 (R.W. de V.W.).

1 Cooperative Network for Molecular and Genetic Markers for Prostate Cancer, National Cancer Institute. Back

Received for publication September 3, 1996. Revision received December 16, 1996. Accepted for publication January 15, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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