Androgen Receptor Expression and Cellular Proliferation During Transition from Androgen-Dependent to Recurrent Growth after Castration in the CWR22 Prostate Cancer Xenograft
2002; Elsevier BV; Volume: 160; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64365-9
ISSN1525-2191
AutoresDesok Kim, Christopher W. Gregory, Frank S. French, Gary J. Smith, James L. Mohler,
Tópico(s)Prostate Cancer Diagnosis and Treatment
ResumoAndrogen receptor expression was analyzed in the CWR22 human prostate cancer xenograft model to better understand its role in prostate cancer recurrence after castration. In androgen-dependent tumors, 98.5% of tumor cell nuclei expressed androgen receptor with a mean optical density of 0.26 ± 0.01. On day 2 after castration androgen deprivation decreased immunostained cells to 2% that stained weakly (mean optical density, 0.16 ± 0.08). Cellular proliferation measured using Ki-67 revealed <1% immunostained cells on day 6. Androgen receptor immunostained cells increased to 63% on day 6 and 84% on day 32 although immunostaining remained weak. Cellular proliferation was undetectable beyond day 6 after castration until multiple foci of 5 to 20 proliferating cells became apparent on day 120. These foci expressed increased levels of prostate-specific antigen, an androgen receptor-regulated gene product. In tumors recurrent 150 days after castration androgen receptor-immunostaining intensity was similar to CWR22 tumors from intact mice although the percentage of cells immunostained was more variable. The appearance of proliferating tumor cells that expressed androgen receptor and prostate-specific antigen 120 days after castration suggests that these cells represent the origin of recurrent tumors. Androgen receptor expression was analyzed in the CWR22 human prostate cancer xenograft model to better understand its role in prostate cancer recurrence after castration. In androgen-dependent tumors, 98.5% of tumor cell nuclei expressed androgen receptor with a mean optical density of 0.26 ± 0.01. On day 2 after castration androgen deprivation decreased immunostained cells to 2% that stained weakly (mean optical density, 0.16 ± 0.08). Cellular proliferation measured using Ki-67 revealed <1% immunostained cells on day 6. Androgen receptor immunostained cells increased to 63% on day 6 and 84% on day 32 although immunostaining remained weak. Cellular proliferation was undetectable beyond day 6 after castration until multiple foci of 5 to 20 proliferating cells became apparent on day 120. These foci expressed increased levels of prostate-specific antigen, an androgen receptor-regulated gene product. In tumors recurrent 150 days after castration androgen receptor-immunostaining intensity was similar to CWR22 tumors from intact mice although the percentage of cells immunostained was more variable. The appearance of proliferating tumor cells that expressed androgen receptor and prostate-specific antigen 120 days after castration suggests that these cells represent the origin of recurrent tumors. High-affinity binding of dihydrotestosterone to androgen receptor (AR) causes AR to function as a transcription factor1Quigley CA De Bellis A Marschke KB el-Awady MK Wilson EM French FS Androgen receptor defects: historical, clinical, and molecular perspectives.Endocr Rev. 1995; 16: 271-321Crossref PubMed Google Scholar, 2Roy AK Lavrovsky Y Song CS Chen S Jung MH Velu NK Bi BY Chatterjee B Regulation of androgen action.Vitam Horm. 1999; 55: 309-352Crossref PubMed Scopus (184) Google Scholar that regulates a network of androgen response genes.3Gregory CW Hamil KG Kim D Hall SH Pretlow TG Mohler JL French FS Androgen receptor expression in androgen-independent prostate cancer is associated with increased expression of androgen-regulated genes.Cancer Res. 1998; 58: 5718-5724PubMed Google Scholar, 4Eid MA Kumar MV Iczkowski KA Bostwick DG Tindall DJ Expression of early growth response genes in human prostate cancer.Cancer Res. 1998; 11: 2461-2468Google Scholar Prostate cancer (CaP) is androgen-dependent and its growth is mediated by this AR-regulated gene network. Androgen deprivation causes reduced AR expression,5Prins GS Birch L Immunocytochemical analysis of androgen receptor along the ducts of the separate rat prostate lobes after androgen withdrawal and replacement.Endocrinology. 1993; 132: 169-178PubMed Google Scholar apoptosis, decreased cell volume,6Kyprianou N Isaacs JT Activation of programmed cell death in the rat ventral prostate after castration.Endocrinology. 1988; 122: 552-562Crossref PubMed Scopus (634) Google Scholar and decline of serum prostate-specific antigen (PSA). However, most CaPs eventually develop the capacity for recurrent growth in the absence of testicular androgen. All of 22 specimens of testicular androgen-independent metastatic CaP showed positive immunohistochemical staining for AR protein.7Hobisch A Culig Z Radmayr C Bartsch G Klocker H Hittmair A Androgen receptor status of lymph node metastases from prostate cancer.Prostate. 1996; 28: 129-135Crossref PubMed Scopus (128) Google Scholar Transurethral resection of prostate specimens from 10 untreated CaP patients and 20 patients with CaP recurrent after androgen deprivation were compared and no significant difference in the percentage of AR-positive cells was found.8Ruizeveld de Winter JA Janssen PJA Sleddens HMEB Verleun-Mooijman MCT Trapman J Brinkmann AO Santerse AB Schröder FH van der Kwast TH Androgen receptor status in localized and locally progressive hormone refractory human prostate cancer.Am J Pathol. 1994; 144: 735-746PubMed Google Scholar, 9DeVere White RW Myers F Chi S-G Chamberlain S Siders D Lee F Stewart S Gumerlock PH Human androgen receptor expression in prostate cancer following androgen ablation.Eur Urol. 1997; 31: 1-6Google Scholar Because AR expression is similar in androgen-dependent and recurrent CaP, we sought to understand how AR expression changes in relation to cellular proliferation in the interval between androgen deprivation and tumor recurrence. CWR22 is an androgen-dependent human CaP xenograft propagated subcutaneously in nude mice. CWR22 resembles the majority of human CaPs; CWR22 secretes PSA, undergoes tumor regression after androgen deprivation, and recurs as a palpable, growing and ultimately lethal tumor after several months in the absence of testicular androgen.10Pretlow TG Wolman SR Micale MA Pelley RJ Kursh ED Resnick MI Bodner DR Jacobberger JW Delmoro CM Giaconia JM Xenografts of primary human prostatic carcinoma.J Natl Cancer Inst. 1993; 85: 394-398Crossref PubMed Scopus (136) Google Scholar, 11Wainstein MA He F Robinson D Kung H-J Schwartz S Giaconia JM Edgehouse NL Pretlow TP Bodner DR Kursh ED Resnick MI Seftel A Pretlow TG CWR22: androgen-dependent xenograft model derived from a primary human prostatic carcinoma.Cancer Res. 1994; 54: 6049-6052PubMed Google Scholar, 12Nagabhushan M Miller CM Pretlow TP Giaconia JM Edgehouse NL Schwartz S Kung HJ de Vere White RW Gumerlock PH Resnick MI Amini SB Pretlow TG CWR22: the first human prostate cancer xenograft with strongly androgen-dependent and relapsed strains both in vivo and in soft agar.Cancer Res. 1996; 56: 3042-3046PubMed Google Scholar, 13Tan J-A Sharief Y Hamil KG Gregory CW Zang D-Y Sar M Gumerlock PH deVere White RW Pretlow TG Harris SE Wilson EM Mohler JL French FS Dehydroepiandrosterone activates mutant androgen receptors expressed in the androgen-dependent human prostate cancer xenograft CWR22 and LNCaP cells.Mol Endocrinol. 1997; 11: 450-459Crossref PubMed Scopus (266) Google Scholar We demonstrated that recurrence of CWR22 tumor after androgen deprivation was associated with re-expression of a network of androgen-regulated genes including PSA, human kallikrein-2, Nkx 3.1, AR co-activator ARA-70, cell cycle genes Cdk1 and Cdk2,3Gregory CW Hamil KG Kim D Hall SH Pretlow TG Mohler JL French FS Androgen receptor expression in androgen-independent prostate cancer is associated with increased expression of androgen-regulated genes.Cancer Res. 1998; 58: 5718-5724PubMed Google Scholar and insulin-like growth factor binding protein-5.14Gregory CW Kim D Ye P D'Ercole AJ Mohler JL French FS Androgen receptor up-regulates insulin-like growth factor binding protein-5 (IGFBP-5) expression in a human prostate cancer xenograft.Endocrinology. 1999; 140: 2372-2381Crossref PubMed Scopus (51) Google Scholar Recently, Amler and associates15Amler LC Agus DB LeDue C Sapinosa ML Fox WD Kern S Lee D Wang V Leysens M Higgins B Martin J Gerald W Dracopoli N Cordon-Cardo C Scher HI Hampton GM Dysregulated expression of androgen-responsive and nonresponsive genes in the androgen-independent prostate cancer xenograft model CWR22-R.Cancer Res. 2000; 60: 6134-6141PubMed Google Scholar have reported incomplete reactivation of the androgen response pathway despite androgen absence in recurrent CWR22 using microarray analysis. Similar expression of AR and these androgen-regulated genes in androgen-dependent and recurrent CWR22 tumors suggested a role for AR regulation of gene expression in the development of recurrent CWR22 despite the absence of testicular androgen. Video image analysis has been used to quantitate AR expression more precisely than visual scoring.16Sadi MV Barrack ER Image analysis of androgen receptor immunostaining in metastatic prostate cancer. Heterogeneity as a predictor of response to hormonal therapy.Cancer. 1993; 71: 2574-2580Crossref PubMed Scopus (107) Google Scholar, 17Tilley WD Lim-Tio SS Horsfall DJ Aspinall JO Marshall VR Skinner JM Detection of discrete androgen receptor epitopes in prostate cancer by immunostaining: measurement by color video image analysis.Cancer Res. 1994; 54: 4096-4102PubMed Google Scholar, 18Prins GS Sklarew RJ Pertschuk LP Image analysis of androgen receptor immunostaining in prostate cancer accurately predicts response to hormonal therapy.J Urol. 1998; 159: 641-649Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 19Magi-Galluzzi C Xu X Hlatky L Hahnfeldt P Kaplan I Hsiao P Chang C Loda M Heterogeneity of androgen receptor content in advanced prostate cancer.Mod Pathol. 1997; 10: 839-845PubMed Google Scholar We developed an automated method for measuring AR expression in individual cells that was used to demonstrate the dependence of AR protein levels on serum androgen levels in the CWR22 model.20Kim D Gregory CW Smith GJ Mohler JL Immunohistochemical quantitation of androgen receptor expression using color video image analysis.Cytometry. 1999; 35: 2-10Crossref PubMed Scopus (35) Google Scholar In CWR22 tumor-bearing mice castrated for 6 days, AR mean optical density (MOD) decreased to 57% of levels in tumors from intact mice. After 72 hours of exogenous testosterone administration to 6-day castrated mice, AR MOD in CWR22 returned to the level found in tumors from intact mice. Cellular proliferation of testosterone-treated tumors reached ∼50% of the original androgen-stimulated CWR22 tumors from intact mice.14Gregory CW Kim D Ye P D'Ercole AJ Mohler JL French FS Androgen receptor up-regulates insulin-like growth factor binding protein-5 (IGFBP-5) expression in a human prostate cancer xenograft.Endocrinology. 1999; 140: 2372-2381Crossref PubMed Scopus (51) Google Scholar These data suggested that the majority of CWR22 cells on day 6 after castration had functional AR. In archived radical prostatectomy specimens, AR protein content was higher in androgen-dependent, clinically localized CaP and lower in prostate intraepithelial neoplasia than benign prostatic hyperplasia (BPH).19Magi-Galluzzi C Xu X Hlatky L Hahnfeldt P Kaplan I Hsiao P Chang C Loda M Heterogeneity of androgen receptor content in advanced prostate cancer.Mod Pathol. 1997; 10: 839-845PubMed Google Scholar, 20Kim D Gregory CW Smith GJ Mohler JL Immunohistochemical quantitation of androgen receptor expression using color video image analysis.Cytometry. 1999; 35: 2-10Crossref PubMed Scopus (35) Google Scholar AR immunostaining intensity was similar in androgen-stimulated and recurrent tumors from the CWR22 xenograft and transurethral resection of the prostate specimens of BPH; all tissues were small volume and fixed immediately after procurement.20Kim D Gregory CW Smith GJ Mohler JL Immunohistochemical quantitation of androgen receptor expression using color video image analysis.Cytometry. 1999; 35: 2-10Crossref PubMed Scopus (35) Google Scholar Finally, 12 specimens of recurrent CaP and 16 specimens of BPH, all acquired by transurethral resection of the prostate and fixed immediately, exhibited similar AR immunostaining (unpublished data). Taken together, these findings suggest that AR is expressed in androgen-stimulated CaP, diminished but recoverable after castration, and re-expressed despite androgen absence on CaP recurrence. We sought to test the hypothesis that re-expression of AR coincided with the onset of androgen-independent cellular proliferation in CaP. To test this hypothesis, the temporal relationship between AR protein expression and cellular proliferation was determined using the CWR22 xenograft model during tumor regression and recurrence after castration. Quantitative immunohistochemistry and color video image analysis were used to measure precisely the proportion of cells expressing AR and Ki-67 and the intensity of expression of AR associated with response to androgen deprivation and emergence of the recurrent phenotype. Nude/nude athymic mice were purchased from Harlan Sprague-Dawley, Inc., Indianapolis, IN. The CWR22 tumor model has been maintained by continuous passage since December of 1995 from CWR22 cells that were a gift from Thomas A. Pretlow, MD, PhD, Case Western Reserve University). CWR22 tumors were transplanted as 1 million dissociated cells suspended in Matrigel (Collaborative Research Inc., Bedford, MA) injected subcutaneously into nude mice 4 to 5 weeks of age.11Wainstein MA He F Robinson D Kung H-J Schwartz S Giaconia JM Edgehouse NL Pretlow TP Bodner DR Kursh ED Resnick MI Seftel A Pretlow TG CWR22: androgen-dependent xenograft model derived from a primary human prostatic carcinoma.Cancer Res. 1994; 54: 6049-6052PubMed Google Scholar, 12Nagabhushan M Miller CM Pretlow TP Giaconia JM Edgehouse NL Schwartz S Kung HJ de Vere White RW Gumerlock PH Resnick MI Amini SB Pretlow TG CWR22: the first human prostate cancer xenograft with strongly androgen-dependent and relapsed strains both in vivo and in soft agar.Cancer Res. 1996; 56: 3042-3046PubMed Google Scholar A 12.5-mg sustained-release testosterone pellet (Innovative Research of America, Sarasota, FL) was placed subcutaneously in each animal 2 days before tumor injection and every 3 months thereafter to maintain consistent serum levels of testosterone of ∼4 ng/ml. After tumors reached a volume of 1 cm3, animals were anesthetized with methoxyflurane, castrated, and the testosterone pellets removed. Intact mice bearing tumors and castrated animals with either regressed or recurrent CWR22 tumors were exposed to methoxyflurane and sacrificed by cervical dislocation. Tumor height, width, and depth were measured using calipers and tumor volume was calculated by multiplying these three measurements and 0.5234. Tumors were excised and cut into several pieces (∼125 mm3); half was frozen in liquid nitrogen and half was fixed in 10% buffered formalin for 24 to 48 hours, washed in phosphate-buffered saline (PBS) for 24 hours, and paraffin-embedded. Specimens of BPH prepared identically were used as positive controls. Blood was obtained on sacrifice of all tumor-bearing mice for measurement of serum PSA. The avidin-biotin-immunoperoxidase technique21Sar M Application of avidin-biotin complex technique for the localization of estradiol receptor in target tissues using monoclonal antibodies.in: Bullock GR Petrusz P Techniques in Immunocytochemistry. vol 3. Academic Press, New York1985: 43-54Google Scholar was modified for use in paraffin-embedded tissues that were immunostained using capillary action with a MicroProbe staining station (Fisher Scientific, Pittsburgh, PA).22Brigati DJ Budgeon LR Unger ER Koebler D Cuomo C Kennedy T Perdomo JM Immunocytochemistry is automated: development of a robotic workstation based upon the capillary action principle.J Histotechnol. 1988; 11: 165-183Crossref Scopus (91) Google Scholar Monoclonal antibody (mAb) F39.4.1 (BioGenex, San Ramon, CA) recognizes an epitope in the N-terminal region of human AR.23Zegers ND Claassen E Neelen C Mulder E van Laar JH Vorrhorst MM Berrevoets CA Brinkmann AO van der Kwast TH Ruizeveld de Winter JA Trapman J Boersma WJA Epitope prediction and confirmation for the human androgen receptor: generation of monoclonal antibodies for multi-assay performance following the synthetic peptide strategy.Biochim Biophys Acta. 1991; 1073: 23-32Crossref PubMed Scopus (96) Google Scholar mAb MIB-1 (Oncogene, Cambridge, MA) and polyclonal antibody MIB-5 (DAKO Corp., Carpinteria, CA) react with the cell cycle-associated antigen Ki-67 expressed during the proliferative phases (G1, S, G2, and M) but absent in the resting phase (G0) of the cell cycle.24Gerdes J Lemke H Baisch H Wacher HH Schwab U Stein H Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67.J Immunol. 1984; 133: 1710-1715PubMed Google Scholar mAb A67-B/E3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) corresponds to amino acids 1 to 261 representing full length PSA p30 of human origin.25Lundwall A Characterization of the gene for prostate-specific antigen: a human glandular kallikrein.Biochem Biophys Res Commun. 1989; 161: 1151-1159Crossref PubMed Scopus (102) Google Scholar Paraffin-embedded CWR22 tumor specimens were cut into 6-μm-thick histological sections. After deparaffinization and rehydration, tissue sections were heated to 100°C for 30 minutes in a vegetable steamer in the presence of antigen retrieval solution (CITRA, pH 6.0; BioGenex) and cooled for 10 minutes. Slides were preincubated with 2% normal horse serum for 5 minutes at 37°C and washed with automation buffer (Fisher Scientific). AR mAb was diluted 1:300 (0.13 μg/ml in PBS containing 0.1% bovine serum albumin, pH 7.4) and sections were stained for 120 minutes at 37°C. Slides were incubated in biotinylated anti-mouse immunoglobulin (IgG) (Vector Laboratories, Inc., Burlingame, CA) for 15 minutes at 37°C (1:200 in PBS, pH 7.4) and in horseradish peroxidase-conjugated avidin-biotin complex (Vector Laboratories, Inc.) for 15 minutes at 37°C (1:100 in PBS, pH 7.4). The immunoperoxidase complexes were visualized using diaminobenzidine tetrahydrochloride (Vector Laboratories, Inc) for 8 minutes at 37°C (0.75 mg/ml in Tris buffer containing 0.03% hydrogen peroxide, pH 7.6). Slides were dehydrated through graded alcohol solutions and cleaned by Hemo-De xylene substitute (Fisher Scientific). Counterstaining was performed with hematoxylin (Gill's formula, 1:6 dilution; Fisher Scientific) for 12 seconds. Slides were mounted with Permount and coverslips. Two representative slides were selected from each time point and stained with the polyclonal AR antibodies, AR52 and PG-21, following protocols reported previously.5Prins GS Birch L Immunocytochemical analysis of androgen receptor along the ducts of the separate rat prostate lobes after androgen withdrawal and replacement.Endocrinology. 1993; 132: 169-178PubMed Google Scholar, 26Sar M Lubahn DB French FS Wilson EM Immunohistochemical localization of the androgen receptor in rat and human tissues.Endocrinology. 1990; 127: 3180-3186Crossref PubMed Scopus (441) Google Scholar AR52 was provided by Dr. Elizabeth M. Wilson (University of North Carolina at Chapel Hill) and PG-21 was provided by Dr. Gail S. Prins (University of Illinois at Chicago). Slides prepared from a CWR22 tumor on day 6 after castration and human BPH were included as external controls to avoid variation of immunostaining intensity caused during staining procedures. Nonimmune mouse IgG (Vector Laboratories, Inc.) was used instead of AR mAb at the same IgG concentration for negative control slides prepared from the same tissue blocks as specimens; negative control slides were nonreactive. MIB-1 mAb staining was performed at an IgG concentration of 0.5 μg/ml (1:50). All other steps were as described for AR immunostaining. Serial sections adjacent to the sections stained for AR were obtained from tumors on day 120 after castration and stained with MIB-1 mAb. Colon cancer tissue served as positive controls and 0.5 μg/ml of nonimmune mouse IgG was used instead of MIB-1 mAb at the same IgG concentration for negative control slides prepared from the same tissue blocks as specimens; negative control slides were nonreactive. PSA mAb (1:50, 4 μg/ml) was biotinylated and blocked in vitro using the Iso-IHC kit (InnoGenex, San Ramon, CA) to avoid background staining caused by infiltrated murine cells in CWR22 tumors harvested from castrated animals. Sections were digested in Proteinase-K (20 μg/ml, DAKO Corp.) for 6 minutes at room temperature. Sections were incubated in the blocking solution and labeled with PSA mAb for 1 hour at 37°C and in streptavidin-peroxidase (InnoGenex) for 5 minutes at 37°C. Immunoreaction was visualized by diaminobenzidine tetrahydrochloride for 8 minutes at 37°C. Double immunohistochemistry was performed on additional CWR22 slides to co-localize PSA expression among Ki-67-positive tumor cells. Sections were eluted by glycine buffer (pH 2.3) for 5 minutes three times at room temperature and antigen-retrieved as described previously. A mixture of normal goat serum (2%) and avidin (1:50 in PBS, Vector Laboratories, Inc.) was used for blocking for 5 minutes at 37°C. Sections were reacted with MIB-5 (1:50, 20 μg/ml) mixed with biotin (1:50 in PBS, Vector Laboratories, Inc.) for 2 hours at 37°C. The same avidin-biotin-peroxidase complex technique used for MIB-1 was performed. Immunoreaction was visualized by 3-amino-9-ethylcarbazole (AEC) (Vector Laboratories, Inc.) for 10 minutes at 37°C. BPH and CaP specimens were used as positive controls. For the negative control slide, nonimmune rabbit IgG (Vector Laboratories, Inc.) was used instead of PSA mAb at the same IgG concentration; appropriate biotinylated IgGs were replaced with PBS in PSA and MIB-5 steps to check against cross-reactions. Negative control slides showed neither nonspecific reaction nor cross-reactions. Automated digital image analysis was performed as described previously.20Kim D Gregory CW Smith GJ Mohler JL Immunohistochemical quantitation of androgen receptor expression using color video image analysis.Cytometry. 1999; 35: 2-10Crossref PubMed Scopus (35) Google Scholar Briefly, imaging hardware consisted of a Zeiss Axioskop microscope, a 3-chip charge-coupled device camera (C5810; Hamamatsu Photonics Inc., Hamamatsu, Japan), a camera control unit (Hamamatsu Photonics Inc.), and a Pentium-based personal computer. Each field of view for AR-stained slides was digitized at total magnification ×1200 using a ×40 objective (numerical aperture, 1.3). For MIB-1- and PSA-stained slides, a ×20 objective (numerical aperture, 0.6) was used for total magnification at ×600. Twenty images that contained ∼200 to 250 nuclei at ×1200 and 400 to 500 nuclei at ×600 provided an adequate sample size for each tumor because the deviation of average intensity values of randomly chosen immunopositive areas became stable (within ±5%). Immunopositivity and immunonegativity were determined using a linear discriminant analysis based on hue, saturation, and intensity of 100 immunostained cells of an intact CWR22 specimen and 100 cells of a negative control slide, respectively. The positivity for AR, Ki-67, and PSA was defined as the total number of pixels from immunopositive areas divided by the total number of pixels from all nuclear areas detected in a given specimen. Differences in MOD and percentage of AR-, Ki-67-, and PSA-positive cells from all images from all tumors at various time points were evaluated using Wilcoxon rank sum tests. Correlations between features were examined using the Pearson's product moment correlation test. F-tests were performed to compare the variances among samples. Statistical significance was achieved if P < 0.05. Lysates were prepared from frozen CWR22 tumors. Tumor tissue (100 mg) was pulverized in liquid nitrogen, thawed on ice, and mixed with 1.0 ml of RIPA buffer with protease inhibitors (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.5 mmol/L phenylmethylsulfonyl fluoride, 10 μmol/L pepstatin, 4 μmol/L aprotinin, 80 μmol/L leupeptin, and 5 mmol/L benzamidine). Tissue was homogenized on ice for 30 seconds using a Biohomogenizer (Biospec Products, Inc., Bartlesville, OK). Two μl of 0.2 mol/L phenylmethylsulfonyl fluoride were added and homogenates incubated 30 minutes on ice. Homogenates were centrifuged at 10,000 × g for 20 minutes; supernatants were collected and centrifuged to prepare final lysates. Supernatant protein (100 μg) from each sample was electrophoresed in 12% sodium dodecyl sulfate-polyacrylamide gels and electroblotting to Immobilon-P membrane (Millipore Corp., Bedford, MA). Immunodetection used AR mAb F39.4.1 at 1:10,000 dilution. Secondary antibody (goat anti-mouse IgG conjugated to horseradish peroxidase; Amersham Corp., Arlington Heights, IL) was used for detection by enhanced chemiluminescence (DuPont-NEN Research Products, Boston, MA). Average MOD and percentage of cells expressing AR (percent AR positivity) were determined in tumors from CWR22-bearing mice before and after castration (Table 1). The majority of nuclei in CWR22 tumors from intact nude mice (98.5 ± 0.2%) showed intense staining of AR (MOD was 0.26 ± 0.01) (Figure 1). On day 1 after castration, AR MOD decreased and remained low on day 4 after castration whereas AR percent positivity declined to a minimum on day 2 (2%) and remained low on day 4 (10%). On day 6 after castration, AR-positive nuclei increased sixfold to 63% and were distributed evenly throughout all tumor sections. AR positivity increased further to 71% on day 12. AR MOD decreased to a low of 0.11 on day 4 and remained low at 0.15 to 0.17 on days 12 through 120 after castration. Recurrent CWR22 tumors obtained ∼150 days after castration exhibited lower percent AR positivity (72.1 ± 7.6%) than CWR22 tumors from intact, androgen-stimulated mice (P = 0.03). However, among malignant nuclei expressing AR, MOD was similar (P = 0.99) in the original CWR22 under androgen stimulation and recurrent CWR22 in the absence of testicular androgens. Immunostaining of CWR22 tumors before and after castration yielded similar results when the polyclonal antibodies AR-52 and PG-21 were used instead of AR mAb F39.4.1 (data not shown). Western blotting (Figure 2) of CWR22 tumor lysates revealed similar AR levels in androgen-dependent and recurrent CWR22 tumors and reduced AR levels after castration until tumor recurrence.Table 1Quantitation of Tumor Volume (cm3), AR Expression (AR MOD and %AR Positivity), Tumor Cellular Proliferation (%Ki-67 Positivity), and PSA Serum Levels (ng/ml) and Tissue Expression (%PSA Positivity) Measured in Androgen-Stimulated, Androgen-Deprived and Recurrent CWR22 Tumors*AR mean optical density (MOD), percent AR positivity, percent Ki-67 positivity, serum PSA level, and percent PSA positivity at all time points after castration decreased significantly (P < 0.01) compared to intact CWR22 except no significant differences were found for AR MOD in recurrent CWR22 and percent AR positivity on days 90 and 120 after castration compared to day 0 (P > 0.05).Days after castrationNo. of tumorsTumor volumeAR MOD%AR positivity%Ki-67 positivitySerum PSA%PSA positivityintact CWR22121.11 ± 0.94†Tumor volume and serum PSA is described by mean ± SD and therefore the data for time points containing only two measurements were not presented.0.26 ± 0.01‡The image analysis data for all nuclei for all tumors at each time point is described by mean ± SD. Each tumor is represented by 20 images containing 200 to 250 nuclei for AR and 400 to 500 nuclei for Ki-67.98.5 ± 0.273.5 ± 4.4246.7 ± 55.3†Tumor volume and serum PSA is described by mean ± SD and therefore the data for time points containing only two measurements were not presented.17.4 ± 3.6Day 120.15 ± 0.1028.5 ± 7.456.9 ± 7.53.4 ± 1.0Day 240.81 ± 0.090.16 ± 0.082.3 ± 5.526.1 ± 5.6169.0 ± 53.75.5 ± 2.8Day 420.11 ± 0.099.9 ± 8.69.4 ± 8.14.8 ± 1.1Day 660.72 ± 0.490.15 ± 0.0662.7 ± 4.8§Percent AR positivity on days 6, 12, 32, 64, 90, and 120 after castration and upon recurrence increased significantly (P < 0.001) compared to days 1, 2, and 4 after castration.0.8 ± 0.5109.9 ± 76.35.6 ± 1.5Day 1260.81 ± 0.270.17 ± 0.0670.9 ± 4.9§Percent AR positivity on days 6, 12, 32, 64, 90, and 120 after castration and upon recurrence increased significantly (P < 0.001) compared to days 1, 2, and 4 after castration.0.3 ± 0.318.8 ± 10.80.8 ± 0.9Day 3240.64 ± 0.250.17 ± 0.1170.4 ± 7.3§Percent AR positivity on days 6, 12, 32, 64, 90, and 120 after castration and upon recurrence increased significantly (P < 0.001) compared to days 1, 2, and 4 after castration.0.1 ± 0.35.6 ± 4.10.6 ± 0.3Day 6420.15 ± 0.0571.7 ± 12.2§Percent AR positivity on days 6, 12, 32, 64, 90, and 120 after castration and upon recurrence increased significantly (P < 0.001) compared to days 1, 2, and 4 after castration.0.4 ± 0.21.1 ± 0.1Day 9040.64 ± 0.300.17 ± 0.0573.4 ± 6.5§Percent AR positivity on days 6, 12, 32, 64, 90, and 120 after castration and upon recurrence increased significantly (P < 0.001) compared to days 1, 2, and 4 after castration.0.8 ± 0.511.1 ± 1.81.5 ± 0.7Day 12040.78 ± 0.370.17 ± 0.0372.3 ± 8.4§Percent AR positivity on days 6, 12, 32, 64, 90, and 120 after castration and upon recurrence increased significantly (P < 0.001) compared to days 1, 2, and 4 after castration.3.3 ± 1.221.3 ± 4.1∥Serum PSA level and percent PSA positivity on day 120 after castration were significantly higher than on day 90 after castration (P < 0.05).3.4 ± 0.1∥Serum PSA level and percent PSA positivity on day 120 after castration were significantly higher than on day 90 after castration (P < 0.05).Recurrent CWR22121.63 ± 0.380.26 ± 0.01¶Recurrent CWR22 showed a significant increase in
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