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First published online 1 September 2005
doi: 10.1242/jcs.02546

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Antiandrogens prevent stable DNA-binding of the androgen receptor

Department of Pathology, Josephine Nefkens Institute, Erasmus MC, University Medical Center Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands

View larger version (30K): [in a new window]   Fig. 1. Schematic representation, expression and transactivating capacity on androgen-regulated promoters of GFP-AR proteins investigated. (A) As well as GFP-tagged wild-type AR, ARs containing a mutation in the DBD disrupting DNA-binding (Brüggenwirth et al., 1998), or in helix 3 (W741C) or helix 12 (T877A) of the AR LBD, which result in altered ligand specificity (Veldscholte et al., 1990; Hara et al., 2003) were studied. TAD, Transactivating domain; DBD, DNA-binding domain; LBD, Ligand binding domain. (B) Hep3B cells containing stably integrated GFP-AR expression constructs and AR expressing prostate cancer cell lines were cultured for 1 day in the absence (lanes 1-6) or presence of 1 nM R1881 (lanes 7-12). Cell lysates were prepared and subjected to western blotting. Western blots of cell lysates from Hep3B cells (AR negative) containing GFP-AR (lanes 1 and 7), GFP-AR(A573D) (lanes 2 and 8), GFP-AR(W741C) (lanes 3 and 9) or GFP-AR(T877A) (lanes 4 and 10) and LNCaP (lanes 5 and 11) and PC346 (lanes 6 and 12) using an anti-AR or ß-actin antibody. ß-actin expression was used as a loading control. (C,D) Co-transfection assays of GFP-AR and the mutants depicted in A with androgen-regulated promoter constructs (ARE)2-TATA-luciferase (C) and mouse mammary tumour virus (MMTV) luciferase (D) in the presence of 10–9 M R1881, 10–6 M bicalutamide (Bic), 10–6 M OH-flutamide (OH-F) or no ligand as indicated. Luciferase activity of the GFP-AR proteins is plotted relative to activity of GFP-AR in presence of 10–9 M R1881. Mean±s.e.m. of at least three independent experiments are shown.

View larger version (45K): [in a new window]   Fig. 2. Combined strip-FRAP and FLIP-FRAP reveal that a fraction of agonist-liganded GFP-ARs is transiently immobilized. (A) The strip-FRAP method. A strip in the centre of a nucleus is bleached (red rectangle) with high laser power. Subsequently, fluorescence in the strip is measured at regular time intervals. Images are shown in false colour to visualize fluorescence differences more clearly. (B) Combined FLIP and FRAP method (FLIP-FRAP). A strip at one pole of the nucleus was bleached for a relatively long period. The difference between fluorescence signals in the bleached region (FRAP, red rectangle) and a distal region at 10 µm from the bleached region of the nucleus (FLIP, yellow rectangle) was determined at regular time intervals. (C,D) Strip-FRAP and FLIP-FRAP experiments of GFP-AR or the non-DNA-binding mutant GFP-AR(A573D) in the presence of 10–9 M R1881. (C) Graph showing fluorescence intensities relative to complete redistribution of the non-DNA-binding mutant GFP-AR(A573D) in the presence of R1881 plotted as a function of time. Mean values of at least ten cells of a representative experiment are plotted. All experiments were performed at least three times. (D) Graph showing the difference between fluorescence intensity in the FLIP and FRAP regions (rectangles in B) relative to the difference directly after bleaching, plotted against time. Mean values±two times the s.e.m. of two independent experiments on at least ten cells are plotted. (E,F) Computer simulations (see Materials and Methods) of strip-FRAP and FLIP-FRAP of freely diffusing molecules do not explain the experimental FRAP data obtained with both methods. D is the effective diffusion coefficient. Experimental strip-FRAP data on wild-type GFP-AR lies in between curves representing indicated scenarios of free diffusion (E), whereas experimental FLIP-FRAP data on wild-type GFP-AR lies outside these boundaries (F). (G,H) Computer simulations representing a model where, next to freely diffusing molecules, a fraction is transiently immobilized, fitted to both strip-FRAP and FLIP-FRAP experimental curves on wild-type GFP-AR. Computer simulations correspond to the average of best fits of FRAP and FLIP-FRAP experiments respectively (Table 1), so are not necessarily the best fits of the individual experiments. Absolute value of residuals of the computer simulation fit and the experimental data on each time point are plotted below the x-axis.

View larger version (28K): [in a new window]   Fig. 3. In the presence of antagonists OH-flutamide and bicalutamide GFP-AR shows no or little DNA-dependent immobilization. Strip-FRAP (A,C) or combined FLIP and FRAP (B,D) of GFP-AR or the non-DNA-binding mutant GFP-AR(A573D) in the presence of 10–6 M OH-flutamide (A,B) or 10–6 M bicalutamide (C,D). Experimental settings were identical to those described in Fig. 2. Lower graphs show computer simulations corresponding to the average of best fits of strip-FRAP and FLIP-FRAP models of wild-type GFP-AR (see Table 1). The absolute values of the residuals of the fit and the experimental data are plotted below the x-axis. Larger residuals in the first second of strip-FRAPs are probably due to larger variation in the beginning of the experiment, when fluorescence changes rapidly.

View larger version (102K): [in a new window]   Fig. 4. Activation of AR by agonistic ligands results in intranuclear localization in foci. Confocal laser-scanning microscope images showing representative nuclei of Hep3B cell lines stably expressing GFP-AR (A-C), the non-DNA-binding mutant GFP-AR(A573D) (D-F) or GFP-AR proteins with mutations in the LBD (which result in altered ligand specificity), GFP-AR(W741C) (G-I) and GFP-AR(T877A) (J-L). Subnuclear localization was observed in the presence of 10–9 M R1881 (A,D,G,J), 10–6 M OH-flutamide (B,E,H,K) or 10–6 M bicalutamide (C,F,I,L). With all ligands, androgen receptors are localized in the nucleus, but are excluded from nucleoli (dark areas in the nucleus). In situations where AR is able to activate transcription intranuclear foci are observed (A,G,I,J,K, see Fig. 1). Bar, 5 µm.

View larger version (32K): [in a new window]   Fig. 5. Prostate cancer-related AR-LBD mutants display reduced mobility in the presence of their agonistic ligands. Nuclear mobility of antiandrogen-resistant prostate cancer mutants AR(T877A) and AR(W741C) was investigated using two complementary FRAP assays (see also Fig. 2): strip-FRAP (A,C,E) and combined FLIP and FRAP (B,D,F). Intranuclear mobility of these mutants in the presence of 10–9 M R1881 (A,B), 10–6 M OH-flutamide (C,D) or 10–6 M bicalutamide (E,F) was studied. Mobility of non-DNA-binding GFP-AR(A573D) is plotted as a reference. Experimental settings were identical to those described in Fig. 2. Lower graphs in C-F show computer simulations corresponding to the average of best fits of strip-FRAP and FLIP-FRAP models (data in Table 1) of the experimental curves of GFP-AR(T877A) (C,D) and GFP-AR(W741C) in the presence of 1 µM OH-flutamide (C,D) or bicalutamide (E,F). Absolute values of the residuals of computer-simulated curves and experimental data are plotted below the x-axis.

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