Androgen receptor decoy molecules block the growth of prostate cancer -- Quayle et al. 104 (4): 1331 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Quayle et al. 10.1073/pnas.0606718104.

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SI Figure 6

Fig. 6. AR and PSA protein in vitro and in vivo. (A) Western blot analysis of LNCaP cells transiently transfected with decoy AR_1-558 (67 kDa) using an antibody to HisTag. All antibodies were from Santa Cruz Biotechnology unless otherwise stated. (B) Expression of endogenous full-length AR and decoy AR_1-558 in cells stably transfected with empty vector and decoy AR_1-558 using an antibody to the AR NTD. Arrows point to full-length endogenous AR (110 kDa) and decoy AR_1-558. The AR441 antibody is specific for an epitope in the AR NTD. (C) Expression of AR protein in tumors stably transfected with either vector or decoy AR_1-558 grown in SCID mice for 38 weeks. Both tumors expressed endogenous AR at 110 kDa. Decoy AR_1-558 expression was maintained until the end of the experiment. (D) Western blot analyses for PSA and cytokeratin 18 (Abcam) in tumors. Mouse prostate tissue was used as a negative control. Tumors were from both intact and androgen-independent hosts (AI). Cytokeratin 18 levels provide an indication of the epithelial component of each sample.

SI Figure 7

Fig. 7. Decoy AR_1-558 altered the morphology of prostate cancer tumors. Hematoxylin and eosin staining of sections of prostate cancer tumors harvested at the duration of the experiment from hosts bearing tumors that stably expressed vector (row 1) or decoy AR_1-558 (row 2). Column 1 displays sections of tumors from intact hosts, while column 2 displays sections from castrated hosts that had become androgen independent.

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Fig. 8. Lentivirus delivery of decoy AR_1-558 to established tumors that express full-length AR. (A) Schematic representation of the structure of lentivirus vectors used in this study. 5' LTR, HIV 5'LTR; Y, HIV-1 psi Packaging sequence; RRE, HIV-1 Rev response element; CMV, CMV promoter; V5, V5 epitope; 3'LTR, DU3 HIV 3'LTR. (B) Western blot analysis of proteins in transduced LNCaP cells maintained in vitro. Viral concentrations used to infect the cells ranged from 0.1-200 ml. Antibody to the AR NTD was used to detect AR_1-558 and GFP-AR_1-558 while an antibody to GFP was used to detect GFP. Control samples are protein lysates from mock-transduced cells. (C) Fluorescent micrograph showing expression of GFP-AR_1-558 in LNCaP s.c. tumors harvested 5 days after injection with lentiviral particles (1 ´ 10⁷ T units/ml).

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Fig. 9. AR, AR_1-558, and PSA protein levels in harvested tumors. (A) Western blot analysis confirmed expression of decoy molecules and full-length AR was maintained in tumors harvested 20 days after the first injection of lentivirus particles. (B) Western blot analyses for PSA and cytokeratin 18 in tumors harvested upon completion of the experiment. (C) Expression of GFP and GFP-AR_1-558 in PC3 tumors harvested 25 days after the first injection of lentivirus particles.

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Fig. 10. Percentage final weight of hosts at the duration of the experiment. Mean weights were for (A) LNCaP experiments n = 33, 25.7 ±2.1 g (initial) and 23.9 ± 1.9 g (final); and (B) PC3 experiments, n = 33, 28.2 ± 1.5 g (initial) and 29.8 ± 1.6 g (final). Student's t test was used for statistical comparison of final weight versus initial weight in each treatment group. *, P £ 0.05.