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Background: We developed non-invasive, cell-based screening assays to rapidly and biologically assess factors that modulate prostate cancer growth and affect androgen receptor (AR) activity., Methods: LNCaP cells, which stably express enhanced green fluorescent protein (EGFP) either constitutively or upon AR activation, were treated with a variety of agents, and then monitored by fluorescence and MTS assays for dose-dependent changes in cell number and AR activity., Results: The assays were validated for rapid, fluorescence-based, quantitative measurement for the presence of growth and AR modulators. Using these assays, we found that osteoblast conditioned media (CM) enhanced prostate cancer cell growth, but not AR activity. After priming with androgen (<1 nM R1881), forskolin or the pesticide dichlorvos enhanced AR activation, whereas interleukin-6 (IL-6) inhibited it., Conclusion: These non-destructive, cell-based assays enable rapid systematic monitoring of the effects of drugs or complex mixtures on prostate cancer cell growth and/or AR activity.

Background: The purpose of this study was to determine the contribution of different transactivating regions of the androgen receptor (AR) to the induction of androgen-regulated promoters in poorly (PC3 cells) and well-differentiated (LNCaP cells) prostate cancer cell lines., Methods: PC3 and LNCaP cells were co-transfected with plasmids expressing full-length AR or deletion mutants together with luciferase reporters linked to the probasin (PB) and PSA promoters; as well as to ARR3tk, a PB-derived recombinant promoter., Results: Androgen induction of the ARR3tk promoter in the presence of AR was 8- to 10-fold higher than that seen with the PB promoter. Activation of ARR3tk was greatest with an androgen-independent construct in which the first 231 amino acids and the ligand binding domain had been removed, indicating that this promoter is more responsive to activating functions in the N-terminal domain than in the ligand binding domain. By comparison, induction of the PB promoter was greatest with the full-length AR, which suggests that the ligand binding domain also makes a major contribution to the activation of this promoter. In similar analyses with the PSA promoter, AR regions required for promoter induction was dependent on the host cell type. In PC3 cells, the predominant AR transactivation function was androgen-independent and resided in the N-terminal domain, whereas in LNCaP cells, the highest level of induction was androgen dependent and also required participation of the ligand binding domain., Conclusions: Our results indicate that the relative utilization of transactivating functions in N-terminal and ligand binding domains of the AR is promoter and cell specific.

With increasing purification of the androgen receptor from nuclei of rat ventral prostate, a receptor-like protein could be demonstrated by chemical staining with silver nitrate. After sonication and digestion of nuclei with micrococcal nuclease, the solubilized receptor was applied to a column of Matrex Gel Green A and eluted with a linear gradient of 0-2 M NaCl. Characterized by specific binding of dihydrotestosterone, this form of the receptor was also androgen dependent and yielded an apparent Mr of 33,000 when analyzed by polyacrylamide gel electrophoresis and silver nitrate staining. To facilitate recovery following chromatography, the receptor was precipitated with 0-40% ammonium sulfate. Analysis of the 15-fold enriched fraction by sucrose density-gradient centrifugation confirmed the presence of a 3S androgen-binding protein. About 200 ng of the precipitated protein was applied to a column of dihydrotestosterone-17 beta-succinyl agarose (ligand concentration, 0.25 mumol/ml). The fractions eluted with 50 microM dihydrotestosterone were electrophoresed and stained as before; again, the presence of a 33,000 Mr protein sensitive to castration was demonstrated. Alternatively, when the precipitated protein was fractionated by fast protein liquid chromatography utilizing a Superose 12 HR 10/30 column, the receptor coeluted with nuclear proteins in the 29,000-36,000 Mr range as determined both by retention time and electrophoresis. In combination, the above methods may be used to obtain a receptor protein purified to near homogeneity with a yield of 5-10%. The amount of receptor afforded by the purification sequence is small but nevertheless sufficient for chemical detection. We anticipate that with modification, the procedures may prove suitable for the recovery of nuclear androgen receptor on a preparative scale.