The subnuclear three-dimensional image analysis of androgen receptor fused to green fluorescence protein.

To establish the novel approach in order to distinguish the transcriptionally active androgen receptor (AR) from the transcriptionally inactive AR, we performed the three-dimensional construction of confocal microscopic images of intranuclear AR. This method clearly distinguished the subnuclear localization of transcriptionally active AR tagged with green fluorescent protein (AR-GFP) from the transcriptionally inactive AR-GFP. Transcriptionally active AR-GFP mainly produced 250-400 fluorescence foci in the boundary region between euchromatin and heterochromatin. Although the AR-GFP bound to such antiandrogens as hydroxyflutamide or bicalutamide translocated to the nucleus, they homogeneously spread throughout the nucleus without producing any fluorescence foci. Antiandrogenic environmental disrupting chemicals, such as 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene, vinclozolin, or nitrofen, also disrupted the intranuclear fluorescence foci. A point mutation (T877A) resulted in the loss of ligand specificity in AR-GFP. Even in this mutant receptor, agonists, such as dihydrotestosterone, hydroxyflutamide, or progesterone, produced the fluorescence foci in the nucleus, whereas the transcriptionally inactive mutant binding bicalutamide was observed to be spread homogeneously in the nucleus. Taken together, our findings suggest that, after nuclear translocation, AR is possibly located in the specific region in the nucleus while demonstrating clustering tightly depending on the agonist-induced transactivation competence.