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Nuclear import of the human androgen receptor.

ABSTRACT: Nuclear import of the human androgen receptor was investigated by immunocytochemical analysis of androgen receptor deletion and substitution mutants, which were transiently expressed in COS-1 cells. The signal responsible for nuclear import is encoded by amino-acid residues 608-625 and is functionally similar to the bipartite nucleoplasmin nuclear-localization signal. Although the subcellular distribution of androgen receptors mutated in the DNA-binding domain was unchanged compared with the wild-type androgen receptor, in the presence of ligand these mutations resulted in part of the receptor population forming clusters. Depending on the presence or absence of the bipartite nuclear localization signal, clusters were formed in the nucleus or in the cytoplasm, respectively. Expression of the wild-type androgen receptor in different cell lines revealed a cell-line-specific subcellular distribution of the unliganded receptor. The androgen receptor was predominantly nuclear when expressed in HeLa cells, whereas mainly cytoplasmic staining was observed when it was expressed in COS-1 cells. In the presence of hormone, the androgen receptor was located in the nucleus, independent of the cell line that was expressing the receptor. Anti-androgens and various steroid hormones induced the nuclear localization of the wild-type androgen receptor in a dose-dependent way, without activating transcription of an androgen-regulated reporter gene. This indicates that the inability of the tested compounds to activate transcription is not due to inhibited nuclear import.

SUBMITTER: Jenster G

PROVIDER: S-EPMC1134432 | biostudies-other | 1993 Aug

REPOSITORIES: biostudies-other

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Project description:BackgroundCastration-resistant prostate cancer can develop resistance to enzalutamide because of androgen receptor (AR) point mutations, AR overexpression, constitutively active AR splice variants, and/or elevated intratumoral androgen synthesis. The point mutation ARF876L was reported to be stimulated, instead of inhibited, by enzalutamide, thus contributing to enzalutamide resistance. We have recently developed JJ-450 as a novel AR antagonist with the potential to treat enzalutamide-resistant castration-resistant prostate cancer (CRPC).MethodsWe employed several assays to determine the impact of JJ-450 and enzalutamide on prostate cancer cell lines expressing green fluorescent protein (GFP)-ARF876L . These assays include a prostate-specific antigen enhancer/promoter-based luciferase assay to determine AR transcriptional activity, a quantitative real-time polymerase chain reaction assay, and Western blot analysis to detect expression of AR-target genes at the messenger RNA and protein level, fluorescence microscopy to show AR subcellular localization, and a 5-bromo-2'-deoxyuridine assay to measure prostate cancer cell proliferation.ResultsAs expected, enzalutamide inhibited wild-type (WT) AR but not ARF876L transcriptional activity in the luciferase assay. In contrast, JJ-450 inhibited both WT-AR and ARF876L transcriptional activity to a similar extent. Also, enzalutamide retarded androgen-induced nuclear import of GFP-AR, but not GFP-ARF876L , whereas JJ-450 retarded nuclear import of both GFP-AR and GFP-ARF876L . To further evaluate JJ-450 inhibition of ARF876L , we stably transfected C4-2 cells separately with GFP-AR or GFP-ARF876L . Enzalutamide inhibited endogenous AR-target gene expression in C4-2-GFP-ARWT , but not in the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited AR-target gene expression in both C4-2 sublines. More importantly, enzalutamide inhibited proliferation of C4-2-GFP-ARWT , but not of the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited proliferation of both C4-2 sublines.ConclusionJJ-450 inhibits enzalutamide-resistant ARF876L mutant nuclear translocation and function. Our findings suggest that JJ-450 and its analogs should be further developed to provide a potential new approach for the treatment of enzalutamide-resistant CRPC.

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Nuclear import of the human androgen receptor.

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