Induction of cell-free, in vitro transcription by recombinant androgen receptor peptides.

Snoek R, Rennie PS, Kasper S, Matusik RJ, Bruchovsky N
J Steroid Biochem Mol Biol. 1996 59 (3-4): 243-50

PMID: 9010316 · DOI:10.1016/s0960-0760(96)00116-1

An in vitro, cell-free transcription system, based on prostate-derived transcriptional machinery and very powerful androgen response elements (AREs), has been developed. Multiple (p(ARR3)LovTATA) AREs from the androgen-regulated probasin gene were linked to G-free cassettes and used in nuclear extracts prepared from prostate carcinoma cell lines (PC3 and LNCaP cells) to test specific induction of transcription by full-length AR and by glutathione-S-transferase (GST)-fusion peptides in which the androgen receptor (AR) DNA-binding domain alone (AR524-649), or together with the ligand-binding domain (AR524-902), or a portion of the NH2-terminal domain (AR232-649) were incorporated. In the presence of AR, nuclear extracts from PC3 cells had greater activity in supporting transcription than those from LNCaP cells; and lower background activity than those from HeLa cells. All of the AR forms correctly initiated in vitro transcription of ARE-templates in an androgen-independent manner. The amount of specific, inducible transcript was dependent on the concentration of AR peptide present. AR524-902 was the most potent transactivator tested, with the maximal level of specific transcript over 900-fold higher than the minimal level. At all concentrations this peptide was three to four times more active than either AR524-649 or AR232-649. In conclusion, we have developed a very specific and sensitive cell-free transcription system for delineating trans-activational regions of the AR.

MeSH Terms (20)

Androgen-Binding Protein Animals Carcinoma Cell-Free System Cell Extracts Cell Nucleus Escherichia coli Glutathione Transferase HeLa Cells Humans Male Peptides Prostatic Neoplasms Rats Receptors, Androgen Recombinant Fusion Proteins Sensitivity and Specificity Transcription, Genetic Transcriptional Activation Tumor Cells, Cultured

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