Induction of cell-free, in vitro transcription by recombinant androgen receptor peptides.
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.
Snoek
R
R
Department of Cancer Endocrinology, B.C. Cancer Agency, Vancouver, Canada.
Rennie
P S
PS
Kasper
S
S
Matusik
R J
RJ
Bruchovsky
N
N
D000727:Androgen-Binding Protein / Q000235:genetics; D000818:Animals; D002277:Carcinoma; D002457:Cell Extracts; D002467:Cell Nucleus; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008297:Male; D010455:Peptides; D011471:Prostatic Neoplasms; D051381:Rats; D011944:Receptors, Androgen / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D000727:Androgen-Binding Protein; D002457:Cell Extracts; D010455:Peptides; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; C027519:probasin; D005982:Glutathione Transferase