Prostate cells require the presence of androgens for differentiation, development and maintenance. The effects of androgens are mediated predominantly by the androgen receptor (AR). Recent studies have demonstrated that expression of the AR gene is regulated by androgens and modulated by other growth regulatory factors. These studies suggest that both androgens and other growth regulatory factors (i.e. retinoic acid (RA), TPA via c-Jun/c-Fos and the cAMP regulated protein, CREB) play roles in regulating the AR gene activity. In order to define these roles the following specific aims will be addressed:
Specific Aim 1 proposes to determine whether androgens and other factors regulate the synthesis and/or stabilization of AR mRNA and protein by a) Nuclear run-on analysis of AR and mRNA in human prostate cells treated with androgen plus/minus RA, TPA and forskolin, b) Measurement of the half-life of AR mRNA in the presence of androgen plus/minus RA, TPA and forskolin c) Northern blot analysis of AR, CREB, c-Jun and c-Fos mRNAs in cells treated with androgen plus/minus RA, TPA and forskolin.
Specific Aim 2 proposes to identify transcriptional regulatory elements in the promoter region of the human AR gene by a) Cloning and sequencing the 5'-flanking region of the human androgen receptor gene, b) Cotransfection of the hAR promoter (full-length and deletion mutants) into prostate cells with vectors expressing hAR, RAR, cJun, cFos and CREB and c) Footprinting analysis of protein binding sites in the promoter region of the hAR gene in prostate cells transfected with hAR, RAR, cJun, cFos, and CREB.
Specific Aim 3 proposes to characterize the mechanism(s) by which transcription factors regulate the human AR gene by a) Cotransfection of hAR, RAR, cJun, cFos and CREB with putative regulatory elements (wild-type and mutated) coupled to a heterologous promoter (tk-CAT) and b) Band-shift analysis of putative regulatory elements with (i) nuclear extracts from prostate cells treated with androgen plus/minus RA, TPA and forskolin and (ii) recombinant nuclear transcription factors (AR, RAR, cJun, cFos, and CREB).
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