The long-term objective of this research is to develop therapies for prostate cancer that will reverse of prevent the molecular consequences of anomalous transcriptional events associated with tumor progression and the emergence of androgen independence. The transcriptional regulation of the prostate-specific antigen (PSA) gene has been shown to correlate well with progression of prostate cancer, with both gene expression and the disease going from an androgen-dependent to an androgen-independent stage. In fact, the earliest detectable changes associated with progression is the loss of androgen-independent state. In fact, the earliest detectable change associated with progression is the loss of androgenic control of PSA gene expression. Accordingly, the major aim of this work is to study the molecular mechanisms of androgen-independent regulation of the PSA gene and to characterize defects in regulation that appear with nascent independent activation of the androgen receptor by PKA and growth factors. The androgen receptor domains involved will be determined by transfecting a series of deletion mutants of the androgen receptor into PKA-activated LNCaP cells. (2) To investigate the function of transcriptional binding sites unrelated to the androgen response element on the PSA promoter/enhancer which may act independently or together with the androgen receptor in androgen-independent malignancy. Methods will include DNase footprinting, electrophoretic mobility shift assays, DNA deletion mapping and an in vitro transcription assay. (3) To identify androgen receptor and non-androgen receptor co-regulators that may be integral to the development of androgen-independent transcriptional activity. Protein- protein interactions will be examined in co-immunoprecipitation and glutathione S-transferase-pulldown experiments and, the biological relevance of these co-regulators, by application of transfection experiments and an in vitro transcription assay. It is anticipated that the proposed research will provide new insight into the molecular basis of progression to androgen-independence and identify new targets for delaying or averting androgen resistance in prostate cancer when first manifested by defective expression of the PSA gene.
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