The overall objective of the proposed research is to gain insight into the regulation and genetics of steroid 5alpha-reductase (5alpha- reductase) in benign prostatic hyperplasia (BPH) and prostate adenocarcinoma and to isolate genes whose expression is altered in prostate cancer. 5alpha-Reductase plays a crucial role in the embryogenesis and maintenance of the prostate and as such is a candidate gene for altered expression in prostate pathologies involving unregulated growth. In addition, other genes must participate in the development of prostate cancer, a process characterized by initiation, progression, metastasis, and hormone dependence of tumor growth. Our initial goal in this new project is to characterize the hormonal regulation of 5alpha- reductase isozyme expression in BPH and prostate cancer. Biopsy material from patients undergoing hormonal ablation therapies (finasteride, antiandrogens) for BPH and prostate cancer will be obtained from the Tissue Core of this Center application. Expression of the type 1 and type 2 isozymes of 5alpha-reductase will be measured in these samples by immunoblotting, immunohistochemistry, enzyme activity and RNA blotting. The goal of this aim is to define the role of androgens in the expression of the 5alpha-reductase genes. Our second objective is to screen for mutations in the 5alpha-reductase genes in subjects with prostate cancer. The methods of Southern blotting and single strand DNA conformation analysis will be used to identify mutations in the genes. Putative mutations will be created in an expressible CDNA and their biochemical effects on enzyme activity determined after transfection into cultured cells. The goal of these studies is the identification of gain- or loss- of-function-mutations in the 5alpha-reductase loci that may contribute to tumor progression or the transition from androgen-dependence to androgen-independence. Our final objective in this proposal is the isolation of genes that are differentially expressed in prostate cancer. A differential polymerase chain reaction method will be used to identify MRNAS whose expression is altered in prostate cancer. Complementary DNAs corresponding to these MRNAS will then be cloned and characterized by DNA sequencing, RNA blotting, expression in transfected cells, and in situ MRNA hybridization. Our ultimate goal is the identification and characterization of prostate specific dominant of recessive oncogenes.
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