In this project, we will focus on the androgen regulation of the human acidic fibroblast growth factor (aFGF) and androgen-induced growth factor (AIGF) in prostatic epithelial cell proliferation. Acidic FGF is one of the best characterized mitogens for endothelial cells and acts as an angiogenic factor in vivo; therefore, it may play a role in tumorigenesis. Studies in the Dunning prostate tumor model in the rat implicated aFGF as the growth factor in prostate cell growth and malignancy, and aFGF was found to suppress growth inhibition of transforming growth factor. In addition, sequence comparison showed that aFGF is identical to prostatropin, a prostate epithelial cell growth factor. We have further shown that over-expression of aFGF in fibroblast cells results in transformation to a malignant phenotype. Recently, our laboratory demonstrated that expression of an aFGF mRNA (designated 1.C) in LNCaP cells, an androgen-dependent lymph node cell line derived from a prostate carcinoma, is markedly induced by testosterone. In contrast, aFGF mRNA is constitutively expressed in PC-3 cells, an androgen-independent prostate carcinoma cell line. Androgen acts on SC-3 mouse mammary carcinoma cells to induce secretion of AIGF, the eighth and the latest member of the FGF family. To date, aFGF and AIGF are the only two growth factors whose mRNA levels have been shown to be up-regulated by androgens. We propose to isolate the promoters of these two genes, to test their androgen- inducibility, and to determine whether expression of aFGF and AIGF in prostate carcinomas correlates with tumor progression. The aFGF and AIGF proteins and mRNAs will be localized in the prostate tumor cells using immunofluorescence analyses and in situ hybridization such that the site(s) of action for aFGF and AIGF can be identified. We also propose to identify the transcriptional start sites of aFGF 1.C and AIGF mRNAs by RNase protection analyses and primer extension analyses. The promoter/enhancer sequences will be identified by constructing a series of recombinant molecules that will direct the expression of the gene coding for luciferase in LNCaP and PC-3 cells. The cis-acting sequences will be further defined by gel retardation assays and DNase I footprinting analyses. A better understanding of the regulatory sequences will provide insight into how the aFGF and AIGF genes are regulated so as to support prostate cell growth and malignant transformation. We also plan to isolate trans-acting factors by a novel transduction method. Briefly, cells will be transfected with the 1.C promoter fused to the guanine phosphoribosyltransferase (gpt) gene and then transduced with a cDNA library derived from cells expressing l .C mRNA. The recipient cells of cDNA coding for the proper trans-acting factors will be conferred growth advantage in hypoxanthine-aminopterin-thymidine medium through trans- activation of the chimeric construct. Finally, a transgenic mouse model will be developed in order to study aFGF-induced tumorigenicity. In light of the findings that transgenic mice bearing FGF-3 present benign prostate hypertrophy, we developed transgenic mouse founders bearing human aFGF gene. We propose to establish transgenic mouse colonies to study aFGF- induced tumorigenicity.
