Despite the prevalence of prostate cancer, basic research regarding the cause and biology of this disease has been severely hampered by the lack of in vitro models of human prostate cancer. This proposal outlines an experimental plan to develop an in vitro model of carcinogenesis in human prostate cancer and to utilize that model and established cell lines to study the mechanisms by which prostate cancer cell growth is controlled. We will intensively examine the role of peptide growth factors in cell growth. The experimental plan will include: 1) procurement of prostate tissue specimens via the University of Wisconsin Clinical Cancer Center (UWCCC) tissue procurement system from UW and VA patients undergoing therapeutic or diagnostic surgical procedures; 2) isolation of epithelial cells from normal, hypertrophic, and cancerous prostate tissue by collagenase digestion and establishment of primary epithelial cell cultures in serum-free medium; 3) through transfection with the SV40 large T antigen gene to extend the lifespan of cells in culture; 4) neoplastic transformation of nontumorigenic prostate epithelial cells by treatment with carcinogens and serial transfection of activated oncogenes, including H-ras and nuclear oncogenes (p53, myc, etc.); 5) assessment of the cell lines at each step from normal epithelial cells to androgen-independent tumorigenic cells for growth factor production and responsiveness, paying particular attention to TGFalpha, TGFbeta, IGF-I, IGF-II, aFGF, and PDGF. We will examine the tumorigenic phenotype of the cell lines at each step in nude mice. Tumor growth will be measured and visceral organs will be histologically examined to determine metastatic potential. Subsequent studies will utilize nude mice tumor models to assess the effects of parentally administered growth factors, conditioned media, biologics (e.g., interferons, interleukin-2 and TNF), and cytotoxic drugs on tumor establishment, growth, and metastases. 6) Continued characterization of existing established cell lines for growth factor production and responsiveness. One androgen-responsive cell line, for growth factor production and responsiveness. One androgen-responsive cell line, LNCaP, has already been transfected with inducible constructs of the activated H-ras gene or the TGFalpha gene. These clones will be examined for alterations in their androgen-responsive and tumorigenic phenotypes in vitro and in vivo.
| Wilding, G; Gelmann, E P; Freter, C E (1990) Phosphoinositide metabolism in human prostate cancer cells in vitro. Prostate 16:15-27 |
| Rago, R; Mitchen, J; Wilding, G (1990) DNA fluorometric assay in 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water. Anal Biochem 191:31-4 |
