Initial growth inhibition assays with the test chemopreventive agent are being used to select appropriate concentration ranges for use in the subsequent transformation-inhibition assay. Five log dilutions are used from 1 mM or the highest soluble concentration in medium or in a solvent such as dimethylsulfoxide without solvent toxicity. primary prostate epithelial cells are seeded into 60 mm dishes and allowed to attach for 24 hours. Then the chemopreventive test agent is added in fresh medium and the cultures allowed to incubate 72 hours then all cultures receive fresh media containing only the chemopreventive agent and incubated for 3-5 more days. A positive control compound such as a retinoid is used in all assays. Then the cultures are then fixed and stained. Three dishes per point are scored and the relative growth determined. The induction of apoptosis is being measured in human prostate epithelial cells. The level of induction of apoptosis is being measured in response to exposure to potential chemopreventive agents. Five nontoxic concentrations are being measured for their ability to induce apoptosis for each agent. The Inhibition of Prostate Specific Antigen (PSA) is being measured. A prostate specific antigen assay is being used to quantitatively measure effects of chemopreventive agents on the production of PSA in cultures of human prostate epithelial cells. At least 5 nontoxic concentrations are being tested for each compound.
