Research Project
2600914
The established human lung tumor cell line, A427 is being used to assess chemopreventive activity of chemicals by measuring anchorage independent growth inhibition. The A-427 cell line grows in soft agarose at a frequency of 0.37 to 2.2%. This anchorage independent growth can be inhibited by 13-cis-retinoic acid. Initial cytotoxicity assays are used to assess the relative toxicity of each chemopreventive test agent and to permit the selection of approximate concentration ranges for the human tumor cell assay. The expression of the anchorage independent phenotype is measured by the ability of A427, the human lung tumor line to form colonies in semi-solid agarose medium. The modulation of the anchorage independent phenotype is determined by the addition of the chemopreventive agent to the semisolid medium at the time of cell seeding. Exponentially growing cells are seeded at 5 x 10(4) cells per 35 mm dish or well in 0.33% semisolid agarose medium. Five concentrations of the chemopreventive test agent are used at half-log dilutions from the highest concentration possible in medium or in a solvent such as dimethylsulfoxide without solvent toxicity. Controls consist of untreated and solvent treated cells, and the A427 cells exposed to an known positive colony inhibiting agent, such as 13-cis retinoic acid. At the end of approximately 14 days the dishes are stained using a tetrazolium salt and the colonies counted. Total colony number per dish shall be recorded on data sheets. Thirty potential chemopreventive agents are being tested.
