We have developed and applied an in vitro assay (The Human Epidermal Cell (HEC) Assay) to screen candidate chemopreventive agents for efficacy (Elmore et al., 1999, Elmore et al., 2000). The basis for this assay is that human keratinocytes are stimulated with a carcinogen, propane sultone, which results in increased growth and decreased involucrin expression which in indicative of reduced differentiation. Chemopreventive efficacy is determined by the ability of candidate agents to suppress this increased growth and decreased involucrin expression. This assay has proven to be an excellent predictor of in vivo efficacy (Elmore et al., 2000). The drawback of the assay is its labor intensive nature and time scale to completion (1 month). In keeping with the purpose of the Cancer Prevention Research Small Grant Program, the goal of this proposal is to determine specific biomarkers which will permit the rapid screening of candidate agents for efficacy. To achieve this goal, we plan to utilize information we have already generated on agent efficacy, or lack thereof, in the HEC Assay. We will use normal, and spontaneously transformed human epidermal keratinoeytes that have been transfected with an NF-kappaB luciferase reporter construct to determine the potential of agents to induce NF-kappa B expression. We will select at least seven agents known to be effective and five agents known to be ineffective and examine their impact on the expression of NF-kappaB expression in human epidermal cells under conditions utilized in the HEC Assay. In addition, using microarray analysis, we will determine gene expression changes induced in these cells by two chemopreventive agents at concentrations that are relevant to clinical trial plasma levels and one agent that has not demonstrated efficacy. Using cluster analysis, we will determine the global gene expression changes induced by each agent. The strength of the proposed approach lies in the high quality data that has already determined from the HEC Assay.
