The ideal method of cancer control will be chemoprevention. Several chemopreventive agents have been identified through extensive screening using either whole animal studies or other correlative properties such as phase 2 detoxifying enzymes induction. However, it is well documented that drug design directed at highly specific targeted protein(s) lead to the discovery of the most potent and specific drug molecules. The search for biochemical targets, that can lend themselves to many avenues in the design of effective chemoprevention agents, is the long-term goal of our research program. Specific target(s) can be obtained through delineation of the signal transduction pathway(s) involved in the mechanism of action of an active agent. Our approach in this research proposal is to employ the well established paradigm that many inducers of phase 2 enzymes are also potent chemopreventive agents. This is logical because phase 2 enzymes play critical roles in the detoxification and removal of potential carcinogens from the body and thereby protect against carcinogenesis. The main questions are how these phase 2 genes are induced and what are the important cellular signaling component(s) that are involved; and these molecules can then be the target(s) of drug design. To answer these critical questions, we will utilize the proven chemopreventive agents, the isothiocyanates (ITCs), sulforaphane and phenethyl isothiocyanate (PEITC) that are found in the commonly consumed vegetables such as broccoli, as model compounds. Both agents have been shown to induce phase 2 genes, and possess potent chemopreventive actions in carcinogenesis models including 7, 12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor model. Furthermore, currently the only known cellular signaling pathway that is affected by both sulforaphane and PEITC is the mitogen-activated protein kinase (MAPK) pathway. Our hypothesis is that MAPK pathway is an important biochemical link in the signaling cascades leading to phase 2 gene induction by ITCs and thus their chemopreventive actions. The following specific aims are designed to identify and characterize the relevant components of the signaling pathway leading to the induction of phase 2 enzymes. 1) To investigate the role of ERK2 or JNK1, the downstream components of the MAPK pathway, in the regulation of phase 2 gene expression by sulforaphane and PEITC, and their chemical mechanisms. 2) To identify the relevant upstream functional signaling components involved in ERK and JNK activation by sulforaphane and PEITC, in the regulation of phase 2 gene expression. 3) To compare and contrast the activated signaling component(s) and phase 2 gene induction in vivo in the mouse after sulforaphane or PEITC administrations with the observed in vitro cell line data from Specific Aims 1 and 2. The results of our works will provide new information on the signaling pathway components that can be useful as chemopreventive agent design and screening targets.