) The overall goal of this proposal is to investigate the enhancement of two mechanisms preventing colon carcinogenesis modulated by dietary differentiating agents: the elevation of detoxification enzyme levels and an increased rate of colonic cell apoptosis subsequent to cellular differentiation. The two dietary differentiating agents to be studied are sodium butyrate produced by microflora from fiber intake and the vitamin D active metabolite, 1,25-dihydroxyvitamin D3. Differentiation and apoptosis protect colon cells against carcinogenesis by regulating against hyperproliferative growth and elimination of damaged colonocytes. The biochemical detoxification provided in the colon by the enzymes glutathione S-transferase (GST) and NAD(P)H:quinone reductase (QR) eliminates electrophilic carcinogens and their reactive redox-cycling species constantly found in the lumen. A normal diet containing fruits and cruciferous vegetables also contains may inducers of detoxification enzymes such as isothiocyantes, fumarates, coumarins, and allyl sulfides. The coordinate induction of QR and GST is through increased DNA binding of c-Jun and c-Fos at the common AP-1 site in their promoter regions. Using the human colon cell model, HT-29 cells, we have demonstrated a 2- to 3-fold basal level elevation of QR and GST by differentiation with NaB and a synergistic elevation of both enzyme levels and apoptosis by the combination of NaB with QR and GST inducers. Whether similar elevation of the basal level of these enzymes can be achieved with other dietary colonic differentiating agents such as Vitamin D is not known. The mechanism of elevation of these enzymes by differentiating agents is also not known. We propose a study of the influence of Vitamin D-related cellular differentiation of the rate of elevation of both detoxification enzymes and rate of apoptosis. We will investigate using the human colon cell, HT-29, as a model: 1) whether differentiation by 1,25-dihydroxyvitamin D3 enhances increases of basal and induced levels of QR and GST enzymatically and transcriptionally; 2) the importance of c-jun and c-fos activation and their binding at the AP-1 sites in expression of elevated levels of QR and GST during differentiation; and 3) if increase by other differentiating agents such as 1,25-dihydroxyvitamin D3 reflects the increase in apoptotic rate by NaB differentiation or can further enhance its effects.
