Human colon cancer remains resistant to most chemotherapeutic agents. In this project we will analyze repair of O(6) alkylguanine-DNA adducts as a specific mechanism of nitrosourea resistance in colon cancer cells. Because this adduct is a major cause of the cytotoxic effects of nitrosoureas, DNA repair of this adduct may be a useful target for biochemical modulation of drug resistance. For this reason, we will study the DNA repair protein O(6) alkylguanine-DNA alkyltransferase (alkyltransferase), which repairs O(6) alkylguanine-DNA adducts. Specifically, we will establish that the alkyltransferase is an important mechanism of nitrosourea resistance in colon cancer and develop ways to modulate nitrosourea resistance in colon cancer first in vitro, then in xenografts and finally in human clinical trials by inactivating the alkyltransferase. To this end, we aim to: (1) Characterize alkyltransferase activity in colon cancer and colon cancer cell lines. (2) Evaluate the ability of alkyltransferase inhibitors to overcome nitrosourea resistance in the cell lines. (3) Determine whether modulators of other drug resistance systems affect the alkyltransferase and/or alter the action of alkyltransferase inhibitors. (4) Establish the ability of alkyltransferase modulators to sensitize colon tumor xenografts to nitrosoureas (5). Determine the myelotoxicity of each modulator/- cytotoxic drug combination studied in this Program Project at doses that look promising in the xenograft model, and (6) During clinical trials with modulators and BCNU, measure modulator blood levels and monitor the modulation of alkyltransferase achieved in tumor biopsy samples. These studies match the major theme of this Program Project, the development of rational treatment strategies to overcome alkylating agent resistance in patients with colon cancer.