Despite many important advances in cancer chemotherapy, complete eradication of tumors is often prevented by the presence or development of resistance to treatment. One mechanism by which resistance can arise occurs when cellular enzymes prevent or repair cytotoxic DNA modifications introduced by therapeutic agents. Thus, previous studies have shown that the formation of one cytotoxic modification by chloroethylnitrosourea, a dC-dG cross-link, is prevented by O6-alkylguanine-DNA alkyltransferase; high cellular levels of this enzyme are associated with resistance. New data indicate that the presence of 3-methyladenine DNA glycosylase also contributes to resistance to chloroethylnitrosoureas. The studies described here will investigate the mechanism by which glycosylases cause this increase in resistance and determine whether it extends to other chloroethylating agents. Glycosylase will be purified from the cloned human gene (L. Samson et al., Proc. Natl. Acad. Sci. USA 88, 9127-9131, 1991). Then, using DNA substrates modified by [3H]CNU and our library of modified bases, we will determine which modified bases are released by this enzyme. This information will relate the biochemical action of the glycosylase to its protective effect and identify modifications which are potentially cytotoxic. More evidence as to which lesions are cytotoxic will be obtained by determining which modified bases are depleted in resistant glial cells. Knowledge of which lesions are cytotoxic can ultimately be used in the design of new antitumor agents that cause these modifications, and in monitoring the effects of known agents. As substrate bases are identified, they will be tested for their ability to inhibit glycosylase action in vitro. Analogs of these substrates will also be tested as inhibitors. Inhibition of the glycosylase will presumably increase the cytotoxicity of the haloethylnitrosoureas and related agents, and this effect will be tested on glycosylase-deficient E. coli carrying plasmids with the human glycosylase and on cultured glial cells which are known to be resistant to chloroethylnitrosourea and to have high glycosylase levels.
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