Acute nonlymphocytic leukemia (ANLL) can occur following exposure to chemical carcinogens. Nonetheless, the mechanisms of chemical leukemogenesis in the bone marrow have not been identified. A clear association exists between ANLL and exposure to alkylating agents, especially, nitrogen mustard derivatives and, more recently, nitrosoureas. Thus, alkylating agent induced damage to the myeloid elements in the marrow may be a useful model of chemical leukemogenesis. The studies proposed here will evaluate the ability of myeloid cells to repair 0(6)-alkylguanine, a DNA adduct formed by alkylating agents. 0(6)-alkylquanine is directly mutagenic: DNA replication of the adduct results in the base pair substitution AT for GC. Certain alkylating agents, such as nitrosoureas commonly produce 0(6)-alkylguanine; production by nitrogen mustards is not established. 0(6)-alkylguanine is repaired in a unique fashion by an alkyltransferase which covalently binds the alkyl group resulting in a dead end complex. Levels of alkyltransferase fall immediately after nitrosourea exposure, thereafter enzyme induction may occur. Enzyme levels vary widely between species, individuals and tissues. Myeloid precursors have not been studied. These studies will test the hypothesis that myeloid cells are relatively deficient in alkyltransferase activity and are more prone to mutagenic damage by alkylating agents than other cells. The objectives are to (1) determine the relative range of methyltransferase activity in human myeloid and lymphoid precursors, (2) characterize the kinetics of depletion, recovery and induction of methyltransferase a) in myeloid cells exposured in vitro to nitrosoureas and nitrogen mustards, and b) in cells from patients undergoing alkylating agent therapy, (3) determine whether myeloblasts in alkylating agent associated ANLL have lower levels of methyltransferase than myeloblasts in idiopathic ANLL, (4) compare the cytotoxicity and mutagenicity of alkylating agents in myeloid cell lines with differing levels of methyltransferase, and (5) measure the capacity of therapeutic nitrogen mustards to produce 0(6)-alkylguanine. Methyltransferase activity in cell extracts is measured as the reduction in [3H] 0(6)-methylguanine following incubation with [3H]methylated DNA. Clonogenic assays of myeloid cell lines are used in cytotoxicity studies and subcloned with 6-thioguanine and oubain in mutagenicity studies. Presence of 0(6)-alkylguanine is determined by chromatographic separation of 32P labeled digests of alkylated DNA.
