The major focus of this proposal is the elucidation of the mechanisms of action of cytosine arabinoside, ara C. This drug, the single most effective agent in the treatment of AML, appears to exert most of its actions through fraudulent incorporation into DNA. We will attempt to define in precise molecular terms the effects of this misincorporation on the functions of DNA; better define the nature and locus of ara C misincorporation; further explore the intermediary metabolism of the principal metabolite uracil arabinoside (ara U); and finally study which of several biochemical parameters may be predictors of clinical response to the drug. The role of DNA in controlling the life and growth of cells is so central that any derangement is likely to be damaging or lethal. The complex, interrelated functions of DNA make assessment of the impact of misincorporated ara C on any one function of DNA in living cells highly problematic. We have thus taken the approach of using synthetic DNA fragments as simplified in vitro models. We have developed methodology for the chemical synthesis of DNA oligomers containing ara C. We will utilize these oligomers for in vitro assays of DNA functions including chain elongation, replication fidelity, chemical stability, fragment ligation, and as a substrate for 3' 5' exonuclease activity. We will look for evidence of sequence specificity at the nearest neighbor and short sequence levels of DNA organization. We will also look for evidence of removal of ara C from DNA by excision repair mechanisms and for its spontaneous deamination to ara U. Although ara U is usually considered an inactive metabolite of ara C, reports indicate that it may have some activity and that it may modulate the activity of ara C. We will examine whether ara UTP cn serve as a substrate for dUTPase, and whether ara U can be excised from DNA by the uracil DNA glycosylase system. During our clinical treatment program for childhood acute myelogenous leukemia we will study the efficacy of ara C as a single agent in producing early cell kill. We will measure biochemical parameters related to the metabolism of ara C and subsequently look for correlations between these parameters in each patient and the degree of early cell kill produced in that patient.
