MOLECULAR MECHANISMS OF ARA C THERAPY IN MAN

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We have previously demonstrated that ara-C incorporates in leukemic cell DNA and that the extent of this incorporation correlates with loss of clonogenic survival. We have also demonstrated that the incorporated arabinosyl residue serves as a poor primer terminus and thereby results in inhibition of DNA synthesis. The (ara-C)DNA is structurally abnormal and undergoes strand scission upon exposure to alkali. Furthermore, inhibition of DNA synthesis by the incorporation of ara-C causes the accumulation of strand breaks in DNA undergoing repair and replicative synthesis. Finally, the formation of (ara-C)DNA results in both the induction of terminal differentiation and lethal cellular events. These findings provide major new insights into the mechanism of action of the most effective agent in the treatment of acute myelogenous leukemia. The proposed studies will extend our work on incorporation of ara-C during replicative synthesis by monitoring ara-C incorporation during DNA repair. The biochemical effects of (ara-C)DNA formation during both replicative and repair synthesis will then be monitored as a result of exposue to 3-aminobenzamide and caffeine. The molecular parameters will be correlated with biologic effect as determined by mutagenesis and loss of clonogenic survival. Finally, we will employ a new in vitro post-labeling assay to monitor formation of (ara-C)DNA in clinical samples. This approach will be applied to speciments obtained from patients receiving low dose continuous infusion area-C as a clinical correlate of our biologic and biochemical studies. These studies will be performed on HL-60 promyeloblasts and density-arrested, plateau phase diploid fibroblasts. The incorporation of ara-C during replicative and repair DNA synthesis will be monitored by equilibrium centrifugation and DNA damage will be monitored using the alkaline elution technique. This approach should provide the molecular basis for understanding the effects of ara-C on the induction of lethal cellular events. This work is important in providing the experimental basis for the design of biochemically rational clinical trials with ara-C and for gaining an understanding of the molecular control of human leukemia.