Although cytarabine, 1-B-D arabinofuranosylcytosine (ara-C) has been used extensively for the treatment of leukemia, its utility has been severely curtailed due to the development of cellular resistance to this anticancer agent. Maintenance of high intracellular levels of ara-CTP appears to be the essential factor in the chemotherapeutic efficacy of ara-C. There appears to be at least three major determinants to the generation and maintenance of inhibitory levels of ara-CTP. The first is activation of ara-C by phosphorylation to the monophosphate by deoxycytidine kinase. A second determinant is the enzyme, nucleoside deaminase. Increased deamination of ara-C to ara-U leads to ineffective ara-C therapy. A third and more recently uncovered determinant to the maintenance of intracellular ara-CTP are the activities of enzymes capable of hydrolyzing ara-CTP. The overall aim of this proposal is to extend our basic understanding of the metabolism of ara-C by; 1) elucidating the physical and kinetic properties of the enzyme(s) involved in the hydrolysis of ara-CTP, 2) characterization of dUPTase's ability to hydrolyze ara-UTP and a study of the enzymes involved in the phosphorylation of ara-U, 3) determination of the molecular events responsible for the increase in nucleoside deaminase activity in cells that become resistant to ara-C by increasing deaminase activity. The methods to be used include; 1) an analysis of various cultured cell types for their ability to hydrolyze ara-CTP and, purification and characterization of ara-CTP hydrolyzing enzyme(s), 2) characterization of the herterogeneity of the dUTPase enzyme from various cell types using monoclonal antibodies and a determination of the kinetic parameters of dUTPase catalyzed hydrolysis of ara-UTP, 3) fractionation of cellular enzymes capable of phosphorylating ara-U to ara-UTP and profiling various cell types in terms of their ability to phosphorylate ara-U, 4) utilizing recombinant DNA techniques, the isolation of the nucleoside deaminase gene will be performed using a plasmid expression vector and probing with monoclonal antibodies generated against the deaminase enzyme. This cloned gene will then be used to determine whether this type of resistance is due to gene amplification. Basic enzymological and molecular research along these lines may elucidate new pathways in the metabolism of ara-C. Findings from the proposed research may provide new diagnostic protocols and may also lead to new rationales for anticancer drug development.
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