The objective is to improve therapy of human disease, primarily cancer, with nucleoside analogs. The proposed studies are directed at aspects of the identification, purification and characterization of human enzymes for the activation of the purine C-nucleoside analogs tiazofurin and its recently synthesized analog selenazofurin and to assess the relationship of these enzyme systems to sensitivity and resistance of human cells to the drugs and to the metabolism of naturally occurring purine nucleosides. We will test the hypothesis that the C-nucleosides are activated in human lymphoid cells via alternate metabolic pathways, including the novel phosphorylation via a cellular nucleotidase. The following specific aims will be sought: i) isolation and characterization of new mutant lines with defective phosphorylation of the named compounds; ii) purification and characterization of the enzyme activities for the phosphorylation of tiazofurin and selenazofurin from human leukemic cells, including those with varied sensitivities to the drugs. Especially important will be attempts to characterize the nucleotidase(s) with respect to specificity for the phosphorylation of naturally occurring nucleosides and analogs. These studies may also uncover whether the nucleotidase serves as an anabolic route for inosine - or guanosine-type compounds that have demonstrated ability to cause differentiation of lymphoid cells. These studies will also examine the mechanism for a synergistic interaction between tiazofurin and the DNA damaging agent etoposide (VP16-213) in human leukemia cells. The goal is to correlate nucleotide pool depletions, repair enzyme activity and DNA strand-breaks and cytotoxicity in leukemia cells. These studies may reveal whether the active dinucleotide derivative of tiazofurin or selenazofurin may act as inhibitor of DNA repair synthesis in cells.
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