The objective of this proposal is to determine and compare the cellular pharmacology and the molecular mechanisms of action of arabinosylcytosine (ara-C), fludarabine (F-ara-A), and 2', 2'-difluorodeoxycytidine (dFdc) with a view to optimizing their anticancer activity alone and in combinations. This application extends ongoing investigations and utilizes new technical capacities that will provide an understanding of the actions of these drugs at a molecular level. First, the molecular action of DNA-directed nucleoside analogues will be determined with respect to the substrate properties of their 5'-triphosphates for purified human DNA polymerase alpha delta, and epsilon using a defined-sequence primer extension assay. In addition, the ability of DNA polymerase delta and epsilon to excise incorporated analogues will be characterized, and the effect of free and incorporated analogues on DNA ligation will be evaluated. Second, the consequences of incorporation of F-ara-A nucleotide into RNA of exponentially growing cells and quiescent cells will be evaluated with respect to premature termination of transcription, translational efficiency, and cell viability. Third, the biochemical and molecular bases for metabolic modulation of nucleoside analogues which results in synergistic cytotoxicity will be studied. These investigations will focus on the mechanisms by which F-ara-A and dFdC enhance the cellular accumulation of ara-C triphosphate, an action that is associated with synergistic cytotoxicity. Finally, mechanisms by which dFdC """"""""self- potentiates"""""""" its own anabolism and action will be defined. The actions of nucleotides of dFdC against dCMP deaminase, ribonucleotide reductase, and DNA polymerases will be investigated to gain an understanding of the substrate and inhibitory properties of the analogues. Together, these investigations of the metabolism and mechanisms of action of clinically useful drugs and related compounds under development will provide new information to be used in the rational design of combination treatment protocols.
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