FdUMP[N] compounds constitute a novel class of fluoropyrimidine (FP) chemotherapeutic that may be useful for the treatment of human malignacies that are refractory to current chemotherapy. The goal of this research project is to understand the unique cytotoxic mechanism of FdUMP[N] compounds. Human prostate cancer (PC) cells will be used as a model system to investigate FdUMP[N] cytotoxicity because of the relative sensitivity of PC cells to FdUMP[10], and because of the need for new and more effective drugs to treat late-stage PC. PC results in nearly 30,000 deaths in the U.S. each year. Aim 1 focuses on evaluating the extent that FdUMP[N] multimers enter PC cells via active transport and identifying proteins expressed by PC cells that are involved in receptor-mediated endocytosis. Cellular uptake kinetics will be determined from the time- and concentration-dependence of radioactivity in PC cellular lysates following exposure to 32P-labeled FdUMP[N] compounds. The contribution of active transport to cellular uptake will be evaluated using metabolic inhibitors. Structure/function analyses will be performed to identify structural features of FdUMP[N] compounds that promote intracellular internalization of FdUMP[N] compounds via active transport. Protein(s) involved in the active transport of FdUMP[N] compounds will be identified by UV cross-linking, and sequences for these protein(s) will be determined using mass spectrometry. In Aim 2, the intracellular metabolism of [6-3H]FdUMP[N] compounds to monomeric FP metabolites will be evaluated, and thymidylate synthase inhibition and nucleotide pool imbalances will be quantified. In Aim 3, the misincorporation of FdUTP into DNA and the extent and type of DMA damage, including DNA damage resulting from topoisomerase I cleavage complex formation, will be quantified using alkaline elution, pulsed-field gel electrophoresis, and an in vivo complex of enzyme bioassay. These studies greatly enrich our understanding of the unique cytotoxic mechanism for FdUMP[N] compounds towards PC cells.