DNA-DIRECTED EFFECTS OF FdUMP(N)

描述（由申请人提供）：FdUMP[N]化合物构成了一类新型氟嘧啶（FP）化疗药物，可用于治疗当前化疗难治性的人类恶性肿瘤。本研究项目的目的是了解FdUMP[N]化合物独特的细胞毒性机制。人类前列腺癌（PC）细胞将被用作研究FdUMP[N]细胞毒性的模型系统，因为PC细胞对FdUMP[10]的相对敏感性，也因为需要新的更有效的药物来治疗晚期前列腺癌。在美国，PC每年导致近3万人死亡。目的1侧重于评估FdUMP[N]多聚体通过主动运输进入PC细胞的程度，并鉴定PC细胞表达的参与受体介导的内吞作用的蛋白质。细胞摄取动力学将根据暴露于32p标记的FdUMP[N]化合物后PC细胞裂解物放射性的时间和浓度依赖性来确定。主动运输对细胞摄取的贡献将使用代谢抑制剂进行评估。将进行结构/功能分析，以确定FdUMP[N]化合物的结构特征，这些结构特征通过主动运输促进FdUMP[N]化合物在细胞内的内化。参与FdUMP[N]化合物主动转运的蛋白质将通过紫外交联鉴定，这些蛋白质的序列将使用质谱法确定。在Aim 2中，将评估[6-3H]FdUMP[N]化合物对单体FP代谢物的细胞内代谢，并量化胸苷酸合成酶抑制和核苷酸池失衡。在Aim 3中，FdUTP与DNA的错误结合以及DMA损伤的程度和类型，包括拓扑异构酶I切割复合物形成导致的DNA损伤，将使用碱性洗脱、脉冲场凝胶电泳和体内酶生物测定复合物进行量化。这些研究极大地丰富了我们对FdUMP[N]化合物对PC细胞独特的细胞毒性机制的理解。

DESCRIPTION (provided by applicant): 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.