DNA-DIRECTED EFFECTS OF FdUMP(N)

描述（由申请人提供）：FdUMP[N]化合物构成一类新的氟嘧啶（FP）化疗剂，可用于治疗目前化疗难治的人类乳腺癌。本研究项目的目标是了解FdUMP[N]化合物独特的细胞毒性机制。人前列腺癌（PC）细胞将用作研究FdUMP[N]细胞毒性的模型系统，因为PC细胞对FdUMP[10]相对敏感，并且因为需要新的和更有效的药物来治疗晚期PC。PC每年在美国造成近30，000人死亡。目的1着重于评估FdUMP[N]多聚体通过主动转运进入PC细胞的程度，并鉴定由PC细胞表达的参与受体介导的内吞作用的蛋白质。将根据暴露于32 P标记的FdUMP[N]化合物后PC细胞裂解物中放射性的时间和浓度依赖性确定细胞摄取动力学。将使用代谢抑制剂评价主动转运对细胞摄取的贡献。将进行结构/功能分析，以鉴定FdUMP[N]化合物的结构特征，这些结构特征通过主动转运促进FdUMP[N]化合物的细胞内内化。将通过UV交联鉴别参与FdUMP[N]化合物主动转运的蛋白质，并使用质谱法测定这些蛋白质的序列。在目标2中，将评价[6- 3 H]FdUMP[N]化合物至单体FP代谢物的细胞内代谢，并将定量胸苷酸合成酶抑制和核苷酸库失衡。在目标3中，FdUTP错误掺入DNA和DMA损伤的程度和类型，包括拓扑异构酶I切割复合物形成引起的DNA损伤，将使用碱性洗脱，脉冲场凝胶电泳和酶生物测定的体内复合物进行定量。这些研究极大地丰富了我们对FdUMP[N]化合物对PC细胞的独特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.