DNA-DIRECTED EFFECTS OF FdUMP(N)

FdUMP(N) 的 DNA 定向效应

• 批准号: 6966318
• 负责人: William H Gmeiner
• 金额: $ 34.79万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-10 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6966318
• 关键词: DNA damage; antineoplastics; clinical research; cooperative study; crosslink; cytotoxicity; fluoropyrimidine; human tissue; mass spectrometry; metabolism; neoplasm /cancer chemotherapy; prostate neoplasms; protein sequence; protein structure function; receptor mediated endocytosis; thymidylate synthase; DNA damage; antineoplastics; clinical research; cooperative study; crosslink; cytotoxicity; fluoropyrimidine; human tissue; mass spectrometry; metabolism; neoplasm /cancer chemotherapy; prostate neoplasms; protein sequence; protein structure function; receptor mediated endocytosis; thymidylate synthase

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.

描述（由申请人提供）：Fdump [n]化合物构成了一种新型的氟吡啶（FP）化学治疗性，可能对治疗当前化学疗法难治的人类恶性肿瘤有用。该研究项目的目的是了解Fdump [N]化合物的独特细胞毒性机制。人类前列腺癌（PC）细胞将用作模型系统，因为PC细胞对FDUMP的相对敏感性[10]，并且需要新的，更有效的药物来治疗后期PC，以研究Fdump [N]细胞毒性。 PC每年在美国导致近30,000人死亡。 AIM 1的重点是评估Fdump [N]多聚体通过主动转运进入PC细胞的程度，并鉴定由受体介导的内吞作用所涉及的PC细胞表达的蛋白质。细胞摄取动力学将取决于暴露于32p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。主动转运向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，以识别fdump [n]化合物的结构特征，这些化合物通过主动转运促进fdump [n]化合物的细胞内内在化。将通过紫外线交叉链接来鉴定参与fdump [n]化合物的活性转运的蛋白质，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]化合物的细胞内代谢，并评估胸苷基合酶抑制和核苷酸池失衡。在AIM 3中，将使用碱性洗脱，脉冲场凝胶电泳和一种酶生物鉴定的体内凝胶凝胶复合物，将FDUTP的不均衡和DMA损伤的程度和类型（包括由拓扑异构酶I裂解复合物造成的DNA损伤）进行定量。这些研究极大地丰富了我们对fdump [N]对PC细胞的独特细胞毒性机制的理解。