RECOGNITION AND REPAIR OF CISPLATIN DNA DAMAGE

描述(改编自《调查者摘要》)：顺铂被认为是 通过形成其化疗药物来传递其疗效 配位共价DNA加合物及其对程序化细胞的诱导 死亡，或细胞凋亡。核苷酸切除修复对DNA损伤的修复 (NER)途径不利于该药物的细胞毒活性。顺铂 临床上也用作电离辐射(IR)的增敏剂，然而， 顺铂如何发挥这种活性的确切机制尚不清楚。这个 申请人的初步数据支持一种机制，涉及抑制 DNA依赖蛋白激酶(DNA-PK)。中描述的研究目标 这项提议是为了表征结合在一起的细胞蛋白质 顺铂损伤的DNA并确定这些相互作用如何促进 顺铂的体内活性。申请人假设两者均为 顺铂的增敏和细胞毒活性可通过 结合顺铂损伤的DNA的蛋白质因素的相互作用。致信地址 提出假设并实现这些目标后，将完成四个具体目标。 申请者的假说预测，屏蔽蛋白质将优先 与NER蛋白相比，与顺铂损伤的DNA的动力学相互作用。 因此，在目标1中，申请人提议研究 NER和HMG1屏蔽对顺铂-DNA损伤的识别和结合 蛋白质。申请人的假说还预测，在用 顺铂，屏蔽蛋白将与顺铂受损的DNA和 阻断NER蛋白进入受损部位。这些实验 在目标2中描述的将评估NER和屏蔽在体内的相互作用 通过间接免疫荧光检测顺铂损伤DNA的蛋白质。为了测试 假设顺铂的增敏活性是由于 DNA-PK抑制，一系列的体外和体内实验被提议在 目标3和4。顺铂-DNA损伤对DNA-PK活性和 双链DNA断裂修复将在体外进行评估，DSB修复和 对IR的敏感性将在体内进行评估。实现中描述的目标 这项提案将提供有关 哺乳动物蛋白质与顺铂损伤的DNA以及这些相互作用如何改变 药物顺铂的体内细胞毒性和致敏活性。一个 对这些相互作用的更好理解将允许开发更多 有效的癌症治疗方案，可能包括抑制DNA修复 获得更大的细胞毒性或增加癌症敏感性的途径 细胞转IR。

DESCRIPTION (As Adapted From the Investigator's Abstract): Cisplatin is thought to impart its chemotherapeutic efficacy via the formation of coordinate-covalent DNA adducts and the subsequent induction of programmed cell death, or apoptosis. Repair of the DNA damage by the nucleotide excision repair (NER) pathways is detrimental to the cytotoxic activity of this drug. Cisplatin is also used clinically as a sensitizer to ionizing radiation (IR), however, the exact mechanism of how cisplatin exerts this activity is unclear. The applicant's preliminary data support a mechanism involving inhibition of the DNA-dependent protein kinase (DNA-PK). The goals of the research described in this proposal are to characterize the cellular proteins that bind cisplatin-damaged DNA and determining how these interactions contribute to the in vivo activities of cisplatin. The applicant hypothesizes that both the sensitization and cytotoxic activity of cisplatin is potentiated by the interplay of protein factors that bind the cisplatin-damaged DNA. To address the hypothesis and achieve these goals, four specific aims will be completed. The applicant's hypothesis predicts shielding proteins will have a preferential kinetic interaction with cisplatin-damaged DNA compared to NER proteins. Therefore, in Aim 1, the applicant proposes to investigate the kinetics of cisplatin-DNA damage recognition and binding by NER and HMG1 shielding proteins. The applicant's hypothesis also predicts that in cells treated with cisplatin, shielding proteins will be associated with cisplatin-damaged-DNA and block the access of NER proteins to the damaged sites. The experiments described in Aim 2 will assess the in vivo interaction of NER and shielding proteins with cisplatin-damaged DNA via indirect immunofluorescence. To test the hypothesis that the sensitization activity of cisplatin is a result of DNA-PK inhibition, a series of in vitro and in vivo experiments are proposed in Aims 3 and 4. The effect of cisplatin-DNA damage on DNA-PK activity and double-strand DNA break repair will be assessed in vitro and DSB repair and sensitivity to IR will be assessed in vivo. Achieving the goals described in this proposal will provide important information on the interaction of mammalian proteins with cisplatin-damaged DNA and how these interactions alter the cytotoxic and sensitization activity of the drug cisplatin in vivo. A better understanding of these interactions will allow the development of more effective cancer treatment protocols that may include inhibiting DNA repair pathways to achieve greater cytotoxicity or increase the sensitivity of cancer cells to IR.