RECOGNITION AND REPAIR OF CISPLATIN DNA DAMAGE

描述（改编自研究者摘要）：顺铂被认为 以通过形成 配位-共价DNA加合物和随后的程序化细胞的诱导 死亡或凋亡。通过核苷酸切除修复来修复DNA损伤 (NER)这对该药物的细胞毒活性是有害的。顺铂 在临床上也用作电离辐射（IR）的敏化剂，然而， 顺铂如何发挥这种活性的确切机制尚不清楚。的 申请人的初步数据支持一种涉及抑制 DNA依赖性蛋白激酶（DNA-PK）。中描述的研究目标 这项建议是为了表征细胞蛋白质， 顺铂损伤的DNA，并确定这些相互作用如何有助于 顺铂的体内活性。申请人假设， 顺铂的致敏和细胞毒活性是由 结合顺铂损伤的DNA的蛋白质因子的相互作用。解决 假设并实现这些目标，四个具体目标将完成。 申请人的假设预测，屏蔽蛋白将具有优先选择性。 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，申请人提出研究以下化合物的动力学： NER和HMG 1屏蔽顺铂-DNA损伤识别和结合 proteins.申请人的假设还预测，在用 顺铂，屏蔽蛋白将与顺铂损伤的DNA和 阻断NER蛋白进入受损部位。实验 目标2中描述的方法将评估NER和屏蔽的体内相互作用 蛋白质与顺铂损伤的DNA通过间接免疫荧光。测试 假设顺铂的致敏活性是 DNA-PK抑制，一系列的体外和体内实验，提出了在 目标3和4。顺铂-DNA损伤对DNA-PK活性及细胞凋亡的影响 将在体外评估双链DNA断裂修复， 将在体内评估对IR的敏感性。实现 这一建议将提供关于以下方面相互作用的重要信息： 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 药物顺铂在体内的细胞毒性和致敏活性。一 更好地理解这些相互作用将有助于开发更多 有效的癌症治疗方案，可能包括抑制DNA修复 实现更大细胞毒性或增加癌症敏感性的途径 细胞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.