RECOGNITION AND REPAIR OF CISPLATIN DNA DAMAGE

顺铂 DNA 损伤的识别和修复

• 批准号: 6633491
• 负责人: JOHN J. TURCHI
• 金额: $ 19.31万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6633491
• 关键词: DNA binding protein; DNA damage; DNA repair; adduct; chemical kinetics; cis platinum compound; crosslink; endonuclease; enzyme activity; immunofluorescence technique; intermolecular interaction; ionizing radiation; nonhistone nucleoprotein; protein kinase; radiation genetics; radiation sensitivity; tissue /cell culture

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.

描述（改编自研究者摘要）：顺铂被认为 通过形成 配位共价 DNA 加合物和随后的编程细胞诱导 死亡，或细胞凋亡。通过核苷酸切除修复修复DNA损伤 (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）的敏化剂，但是， 顺铂如何发挥这种活性的确切机制尚不清楚。这 申请人的初步数据支持涉及抑制的机制 DNA 依赖性蛋白激酶 (DNA-PK)。中描述的研究目标 该提案旨在表征结合的细胞蛋白 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 顺铂的致敏和细胞毒活性通过 结合顺铂损伤的 DNA 的蛋白质因子之间的相互作用。致地址 假设并实现这些目标，将完成四个具体目标。 申请人的假设预测屏蔽蛋白将具有优先权 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，申请人建议研究动力学 NER 和 HMG1 屏蔽对顺铂-DNA 损伤的识别和结合 蛋白质。申请人的假设还预测，在用 顺铂，屏蔽蛋白将与顺铂受损的 DNA 相关，并且 阻止 NER 蛋白进入受损位点。实验 目标 2 中描述的将评估 NER 和屏蔽的体内相互作用 通过间接免疫荧光检测具有顺铂损伤 DNA 的蛋白质。测试 假设顺铂的致敏活性是由于 DNA-PK抑制，提出了一系列体外和体内实验 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 将在体内评估对IR的敏感性。实现中描述的目标 该提案将提供有关互动的重要信息 具有顺铂损伤 DNA 的哺乳动物蛋白质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。一个 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 细胞到红外。