RECOGNITION AND REPAIR OF CISPLATIN DNA DAMAGE

顺铂 DNA 损伤的识别和修复

• 批准号: 6377417
• 负责人: JOHN J. TURCHI
• 金额: $ 26.46万
• 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/6377417
• 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（根据调查员的摘要改编）：顺铂是思想的 通过形成赋予其化学治疗功效 坐标与与交叉的DNA加合物和随后的编程细胞诱导 死亡或凋亡。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性活性。顺铂 但是，在临床上也被用作电离辐射（IR）的敏化器 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）。所描述的研究目标 该建议是表征结合的细胞蛋白 顺铂受损的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 顺铂的致敏和细胞毒性活性受到 结合顺铂受损的DNA的蛋白质因子的相互作用。解决 假设并实现了这些目标，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 与NER蛋白相比，与顺铂受损的DNA的动力学相互作用。 因此，在AIM 1中，申请人建议研究 顺铂DNA损伤识别和NER和HMG1屏蔽的结合 蛋白质。申请人的假设还预测，在接受治疗的细胞中 顺铂，屏蔽蛋白将与顺铂受损的DNA和 阻止NER蛋白进入受损部位的访问。实验 AIM 2中描述的将评估NER和屏蔽的体内相互作用 通过间接免疫荧光与顺铂损伤的DNA的蛋白质。测试 顺铂的敏化活性的结果是 DNA-PK抑制作用，一系列体外和体内实验在 目标3和4。顺铂DNA损伤对DNA-PK活性和 双链DNA断裂修复将在体外和DSB修复中进行评估，并且 对IR的敏感性将在体内评估。实现所描述的目标 该建议将提供有关相互作用的重要信息 具有顺铂受损的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敏化活性。一个 更好地了解这些互动将使更多 有效的癌症治疗方案，可能包括抑制DNA修复 达到更大的细胞毒性或提高癌症敏感性的途径 细胞到IR。