CHROMATIN STRUCTURE AND RADIOSENSITIVITY

染色质结构和放射敏感性

基本信息

批准号：
2096944
负责人：
ANDREW T. VAUGHAN
金额：
$ 14.31万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-01-07 至 1996-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2096944
关键词：
DNA repair DNA topoisomerases chemical structure chromatin enzyme activity flow cytometry immobilized enzymes radiation sensitivity western blottings

项目摘要

The long term objective of this proposal is the design of a rapid predictive assay of tumor radiosensitivity to assist in the design of patient-specific treatments. For example, radioresistant tumors may be best controlled by either higher total doses of radiation or alternative treatment modalities. It has been found that after cellular irradiation, supercoiled DNA from relatively radiosensitive cells is only poorly rewound by the DNA intercalator ethidium bromide. This can be measured within a flow cytometer by monitoring the light scattered from nucleoids extracted from single cells. It is proposed that the reduced ability of DNA supercoils from irradiated radiosensitive cells to maintain supercoil tension is due to increased relative movement between the base of each DNA loop and its coordinating site at the nuclear matrix. This weakness in DNA association with the matrix allows the propagation of loop to loop supercoil relaxation, by the rotation of matrix DNA around its own long axis, driven by the energy released as a damaged supercoiled loop unwinds. It is further proposed that differences in DNA matrix stability are mediated through the structural or enzymatic activity of DNA topoisomerase II. Such a change in structural stability may affect the effectiveness of DNA repair leading to the difference in radiosensitivity seen. This is to be tested by: SITE-SPECIFICITY OF DNA BREAKS AND THEIR REPAIR IN DISTAL vs MATRIX ASSOCIATED DNA. The induction and repair of single and double stranded DNA breaks will be measured, using alkaline elution and pulse field gel methods, analyzing both DNA that is distal to the replication origin and that which is recently synthesized and therefore more closely linked to, and affected by, the nuclear matrix. ALTERATION OF SUPERCOILING TENSION AS A MEDIATOR OF DNA-MATRIX BINDING AFFINITY. Supercoil tension will be modified using both the topoisomerase II specific inhibitors, VP16 and MAMSA, by experimentally exhausting topoisomerase II content by taking the cells out of cycle, and after nucleoid extraction by ethidium bromide intercalation. The effect of these treatments on DNA-nuclear matrix binding will be determined directly, by restriction enzyme digestion of accessible DNA, and by the ability to maintain positive supercoiling as assayed by nucleoid flow cytometry. ROLE OF TOPOISOMERASE II IN MEDIATING CHANGES IN RADIOSENSITIVITY. The amount and activity of DNA topoisomerase II that is a component of the nuclear matrix will be measured by both specific antisera and its ability to unknott plasmid DNA. Variations in structure or activity of the enzyme may provide a biochemical explanation underlying differences in DNA repair and nuclear supercoiling behavior expected from the execution of Aims 1 and 2.

该提案的长期目标是设计一个快速的肿瘤放射敏感性的预测分析，以协助设计患者特定的治疗。例如，放射抗性肿瘤可能是最好通过较高的总辐射剂量或替代方案来控制治疗方式。研究发现，细胞照射后，来自相对放射敏感性细胞的超螺旋 DNA 的效果很差由 DNA 嵌入剂溴化乙锭重绕。这个可以测量在流式细胞仪内通过监测从类核散射的光从单细胞中提取。建议降低能力来自受辐射的放射敏感细胞的 DNA 超螺旋，用于维持超螺旋张力是由于每个底座之间的相对运动增加造成的 DNA 环及其在核基质上的配位位点。这个弱点 DNA 中与基质的结合允许循环到循环的传播超螺旋弛豫，通过基质 DNA 围绕其自身的长轴旋转轴，由损坏的超螺旋释放的能量驱动放松。进一步提出 DNA 基质稳定性的差异通过 DNA 的结构或酶活性介导拓扑异构酶 II。这种结构稳定性的变化可能会影响 DNA修复的有效性导致放射敏感性的差异看到了。这将通过以下方式进行测试：DNA 断裂及其位点特异性远端与基质相关 DNA 的修复。的诱导和修复使用碱性溶液测量单链和双链 DNA 断裂洗脱和脉冲场凝胶方法，分析远端 DNA 复制起点和最近合成的因此与核基质的联系更密切，并受其影响。超螺旋张力的改变作为 DNA-基质结合的中介亲和力。超线圈张力将使用以下两种方法进行修改拓扑异构酶 II 特异性抑制剂 VP16 和 MAMSA，通过实验通过使细胞脱离循环来耗尽拓扑异构酶 II 含量，以及通过溴化乙锭嵌入提取类核后。效果这些处理对 DNA-核基质结合的影响将被确定直接，通过限制性内切酶消化可接近的DNA，并通过通过类核流测定维持正超螺旋的能力细胞计数术。拓扑异构酶 II 在调节变化中的作用辐射敏感性。 DNA拓扑异构酶II的数量和活性是核基质的一个组成部分，将通过两种特定的方法来测量抗血清及其解开质粒 DNA 的能力。结构变化或酶的活性可以提供生化解释 DNA 修复和核超螺旋行为的根本差异目标 1 和 2 的执行预期。