DNA DAMAGE & REPAIR IN IRRADIATED NORMAL & TUMOR CELLS

本提案的总体目标是确定 放射增敏热疗对细胞核 结构和功能。 特别是，我们将确定 影响：1）急性加热（AH; 43至45度）; 2）步进- 降温（SDH; AH），然后慢性暴露于41 3）41度的慢性加热（耐受性发展） 在X射线照射之前;以及4）在41度的慢性加热 X射线照射后诱导和/或修复 辐射诱导DNA双链断裂（dsb），脱嘌呤（AP） 位点、DNA蛋白质交联（DPC）和染色体畸变 在单层生长的CHO细胞中诱导。 假设我们 将测试的是，高血压诱导的诱导增加 频率，和/或抑制修复，一个或多个这些 辐射诱导的DNA损伤与放射增敏相关 在细胞水平上。 到目前为止，这一假设还没有得到证实。 在DNA单链断裂（ssb）的情况下通常如此。 的诱导频率和可修复性（率和程度） 特定DNA序列中的辐射诱导损伤（ssb和dsb） 将在控制和加热单元中测量。 批量DNA将 通过碱性/中性过滤器洗脱或碱性蔗糖分离 梯度沉降从照射的细胞，和细胞在 修复DNA损伤。 将大量DNA收集到硝酸纤维素上 将通过针对核酸的滤膜杂交来探测滤膜 用于转录活性和/或惰性DNA序列的探针。 热诱导的核结构改变的可能作用 和/或组合物在抑制DNA损伤去除中的作用将 也要评估。 的增加和去除的动力学， 核小体和非核小体染色质DNA中的多肽， 在细胞核基质中， 消化DNA，离心分离核组分， 多肽定量和聚丙烯酰胺表征 凝胶电泳 高温暴露的程度 改变核基质DNA的数量和/或构象 结合位点将通过评估DNA“结构域” 控制和加热细胞中的大小和频率。

The overall objective of this proposal is to determine the effect(s) of radiosensitizing hyperthermic treatments on cell nuclear structure and function. In particular we will determine the effect(s) of: 1) acute heating (AH; 43 to 45 degrees); 2) step- down-heating (SDH; AH followed by chronic exposure to 41 degrees); 3) chronic heating at 41 degrees (tolerance development) before X-irradiation; and 4) chronic heating at 41 degrees subsequent to X-irradiation on the induction and/or repair of radiation-induced DNA double strand breaks (dsb), apurinic (AP) sites, DNA protein crosslinks (DPC) and chromosome aberration induction in CHO cells grown in monolayer. The hypothesis we will test is that a hyperthermia-induced increase in the induction frequency, and/or inhibition of repair, of one or more of these radiation-induced DNA lesions correlates with radiosensitization at the cellular level. To date, this hypothesis has not proven generally true in the case of DNA single strand breaks (ssb). The induction frequency and repairability (rate and extent) of radiation-induced lesions (ssb and dsb) in specific DNA sequences will be measured in control and heated cells. Bulk DNA will be isolated by alkaline/neutral filter elution or alkaline sucrose gradient sedimentation from irradiated cells, and cells during the repair of DNA lesions. Bulk DNA collected onto nitrocellulose filters will be probed by filter hybridization against nucleic acid probes for transcriptionally active and/or inert DNA sequences. The possible role heat-induced alterations of nuclear structure and/or composition in the inhibition of DNA damage removal will also be assessed. The kinetics of increase, and removal, of polyeptides in nuclesomal and nunnucleosomal chromatin DNA and in cell nuclear matrices will be determined by exogenous nuclease digestion of DNA, centrifugal isolation of nuclear components and polypeptide quantitation and characterizations by polyacrylamide gel electrophoresis. The extent to which hyperthermic exposure alters the number and/or conformation of nuclear matrix DNA binding sites will be determined by assessing the DNA "domain" size and frequency in control and heated cells.