DNA DAMAGE & REPAIR IN IRRADIATED NORMAL & TUMOR CELLS

这项建议的总体目标是确定效果(S) 放射增敏热疗对细胞核的影响 结构和功能。特别是，我们将确定 效应(S)：1)急性加热(AH；43-45度)；2)阶跃加热 降温(SDH；AH，随后慢性暴露于41 度)；3)41度的长期供暖(容忍度发展) X射线照射前；4)41度慢性加热 在X射线照射后诱导和/或修复 辐射诱导的DNA双链断裂(DSB)、无嘌呤酸(AP) 位点、DNA蛋白交联(DPC)与染色体畸变 单层培养的CHO细胞的诱导。我们的假设是 会考的是，高热诱导的诱导性增加 其中一个或多个的频率和/或修复的抑制 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)的情况下通常是这样的。 诱导频率和可修复性(速度和程度) 特定DNA序列中的辐射损伤(SSB和DSB) 将在对照和加热牢房中进行测量。大量的DNA将是 通过碱性/中性过滤洗脱或碱性蔗糖分离 辐照细胞的梯度沉淀，以及细胞在 DNA损伤的修复。硝酸纤维素膜上收集的大量DNA 过滤器将通过与核酸的过滤器杂交进行探测 转录活性和/或惰性DNA序列的探针。 热诱导核结构改变的可能作用 和/或成分中抑制DNA损伤的去除将 也要接受评估。的增加和去除的动力学 核小体和核小体染色质DNA中的多肽和 细胞核中的基质将由外源核酸酶决定 DNA的消化，核组分的离心分离和 用聚丙烯酰胺对多肽进行定量和表征 凝胶电泳法。高温暴露的程度 改变核基质DNA的数量和/或构象 结合部位将通过评估DNA“结构域”来确定 对照牢房和加热牢房的大小和频率。

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.