HEAT-INDUCED RADIOSENSITIZATION

该提案的总体目标是确定以下方面的影响： 对细胞核结构的放射增敏热处理， 功能 特别是，我们将确定以下因素的影响：1）急性 加热（AH; 43至45度）; 2）降压加热（SDH; AH，然后 长期暴露于41度）; 3）长期加热至41度（耐受性 显影）;以及4）41度的慢性加热 在X射线照射诱导和/或修复辐射之后， 诱导的DNA双链断裂（dsb），脱嘌呤（AP）位点，DNA蛋白 交联（DPC）和染色体畸变诱导的CHO细胞生长在 单层 我们将检验的假设是， 一种或多种神经元的诱导频率增加和/或修复抑制， 更多的辐射诱导的DNA损伤与 细胞水平的放射增敏作用。 到目前为止，这一假设还没有 在DNA单链断裂（ssb）的情况下被证明通常是正确的。 辐射的感应频率和可修复性（速率和程度）- 在特定DNA序列中诱导的损伤（ssb abd dsb）将在 控制室和加热室。 批量DNA将通过碱性/中性 过滤洗脱或碱性蔗糖梯度沉降 细胞，以及DNA损伤修复过程中的细胞。 将大量DNA收集到 硝酸纤维素滤膜将通过滤膜杂交进行探测， 用于转录活性和/或惰性DNA序列的核酸探针。 热诱导的核结构改变和/或 还将评估组合物在抑制DNA损伤去除中的作用。 核小体和核小体中多肽的增加和去除的动力学 将测定非核小体染色质DNA和细胞核基质 通过外源核酸酶消化DNA，离心分离核 组分和多肽定量和表征 聚丙烯酰胺凝胶电泳 体温过高的程度 暴露改变核基质DNA结合的数量和/或构象 位点将通过评估DNA“结构域”的大小和频率来确定， 控制室和加热室。

The overall objective of the proposal is to determine the effect(s) of radiosensitizing hyperthermic treatments on cell nuclear structure and function. In particular we will determine the effects(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 abd dsb) in specific DNA sequences will be measure 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 of 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 polypeptides in nucleosomal and nonnculesomal 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 characterization of 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.