HEAT-INDUCED RADIOSENSITIZATION

该提案的总体目标是确定(S) 放射增敏热疗对细胞核结构和超微结构的影响 功能。我们将特别确定(S)的影响：1)急性 加热(AH；43至45度)；2)降温-加热(SDH；AH，然后 慢性接触41度)；3)41度慢性加热(耐受性 发展)；和4)41度的慢性加热 在对辐射的诱导和/或修复进行x射线照射之后- 诱导DNA双链断裂(DSB)、无嘌呤(AP)位点、DNA蛋白 中国仓鼠卵巢(CHO)细胞的DPC与染色体诱变 单层。我们要检验的假设是体温过高引起的 一个或一个的诱导频率增加和/或修复抑制 这些辐射诱导的DNA损伤中更多的与 在细胞水平上的放射增敏。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。 辐射的诱导频率和可修复性(速率和程度)- 特定DNA序列中的诱导损伤(SSB和DSB)将在 控制和加热牢房。大量DNA将通过碱性/中性方法分离 辐照后的过滤洗脱或碱性蔗糖梯度沉淀 细胞，以及DNA损伤修复过程中的细胞。收集到的大量DNA 硝化棉过滤器将通过过滤器杂交来检测 转录活性和/或惰性DNA序列的核酸探针。 热诱导的核结构和/或改变的可能作用 还将对抑制DNA损伤去除的成分进行评估。 核小体和核小体中多肽增加和去除的动力学 将测定非染色体染色质DNA和细胞内核基质 用外源核酸酶消化DNA，离心分离细胞核 组分和多肽的定量与表征 聚丙烯酰胺凝胶电泳法。体温升高的程度 暴露改变核基质DNA结合的数量和/或构象 将通过评估DNA“结构域”的大小和频率来确定站点 控制和加热牢房。

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.