HSP70 REGULATION IN CELLS RECOVERING FROM HYPERTHERMIA

热疗是一种公认的癌症治疗方法， 肿瘤消退和对辐射杀伤敏感。 是否有用 然而，由于耐热性的发展，热疗的效果受到限制。 耐热性发生在先前加热到42 ℃的电池中 摄氏度（或更高），导致它们变得更热 治疗。 在实验上，它与诱导相关，并继续 细胞中存在热休克蛋白，特别是70，000 道尔顿蛋白 本研究旨在探讨热休克蛋白70调控的分子机制 热休克后立即表达，最大的时期 HSP 70蛋白的合成比例。 两个关键过程控制 将研究HSP 70的转录后水平-优先 HSP 70 mRNA的翻译和HSP 70 mRNA的周转率。 的方法 将缺失和突变引入两个克隆的HSP 70基因， 然后通过北方印迹法测定转录物的半衰期 通过蛋白质凝胶分析或翻译效率。 RNA 由该分析描绘的调控序列将与 对任何接触的蛋白质进行UV照射，并通过 凝胶电泳 因为这些蛋白质可能对HSP 70有贡献 合成，将产生抗体并用于筛选cDNA表达 文库的调节蛋白克隆。 第二种方法将试图 通过将RNA元件与编码的cDNA直接结合来获得克隆 缩氨酸 除了直接参与热耐受性， 热休克蛋白70 RNA可能是引起热休克稳定的主要原因。 一些原癌基因的转录本 在未来，这将是有趣的， 检查癌基因表达的增加是否在 在高温后的细胞生长或存活中。

Hyperthermia is an accepted approach to cancer therapy where it can cause tumor regression and sensitization to radiational killing. The usefulness of hyperthermia is limited, however, by the development of thermotolerance. Thermotolerance occurs in cells which have been previously heated to 42 degrees C (or higher), causing them to become refractile to further heat treatments. Experimentally, it correlates with the induction and continued presence in the cell of heat-shock proteins, in particular the 70,000 dalton protein (HSP70) This study will examine the molecular mechanisms regulating HSP70 expression immediately after heat-shock, the period when the greatest proportion of HSP70 protein is synthesized. Two key processes controlling the post-transcriptional levels of HSP70 will be studied - preferential translation of HSP70 mRNA and the HSP70 mRNA turnover rate. The approach is to introduce deletions and mutations into two clones HSP70 genes, mouse and human, then determine the transcripts half-life by Northern blot analysis or the translational efficiency by protein gel analysis. RNA regulatory sequences delineated by this analysis will be cross-linked with UV irradiation to any contacting proteins and the proteins identified by gel electrophoresis. Because these proteins may contribute to HSP70 synthesis, antibodies will be produced and used to screen a cDNA expression library for regulatory protein clones. A second approach will attempt to obtain clones by directly binding the RNA elements to the cDNA encoded peptides. Beyond its direct involvement in thermotolerance, the mechanisms regulating HSP70 RNA may be responsible for the observed heat-shock stabilization of some proto-oncogene transcripts. In the future, it will be interesting to examine whether the resulting increase in oncogene expression plays a role in post-hyperthermic cell growth or survival.