HSP70 REGULATION IN CELLS RECOVERING FROM HYPERTHERMIA

热疗是一种公认的癌症治疗方法，它可能会导致 肿瘤消退和对辐射致死的增敏。有用性 然而，热疗的发展受到耐热性的限制。 耐热性发生在先前被加热到42℃的细胞中 摄氏度(或更高)，导致它们对进一步加热变得折射 治疗。在实验上，它与归纳和继续相关 细胞中存在热休克蛋白，特别是70,000 道尔顿蛋白(HSP70) 本研究将探讨热休克蛋白70的分子调控机制。 热休克后立即表现为最大值的时期 合成了一定比例的HSP70蛋白。两个关键过程控制 将研究HSP70转录后水平-优先 HSP70信使核糖核酸的翻译和HSP70信使核糖核酸的转化率。该方法 是将缺失和突变引入两个克隆的HSP70基因，小鼠 和人类，然后用Northern杂交确定转录本的半衰期 蛋白质凝胶分析或翻译效率分析。核糖核酸 这一分析所描述的调控序列将与 紫外线照射对任何接触蛋白质和经鉴定的蛋白质的影响 凝胶电泳法。因为这些蛋白质可能与热休克蛋白70有关 合成，将产生抗体并用于筛选cdna表达。 调控蛋白克隆文库。第二种方法将尝试 通过将RNA元件直接结合到编码的cDNA来获得克隆 多肽。 除了直接参与耐热性外，调节机制 HSP70 RNA可能与观察到的热休克稳定化有关 一些原癌基因转录本。在未来，它将是有趣的 检查由此导致的癌基因表达的增加是否在 在高温后细胞生长或存活的过程中。

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.