PROTEIN DENATURATION--ITS ROLE IN HYPERTHERMIC BIOLOGY

拟议研究的工作假设是，热变性 蛋白质代表基本的高温损伤，导致细胞 杀戮并诱导耐热性。变性蛋白质启动了 形成蛋白质聚集体，随后破坏正常 细胞结构和功能。最终变性聚集 相关的损伤会阻止细胞进行成功的有丝分裂， 它们会在克隆基因上死亡。耐热性是由 变性和/或聚集蛋白的存在并保护细胞免受 抑制蛋白质变性-聚集和高温热致死 促进解体过程。 这些实验旨在明确地证明，蛋白质 变性-聚集是一种致命性的高温损伤，并可确定 它如何导致细胞死亡。变性蛋白的细胞毒性研究 是否存在其他高热损伤将由以下因素确定 将热变性蛋白质电穿孔到未加热的细胞中 对由此产生的细胞毒性进行量化。互补性实验将 包括用不耐热的、非哺乳动物的电穿孔哺乳动物细胞 蛋白质。不耐热的蛋白质能够敏化 加热或降低细胞杀灭的门槛温度将 支持蛋白质变性为致命性高温损伤。 然后将进行实验以确认蛋白质聚集是一种 蛋白质变性的直接后果，并确定 蛋白质聚集的大小，在两个加热时间内积分 而后加热恢复可作为细胞毒性剂量的测量。 然后，将努力识别特定的细胞 表现出致命性高温伤害的目标。实验方法 将包括细胞去核和再融合技术，以确定 细胞毒性与加热胞浆或胞核有关。我们会 确定聚集的蛋白质是否干扰正常的核功能 以导致微核或染色体异常的方式。热度 中心体的损伤也将被研究其对细胞的影响。 生存能力。最后，我们将确定通过哪些机制 耐热性保护细胞免受蛋白质变性/聚集的影响。 这将包括实验，以确定 主要的热休克蛋白抑制聚集体的形成和 促进解体过程。 这项研究的结果将提供有关以下方面的基础知识 热疗与可利用的生物细胞的相互作用 热疗作为一种临床治疗方式的发展 人类癌症。蛋白质变性/聚集也与此有关 作为其他临床应激反应的致命性损害，例如，缺血。 耐热性可以保护自己免受其他压力的影响 诱导耐热性。这种交叉阻力由 耐热性表明这是一种更普遍的机制的一部分 已经进化为保护细胞免受压力。因此，所获得的信息 这项研究可能比狭义的 热疗肿瘤学的应用。

The working hypothesis of the proposed study is that heat-denatured proteins represent the rudimentary hyperthermic lesion that causes cell killing and induces thermotolerance. Denatured proteins initiate the formation of protein aggregates which subsequently disrupt normal cellular structure and function. Ultimately denaturation aggregation related damage prevents cell from undergoing a successful mitosis and they are rendered clonogenically dead. Thermotolerance is induced by the presence of denatured and/or aggregated proteins and protects cells from hyperthermic killing by inhibiting protein denaturation-aggregation and facilitating the disaggregation process. The experiments are designed to demonstrate unequivocally that protein denaturation-aggregation is a lethal hyperthermic lesion and to determine how it causes cell death. The cytotoxicity of denatured proteins in the absence of other hyperthermic damage will be determined by electroporating heat-denatured proteins into nonheated cells and quantitating the resultant cytotoxicity. Complementary experiments will involve electroporating mammalian cells with thermolabile, nonmammilian proteins. The ability of the thermolabile proteins to either sensitize cells to heat or lower the threshold temperature for cell killing will support protein denaturation as a lethal hyperthermic lesion. Experiments will then be performed to confirm protein aggregation as a direct consequence of protein denaturation and to determine if the magnitude of protein aggregation, integrated over both the heating time and post heating recovery serves as a measurement of the cytotoxic dose. Efforts will then be directed towards identifying specific cellular targets that express lethal hyperthermic damage. Experimental approaches will include cell enucleation and refusion techniques to determine the cytotoxity associated heating the cytoplasm nor nucleus. We will determine if aggregated proteins interfere with normal nuclear function in a manner that results in micronuclei or aberrant chromosomes. Heat damage to the centrosome will also be investigated for its effect on cell viability. Finally, we will determine the mechanisms by which thermotolerance protects cells against protein denaturation/aggregation. This will include experiments to determine the relative efficacy of the major heat shock proteins in inhibiting aggregate formation and facilitating the disaggregation process. The results of this study will provide fundamental knowledge concerning the interactions of hyperthermia with biological cells which can be used in the development of hyperthermia as a clinical modality for treating human cancers. Protein denaturation/aggregation has also been implicated as a lethal lesion in other stresses of clinical import, e.g., ischemia. Thermotolerance protects against these other stresses which themselves induce thermotolerance. This cross-resistance provided by thermotolerance suggest that is part of a more generalized mechanism that has evolved to protect cells from stress. Thus, the information obtained from this study may have broader medical implications than the narrow application of hyperthermic oncology.