PROTEIN DENATURATION--ITS ROLE IN HYPERTHERMIC BIOLOGY

这项研究的工作假设是， 蛋白质代表了导致细胞 杀死并诱导耐热性。 变性蛋白质启动 形成蛋白质聚集体，随后破坏正常的 细胞结构和功能。 最终变性聚集 相关的损伤阻止细胞进行成功的有丝分裂， 它们被克隆性地死亡。 耐热性是由 存在变性和/或聚集的蛋白质，并保护细胞免受 通过抑制蛋白质变性-聚集的高温杀伤， 促进分解过程。 这些实验旨在明确证明蛋白质 变性-聚集是一种致命的高温损伤， 如何导致细胞死亡 变性蛋白在细胞中的细胞毒性 是否存在其他高热损伤将由以下因素确定： 将热变性的蛋白质电穿孔到未加热的细胞中， 量化所得细胞毒性。 补充实验将 涉及用不耐热的、非乳头状的 proteins. 热不稳定蛋白质的能力， 加热细胞或降低细胞杀伤的阈值温度将 支持蛋白质变性作为一种致命的热损伤。 然后将进行实验以确认蛋白质聚集作为一种生物学行为。 蛋白质变性的直接后果，并确定 蛋白质聚集的幅度，在加热时间和 并且加热后恢复用作细胞毒性剂量的测量。 然后将努力确定特定的细胞 会造成致命的高温伤害 实验方法 将包括细胞去核和再融合技术，以确定 细胞毒性与加热细胞质或细胞核有关。 我们将 确定聚集的蛋白质是否干扰正常的核功能 以导致微核或异常染色体的方式。 热 中心体的损伤也将研究其对细胞的影响。 生存能力 最后，我们将确定 耐热性保护细胞免于蛋白质变性/聚集。 这将包括实验，以确定相对功效的 抑制聚集体形成的主要热休克蛋白， 促进分解过程。 这项研究的结果将提供有关的基本知识 高温与生物细胞的相互作用， 在发展热疗作为一种治疗 人类癌症 蛋白质变性/聚集也有牵连 作为临床重要的其他应激中的致命损伤，例如，缺血 耐热性保护免受这些其他压力， 诱导耐热性。 这种交叉电阻由 耐热性表明，这是一个更普遍的机制， 已经进化到可以保护细胞免受压力。 因此，获得的信息 这项研究可能有更广泛的医学意义，而不是狭隘的 肿瘤热疗的应用

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.