DETERMINATION OF HYPERTHERMIC CELL KILLING MECHANISMS

初步数据显示，体温过高导致的 细胞内游离钙浓度((Ca+2)i)具有细胞毒性 并促成了由此产生的细胞杀伤。体温过高 (Ca+2)i和细胞内总钙的变化 分别用荧光钙染料Indo 1和Indo 1进行测量 原子吸收。钙离子的细胞毒性将被诱导成 使用钙离子载体离子霉素的非加热细胞，这样 细胞杀伤水平与下面观察到的相似 体温过高。如果细胞内钙离子的变化与 受热和不受热的细胞在等存活期，改变 细胞内钙离子将被牵连为，或其中之一， 细胞对高温的反应，使细胞 繁殖不可行的。生存水平的差距将 指出钙的细胞毒性作用的程度(如果有的话) 致高热致死。钙拮抗剂和钙拮抗剂 将利用钙调节功能进一步建立 钙在热性细胞死亡中的作用及其机制 钙是其细胞毒作用的中介。整体和渗透性 加热的细胞将被用来确定(Ca+2)i是否增加，因为 从细胞外介质中钙的内流或从细胞外介质中释放 细胞内存储。将使用相同的系统来确定 经过处理的细胞如何尝试将钙重新调节到动态平衡 级别。(CA+2)I将在单个单元格中测量，也将 因形成殖民地的能力而得分，以便建立 杀死一个细胞所需的(Ca+2)i的阈值增加。变化 在内质网的形态和胞质分布中 将监测网状细胞、线粒体和细胞骨架，并 在这些相同的单元格中量化，以确定 细胞被改变的(Ca+2)i损伤，代表致命性损伤 网站。对这项研究提出的问题的回答将有所帮助 确定高温如何与细胞相互作用。这反过来又会 使这种模式在临床上得到更好的应用。

Preliminary data suggests that hyperthermia-induced increases in intracellular free calcium concentrations ((Ca+2)i) are cytotoxic and contribute to the resultant cell killing. Hyperthermia induced changes in (Ca+2)i and total intracellular calcium will be measured, respectively, with the fluorescent calcium dye indo 1 and atomic absorption. Calcium cytotoxicity will be induced into nonheated cells using the calcium ionophore ionomycin, such that the cell killing level is similar to that observed following hyperthermia. If changes in intracellular calcium are similar in the heated and nonheated cells at isosurvival, altered intracellular calcium will be implicated as the, or one of the, cellular responses to hyperthermia that renders cells reproductively nonviable. A disparity in survival levels will indicate to what degree (if any) calcium cytotoxicity contributes to hyperthermic killing. Agonists and antagonists of calcium and calcium-regulated functions will be used to further establish the role of calcium in hyperthermic cell death and elucidate how calcium mediates its cytotoxic effects. Whole and permeabilized heated cells will be used to determine if (Ca+2)i increases because of a calcium influx from the extracellular medium or a release from intracellular stores. The same system will be used to determine how treated cells attempt to re-regulate calcium to homeostatic levels. (Ca+2)i will be measured in individual cell that will also be scored for the ability to form a colony in order to establish the threshold increase in (Ca+2)i required to kill a cell. Changes in the morphology and cytoplasmic distribution of the endoplasmic reticulum, mitochondria, and cytoskeleton will be monitored and quantified in these same cells to establish which aspects of the cell are damaged by altered (Ca+2)i and represent lethal lesion sites. Answers to the questions posed by this study will help determine how hyperthermia interacts with cells. This in turn will lead to a more educated application of this modality in the clinic.