Analysis of alterations in Hsp70 protein synthesis of heat-resistant Jurkat subline

每7d暴露一次43℃，并逐渐延长暴露时间以建立耐热亚系(43℃-120min)。当43℃作用120min时，该亚系没有表现出正常Jurkat细胞那样的凋亡。因此，我们分析了该亚系需要多少天才能恢复凋亡潜能，还分析了Hsp70蛋白的产生和半胱氨酸天冬氨酸氨基转移酶的激活，以了解热休克诱导凋亡的机制。按照本实验方法建立的耐热无性系，50%的恢复率约为260天，80%的恢复率约为300天。人们认为Hsp70蛋白在耐热亚系中的积累对热激诱导的细胞凋亡具有抗性。作为热休克蛋白70蛋白积聚的原因，热休克蛋白70蛋白…的降解途径虽然耐热亚系逐渐恢复了细胞的凋亡潜能，但HSP70基因的表达逐渐减少。当用Western印迹分析caspase1、2、3、4、6、7、8、10的激活时，在耐热亚系中caspase的激活几乎完全被抑制。为了了解Hsp70蛋白是如何抑制细胞凋亡的，我们观察了线粒体。在以DiOC6(3)为探针的流式细胞仪分析中，正常Jurkat细胞显示线粒体膜电位降低。相反，耐热亚系没有表现出任何凹陷。然而，正常Jurkat细胞膜电位的降低被认为是细胞凋亡变化的结果，因为膜电位降低的动力学与Annexin V结合的动力学非常相似。用线粒体片段进一步分析与细胞凋亡相关的蛋白质。因此，Bax蛋白在耐热亚系中的表达水平很低。我们正在计划评估耐热亚系中低水平的Bax蛋白是否与抗热休克细胞凋亡有关。较少

Jurkat cells were exposed to 43℃ every 7 days and exposure time was gradually prolonged to establish heat-resistant subline (43℃-120 min). This subline did not show apoptosis in such a fashion as normal Jurkat cells did, when they were exposed to 43℃ for 120 min. Therefore, we analyzed how many days this subline required to recover the apoptotic potential, and Hsp70 protein production and the activation of caspases were also analyzed to understand mechanisms of heat-shock apoptosis.For the recovery of apoptotic potential in heat-resistant subline, about 120 days for 50% recovery of normal Jurkat and 220 days for 80% recovery were required. The heat-resistant clone which was established on the way of this experiment required about 260 days for 50% recovery and about 300 days for 80% recovery.It was thought that accumulation of Hsp70 protein in heat-resistant subline gave the resistancy to heat-shock apoptosis. As a cause of Hsp70 protein accumulation, the degradation pathway of Hsp70 pr … More otein was probably impaired by heat.Although heat-resistant subline recovered apoptotic potential gradually, hsp70 mRNA production decreased gradually. Alterations in phosphorylation and dephosphorylation mechanisms of HSF-1 probably brought out this phenomenon.When caspases 1, 2, 3, 4, 6, 7, 8, 10 were analyzed for their activation by Western blot, the activation of caspases was almost completely suppressed in heat-resistant subline.To understand how Hsp70 protein suppressed apoptosis, we examined mitochondria. In FACS analyses using DiOC6 (3) as a probe, normal Jurkat cells showed depression of mitochondrial membrane potential. On the contrary, heat-resistant subline did not show any depression. However, depression of membrane potential in normal Jurkat cells was thought a consequence of apoptotic changes, since the kinetics of membrane potential depression was very similar to that of Annexin V binding. Further analysis of proteins related to apoptosis were made using mitochondrial fractions. Consequently, Bax protein expression level was very low in heat-resistant subline. We are now planning to evaluate whether low level of Bax protein in heat-resistant subline relates to the resistancy in heat-shock apoptosis. Less