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

Jurkat细胞每7 d暴露于43℃，并逐渐延长暴露时间，建立耐热亚系（43℃-120 min）。当Jurkat细胞在43℃下暴露120 min时，该亚系未表现出正常Jurkat细胞的凋亡方式。因此，我们分析了该亚系恢复凋亡潜能需要多少天，并分析了Hsp70蛋白的产生和caspase的激活，以了解热休克细胞凋亡的机制。正常Jurkat恢复50%需要120天左右，恢复80%需要220天左右。通过这种方式建立的耐热无性系需要260天左右的时间才能恢复50%，300天左右的时间才能恢复80%。认为耐热亚系中Hsp70蛋白的积累使其对热休克细胞凋亡具有抗性。作为Hsp70蛋白积累的原因之一，Hsp70蛋白的降解途径可能受到高温的破坏。虽然耐热亚系逐渐恢复凋亡电位，但hsp70 mRNA的表达量逐渐减少。HSF-1的磷酸化和去磷酸化机制的改变可能导致了这种现象。Western blot分析caspases 1、2、3、4、6、7、8、10的激活情况，发现caspases在耐热亚系中几乎完全被抑制。为了了解Hsp70蛋白如何抑制细胞凋亡，我们检查了线粒体。在以DiOC6(3)为探针的FACS分析中，正常Jurkat细胞显示线粒体膜电位下降。相反，耐热品系没有表现出任何下降。然而，正常Jurkat细胞的膜电位下降被认为是凋亡变化的结果，因为膜电位下降的动力学与膜联蛋白V结合的动力学非常相似。进一步分析与细胞凋亡相关的蛋白使用线粒体组分。因此，Bax蛋白在耐热亚系中的表达量很低。我们正计划评估耐热亚系中Bax蛋白的低水平是否与热休克细胞凋亡的抗性有关。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