| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1999: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1998: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
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
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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
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