| Co-Investigator(Kenkyū-buntansha) |
FURUTA Isao University of Toyama, Faculty of medicine, Professor, 医学部, 教授 (10014268)
OGAWA Ryouhei University of Toyama, Faculty of medicine, Lecturer, 医学部, 講師 (60334736)
KONDO Takashi University of Toyama, Faculty of medicine, Professor, 医学部, 教授 (40143937)
TABUCHI Yoshiaki Life science research center, Associate professor, 生命科学先端研究センター, 助教授 (20322109)
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| Research Abstract |
The enhancement of heat-induced apoptosis by 6-formylpterin, an intracellular generator of hydrogen peroxide (H_2O_2), was examined in human myelomonocytic lymphoma U937 cells. The cells were treated with either 6-formylpterin alone at a nontoxic concentration of 300 μM (37℃), heat shock (44℃/20min) alone, or a combination of the two, then incubated at 37℃ for 6 h. Assessments of apoptosis, mitochondrial membrane potential, and caspase-3 activation were performed by flow cytometry. Moreover, caspase-8 activation, and changes in the intracellular Ca^<2+> concentration ([Ca^<2+>]i) were examined. Bax,Bcl-2,Bcl-XL,Bid, cytochrome c, and PKCδ were detected by Western blotting. The induction of heat-induced apoptosis evaluated by morphological observation and DNA fragmentation were promoted by the addition of 6-formylpterin. Mitochondrial membrane potential was decreased and the activation of caspase-3 and -8 was enhanced in the cells treated with the combination. A decreased-expression of
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Bid was noted, although no significant changes in Bax,Bcl-2, and Bcl-XL expression were observed after the combined treatment. Furthermore, both the release of cytochrome c from mitochondria to cytosol and the translocation of PKCδ from cytosol to mitochondria, which were induced by heat shock, were enhanced by the addition of 6-formylpterin. The number of cells with a higher [Ca^<2+>]i was also increased by the addition of 6-formylpterin. These findings suggest that the increase in [Ca^<2+>]i, the activation of the mitochondria-caspase dependent pathway, and the translocation of PKCδ to mitochondria play principal roles in the enhancement of heat-induced apoptosis by 6- formylpterin. These results were confirmed in the cells exposed to X-ray at a dose of 10Gy, too. When cells were exposed to hyperthermia alone (44℃ for 10 min, 15% apoptosis level), 39 up-regulated genes, such as BAG3,DNAJA1,DNAJB1,HSPA1B,HSPA6,HSPH1,SEPW1, and HO-1,and 3 down-regulated gene, such as CCL2,were identified. In combining heat and 6- formylpterin, two up-regulated genes (LOC219962 and NEUROD4) were identified. The expression levels of these genes were confirmed by real-time quantitative polymerase chain reaction. Less
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