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Title: 放射線誘導アポトーシスにおけるサバイビンの役割とレドックス制御
Other Titles: Role of survivin in radiation-induced apoptosis and its redox regulation
Authors: 小倉, 亜希1 Browse this author
Authors(alt): Ogura, Aki1
Issue Date: 25-Mar-2009
Publisher: Hokkaido University
Abstract: Ionizing radiation is a useful tool for cancer therapy. In hematopoietic cell lines such as malignant lymphoma and leukemia cells, apoptotic cell death is prone to be induced by ionizing radiation. In these cell lines, ionizing radiation is known to easily promote apoptotic signaling such as cytochrome c release from mitochondria, followed by activation of caspase and caspase-activated DNase. However, in solid tumor cell lines derived from adenocarcinoma, squamous cell carcinoma or melanoma, the constitutive or inducible antiapoptotic proteins such as Bcl-2 family and the inhibitor of apoptosis protein (IAP) family proteins seem to inhibit this apoptotic signaling. Survivin is a IAP family, which is intensively expressed in the G2/M phase and phosphorylated by cell division cycle 2 (CDC2), and known to have strongly antiapoptotic activity through interaction with Smac/DIABLO which acts as a proapoptotic protein. Its expression is prominently upregulated in most human cancer cells, but is undetectable or very low in normal tissues. Therefore, survivin seems to be an ideal target for radiosensitization in solid tumor cells. In this study, I firstly examined whether dominant negative vectors encoding survivin mutants, T34A (phosphorylation site by CDC2) and D53A (binding domain for smac/DIABLO) abrogate the function of constitutive survivin and facilitate radiation-induced apoptotic cell death in tumor cells. In first experiment, we prepared two adenoviral vectors for pAd-T34A and pAd-D53A to investigate the mechanism of radioresistance of solid tumor cells. When T34A and D53A were overexpressed in human lung carcinoma A549 and human cervical carcinoma HeLa cells, radiation-induced apoptosis was significantly enhanced. Furthermore, we examined the binding capability of survivin with Smac/DIABLO in the cells overexpressing these mutants. Coimmunoprecipitation analysis revealed that mutant forms of survivin, D53A and T34A, could bind to Smac/DIABLO but with much less affinity compared to the authentic form. These results suggest that radiation-induced apoptosis of tumor cells is increased by inhibition of the interaction between survivin and Smac/DIABLO through overexpression of T34A and D53A. These results indicated that radiation-induced apoptosis of tumor cells is increased by inhibition between survivin and Smac/DIABLO through overexpression of T34A and D53A, suggesting that survivin may be effective for therapeutic treatment in radioresistant solid tumors. Recently, it has been reported that apoptosis in human leukemia Molt-4 cells is remarkably inhibited if antioxidants such as N-acetyl-L-cystein (NAC) and Trolox are added to the medium within several minutes after X irradiation. This suggests that the secondary production of ROS occurs as a late event after irradiation and that these secondary ROS play an important role in radiation-induced apoptotic signaling. Mitochondria in mammalian cells are well-known to play an important role in the intrinsic pathway of genotoxic-agent-induced apoptosis by releasing cytochrome c into cytosol and to be a major source of reactive oxygen species (ROS). The second aim of this study was to examine whether mitochondrial ROS are involved in radiation-induced apoptotic signaling in A549 cells. Post-irradiation treatment with NAC inhibited cytochrome c release from mitochondria but did not affect expression levels of Bcl-2, Bcl-XL and Bax, suggesting that the late production of ROS triggered cytochrome c release. This treatment also inhibited radiation-induced apoptosis in T34A- and D53A-survivin-overexpressed A549 cells. Experiments using DCFDA (a classical ROS fluorescence probe) and MitoAR (a novel mitochondrial ROS probe) demonstrated that intracellular and mitochondrial ROS were enhanced 6 h after X irradiation. Furthermore, the ROS production ability of mitochondria isolated from A549 cells was evaluated by ESR spectroscopy combined with a spin-trapping reagent (CYPMPO). When isolated mitochondria were incubated with NADH, succinate and CYPMPO, an ESR spectrum due to CYPMPO–OOH was detected. This NADH/succinate-dependent ROS production from mitochondria of irradiated cells was significantly increased in comparison with that of unirradiated cells. Oxymetry using litium 5,9,14,23,27,32,36-octa-n-butoxy-2,3-naphthalocyanine (LiNc-BuO) and ESR spectroscopy showed that oxygen consumption in irradiated A549 cells, which was inhibited by rotenone, a mitochondrial complex I inhibitor, was increased in comparison with that of unirradiated cells. These results indicate that ionizing radiation enhances the ROS production from mitochondria to trigger cytochrome c release in A549 cells. In summary, present experiments clearly demonstrated that abrogation of the function of constitutive survivin facilitate radiation-induced apoptotic cell death in radioresistant solid tumor cells such as A549 and HeLa cells and radiation-induced secondary ROS from mitochondria play as an essential role in cytochrome c release from mitochondria in their apoptotic signal pathways.
Conffering University: 北海道大学
Degree Report Number: 甲第9039号
Degree Level: 博士
Degree Discipline: 獣医学
Type: theses (doctoral)
Appears in Collections:学位論文 (Theses) > 博士 (獣医学)

Submitter: 小倉 亜希

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