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Studies on the mechanism of Drs-mediated apoptosis and tumor suppression by gene-knockout mouse

基因敲除小鼠Drs介导的细胞凋亡及抑癌机制研究

基本信息

批准号：
17590341
负责人：
INOUE Hirokazu
金额：
$ 2.3万
依托单位：
Shiga University of Medical Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590341/
关键词：
Tumor suppressor gene Apoptosis Knockout mouse Drs Autophagy GADD34

项目摘要

Drs protein associates with ASY/Nogo-B, an apoptosis-inducing protein in the endoplasmic reticulum, and sequentially activate caspases (-12,-9, and -3) to induce apoptosis in human cancer cells. Malignant tumors were generated in about 30% of the drs KO mice, indicating that drs contributes to the suppression of malignant tumor formation. In this study, we investigated the physiological roles of drs and the mechanism of tumor suppression by this gene by using drs KO mice and cells. The following results were obtained.1.Re-introduction of drs by retrovirus vector into a lung cancer cell line derived from a lung adenocarcinoma of a drs KO mouse caused suppression of tumorigenicity in nude mouse and elevation of apoptosis mediated by activation of caspase-12,-9, and -3.2.drs KO embryonic fibroblasts (MEF) also showed enhanced sensitivity to transformation by v-src oncogene.3.Autophagy induced by low serum conditions was suppressed in drs KO MEF cells. Analyses with electron microscope and GFP-LC3 showed that drs is involved in the maturation process from autophagosomes to autolysosomes.4.Drs associates with Rab24, which is involved in the autophagosome maturation, and colocalized with Rab24 on the autolysosomes during the late phase of autophagy induced by low serum.5.Drs interacts with stress-inducible GADD34. Experiments with KO MEF cells, showed that drs and GADD34 are involved in survival in energy-depletion conditions and anti-viral defense via suppression of mTOR pathway.These results idicate that drs is involved in the regulatuion of autophagy as well as apoptosis. Furthermore, drs plays a role in cell survival in stress conditions and anti-viral defense. By using KO mice and KO cells, we are going to clarify the physiological function of drs and the molecular mechanism of tumor suppression by drs.

Drs蛋白与内质网中的凋亡诱导蛋白ASY/Nogo-B结合，并依次激活半胱天冬酶（-12、-9和-3）以诱导人癌细胞的凋亡。在约30%的drs KO小鼠中产生恶性肿瘤，表明drs有助于抑制恶性肿瘤的形成。在本研究中，我们通过使用drs基因敲除小鼠和细胞来研究drs的生理作用和该基因抑制肿瘤的机制。1.通过逆转录病毒载体将drs重新导入来源于drs KO小鼠肺腺癌的肺癌细胞系中，引起裸鼠致瘤性的抑制和由caspase-12，-9活化介导的凋亡的增加，和-3.2.drsKO胚胎成纤维细胞（MEF）也显示对v-src癌基因。3.在drs KO MEF细胞中，低血清条件诱导的自噬被抑制。电镜和GFP-LC 3分析表明，drs参与了自噬体向自溶酶体的成熟过程; 4.Drs与Rab 24结合，Rab 24参与自噬体的成熟，并在低血清诱导的自噬后期与Rab 24共定位于自溶酶体上; 5.Drs与应激诱导的GADD相互作用34。利用KO MEF细胞进行的实验表明，drs和GADD 34通过抑制mTOR通路参与能量耗竭条件下的生存和抗病毒防御，这些结果表明drs参与自噬和凋亡的调节。此外，drs在应激条件下的细胞存活和抗病毒防御中发挥作用。本研究拟通过KO小鼠和KO细胞，阐明drs的生理功能和drs抑瘤的分子机制。