辐射或药物损伤可致肿瘤细胞DNA双链断裂,而DNA依赖性蛋白激酶(DNA-depended protein kinase,DNA-PK)是DNA损伤修复的关键酶,决定着受损肿瘤细胞的转归。因此,DNA-PK与肿瘤放化疗敏感性的关系成为研究热点。近年来,关于DNA-PK的结构与...辐射或药物损伤可致肿瘤细胞DNA双链断裂,而DNA依赖性蛋白激酶(DNA-depended protein kinase,DNA-PK)是DNA损伤修复的关键酶,决定着受损肿瘤细胞的转归。因此,DNA-PK与肿瘤放化疗敏感性的关系成为研究热点。近年来,关于DNA-PK的结构与功能、在肿瘤细胞株中的活性、在肿瘤组织中的表达的研究日益深入,以DNA-PK为靶点的DNA修复抑制剂作为肿瘤放化疗增敏药已陆续研发并进入临床前试验。该综述简要回顾DNA-PK的生物学作用及其研究进展。展开更多
Objective： Radiosensitivity is mainly determined by the number of DNA double-strand breaks （DSBs） induced by ionizing radiation and the extent of its repair. The DNA-PK complex formation is one of the major pathway...Objective： Radiosensitivity is mainly determined by the number of DNA double-strand breaks （DSBs） induced by ionizing radiation and the extent of its repair. The DNA-PK complex formation is one of the major pathways by which the mammalian cells respond to DSBs repairing. Our previous study suggested that CNE1 is more radioresistant than CNE2. This study was designed to answer whether the radiosensitive difference of Nasopharyngeal Carcinoma cell lines CNE1/CNE2 was related to the expression and localization of Ku70/Ku80/DNA-PKcs. Methods： Immunohistochemistry was performed to detect the subcellular localization of Ku70/Ku80/DNA-PKcs in NPC cells lines CNE1 and CNE2. Western-blot was used to determine the expression of Ku protein in total extract of CNE1 and CNE2 and semi-quantitative assay of protein expression was performed to estimate the optic density （OD） value of each band using automatic image analysis system. Results： Ku70/Ku80/DNA-PKcs primarily located in the nuclei. A part of nucleolus in CNE1 and CNE2 showed positive dyeing of DNA-PKcs. Protein expression of Ku70/Ku80/DNA-PKcs was detected in CNE1 and CNE2, and the integral optical density （IOD） of Ku70 protein was 22.03 ± 7.56 and 19.98 ± 6.04 respectively （t=0.021, P〉0.05）, while the IODs of Ku80 protein in the two cell lines were 33.44 ± 12.87 and 28.98 ± 9.24 respectively （t=0.24, P〉0.05）, and the IODs of DNA-PKcs protein were 45.03 ± 1.77 and 40.87 ± 4.19 （t=1.58, P〉0.05）. The above results suggested that the basic expression of Ku70/Ku80/DNA-PKcs had no statistic difference between the different radiosensitive NPC cell lines CNE1 and CNE2. Conclusion： The variation of radiosensitivity in NPC cell lines CNE1 and CNE2 has no obviously correlation with the subcellular localization and basic expression of DNA-PK protein. So we presumed that the difference of radiosensitivity between CNE1 and CNE2 may be on account of some other factors than subcellular localization and basic expression of DNA-PK.展开更多
The partitioning-defective 3 （Par3）, a key component in the conserved Par3/Par6/aPKC complex, plays fundamental roles in cell polarity. Herein we report the identification of Ku70 and Ku80 as novel Par3-interacting ...The partitioning-defective 3 （Par3）, a key component in the conserved Par3/Par6/aPKC complex, plays fundamental roles in cell polarity. Herein we report the identification of Ku70 and Ku80 as novel Par3-interacting proteins through an in vitro binding assay followed by liquid chromatography-tandem mass spectrometry. Ku70/Ku80 proteins are two key regulatory subunits of the DNA-dependent protein kinase （DNA-PK）, which plays an essential role in repairing double-strand DNA breaks （DSBs）. We determined that the nuclear association of Par3 with Ku70/Ku80 was enhanced by γ-irradiation （IR）, a potent DSB inducer. Furthermore, DNA-PKcs, the catalytic subunit of DNA-PK, interacted with the Par3/Ku70/Ku80 complex in response to IR. Par3 over-expression or knockdown was capable of up- or downregulating DNA-PK activity, respectively. Moreover, the Par3 knockdown cells were found to be defective in random plasmid integration, defective in DSB repair following IR, and radiosensitive, phenotypes similar to that of Ku70 knockdown cells. These findings identify Par3 as a novel component of the DNA-PK complex and implicate an unexpected link of cell polarity to DSB repair.展开更多
The author affiliations were mixed up in the previous published version. The third fund number of National NaturalScience Foundation of China in the Acknowledgments was wrong, it should be "30270335". The Sh...The author affiliations were mixed up in the previous published version. The third fund number of National NaturalScience Foundation of China in the Acknowledgments was wrong, it should be "30270335". The Shanghai MunicipalCouncil for Science and Technology (No.06DZ22032) was missed in the Acknowledgments. There are some labelingand production errors in Figure 2A, Figure 3B and 3C, Figure 5C, Figure 6B and 6E, Figure 7B and 7D. In Figure 2A,left panel "A431" should be "Par3". In Figure 3B and 3C, "anti-Par3CT" should be "anti-Par3LCT", "GST-Par3CT"should be "GST-Par3LCT". In Figure 5C, the second arrow indicating "Lamin B" should be "[3-tubulin". In Figure 6Bright panel, the molecular weight for ?-actin should be "43" instead of"200". In Figure 6E, "Par3" should be "Par3i". Themolecular weight for the DNA-PKcs panel should be the same as the p-DNA-PKcs. In Figure 7B, the time point "240"in the left panel should be "120"; in the right panel of Figure 7B, the title for the y axis should be "DNA released (%)".In Figure 7D, the title for the y axis should be "Survival (%)", and the scale for the y axis should be "100, 10 and 1". These corrections do not affect the conclusions of the study. We apologize for any inconvenience this may havecaused.展开更多
目的:总结国内外对DNA依赖性蛋白激酶(DNA-dependent protein ki-nase,DNA-PK)作为放化疗增敏靶点研究的现状。方法:检索Medline及CHKD期刊全文数据库检索系统,以"DNA-PK、放射敏感性和化疗敏感性"为关键词,检索1998-01-2008...目的:总结国内外对DNA依赖性蛋白激酶(DNA-dependent protein ki-nase,DNA-PK)作为放化疗增敏靶点研究的现状。方法:检索Medline及CHKD期刊全文数据库检索系统,以"DNA-PK、放射敏感性和化疗敏感性"为关键词,检索1998-01-2008-03关于DNA-PK研究的文献,共检索到英文文献1660篇,中文文献115篇。纳入标准:1)DNA-PK的功能;2)DNA-PK作为肿瘤治疗靶点的研究;3)DNA-PK抑制剂研究。根据纳入标准,精选82篇文献,最后纳入分析35篇文献。结果:放射线和类放射线药物均可导致肿瘤细胞DNA双链断裂(DSB),从而杀伤肿瘤细胞,DNA-PK具有包括促进DSB修复在内的诸多功能。抑制DNA-PK的功能和活性,就可抑制DSB的修复,提高肿瘤细胞对放化疗的敏感性。结论:DNA-PK可以成为肿瘤治疗的理想靶点,但在临床应用方面仍有很多问题需要解决。展开更多
文摘辐射或药物损伤可致肿瘤细胞DNA双链断裂,而DNA依赖性蛋白激酶(DNA-depended protein kinase,DNA-PK)是DNA损伤修复的关键酶,决定着受损肿瘤细胞的转归。因此,DNA-PK与肿瘤放化疗敏感性的关系成为研究热点。近年来,关于DNA-PK的结构与功能、在肿瘤细胞株中的活性、在肿瘤组织中的表达的研究日益深入,以DNA-PK为靶点的DNA修复抑制剂作为肿瘤放化疗增敏药已陆续研发并进入临床前试验。该综述简要回顾DNA-PK的生物学作用及其研究进展。
基金This work was supported by a grant fromthe National Natural Science Foundation of China (No.30070237).
文摘Objective： Radiosensitivity is mainly determined by the number of DNA double-strand breaks （DSBs） induced by ionizing radiation and the extent of its repair. The DNA-PK complex formation is one of the major pathways by which the mammalian cells respond to DSBs repairing. Our previous study suggested that CNE1 is more radioresistant than CNE2. This study was designed to answer whether the radiosensitive difference of Nasopharyngeal Carcinoma cell lines CNE1/CNE2 was related to the expression and localization of Ku70/Ku80/DNA-PKcs. Methods： Immunohistochemistry was performed to detect the subcellular localization of Ku70/Ku80/DNA-PKcs in NPC cells lines CNE1 and CNE2. Western-blot was used to determine the expression of Ku protein in total extract of CNE1 and CNE2 and semi-quantitative assay of protein expression was performed to estimate the optic density （OD） value of each band using automatic image analysis system. Results： Ku70/Ku80/DNA-PKcs primarily located in the nuclei. A part of nucleolus in CNE1 and CNE2 showed positive dyeing of DNA-PKcs. Protein expression of Ku70/Ku80/DNA-PKcs was detected in CNE1 and CNE2, and the integral optical density （IOD） of Ku70 protein was 22.03 ± 7.56 and 19.98 ± 6.04 respectively （t=0.021, P〉0.05）, while the IODs of Ku80 protein in the two cell lines were 33.44 ± 12.87 and 28.98 ± 9.24 respectively （t=0.24, P〉0.05）, and the IODs of DNA-PKcs protein were 45.03 ± 1.77 and 40.87 ± 4.19 （t=1.58, P〉0.05）. The above results suggested that the basic expression of Ku70/Ku80/DNA-PKcs had no statistic difference between the different radiosensitive NPC cell lines CNE1 and CNE2. Conclusion： The variation of radiosensitivity in NPC cell lines CNE1 and CNE2 has no obviously correlation with the subcellular localization and basic expression of DNA-PK protein. So we presumed that the difference of radiosensitivity between CNE1 and CNE2 may be on account of some other factors than subcellular localization and basic expression of DNA-PK.
基金This work was supported by the grants from National Natural Science Foundation of China(Nos.30170208,30623003 and 30170208)from the Ministry of Science and Technology,China(No.2001AA233031 and No.2001CB510205)from US NIH(CA50519)(to DJ Chen).
文摘The partitioning-defective 3 （Par3）, a key component in the conserved Par3/Par6/aPKC complex, plays fundamental roles in cell polarity. Herein we report the identification of Ku70 and Ku80 as novel Par3-interacting proteins through an in vitro binding assay followed by liquid chromatography-tandem mass spectrometry. Ku70/Ku80 proteins are two key regulatory subunits of the DNA-dependent protein kinase （DNA-PK）, which plays an essential role in repairing double-strand DNA breaks （DSBs）. We determined that the nuclear association of Par3 with Ku70/Ku80 was enhanced by γ-irradiation （IR）, a potent DSB inducer. Furthermore, DNA-PKcs, the catalytic subunit of DNA-PK, interacted with the Par3/Ku70/Ku80 complex in response to IR. Par3 over-expression or knockdown was capable of up- or downregulating DNA-PK activity, respectively. Moreover, the Par3 knockdown cells were found to be defective in random plasmid integration, defective in DSB repair following IR, and radiosensitive, phenotypes similar to that of Ku70 knockdown cells. These findings identify Par3 as a novel component of the DNA-PK complex and implicate an unexpected link of cell polarity to DSB repair.
基金supported by the grants from National Natural Science Foundation of China(No.30170208,30623003 and 30270335)from the Ministry of Science and Technology,China(No.2001AA233031 and No.2001CB510205)+1 种基金from the Shanghai Municipal Council for Science and Technology(No.06DZ22032)from US NIH(CA50519)(to DJ Chen).
文摘The author affiliations were mixed up in the previous published version. The third fund number of National NaturalScience Foundation of China in the Acknowledgments was wrong, it should be "30270335". The Shanghai MunicipalCouncil for Science and Technology (No.06DZ22032) was missed in the Acknowledgments. There are some labelingand production errors in Figure 2A, Figure 3B and 3C, Figure 5C, Figure 6B and 6E, Figure 7B and 7D. In Figure 2A,left panel "A431" should be "Par3". In Figure 3B and 3C, "anti-Par3CT" should be "anti-Par3LCT", "GST-Par3CT"should be "GST-Par3LCT". In Figure 5C, the second arrow indicating "Lamin B" should be "[3-tubulin". In Figure 6Bright panel, the molecular weight for ?-actin should be "43" instead of"200". In Figure 6E, "Par3" should be "Par3i". Themolecular weight for the DNA-PKcs panel should be the same as the p-DNA-PKcs. In Figure 7B, the time point "240"in the left panel should be "120"; in the right panel of Figure 7B, the title for the y axis should be "DNA released (%)".In Figure 7D, the title for the y axis should be "Survival (%)", and the scale for the y axis should be "100, 10 and 1". These corrections do not affect the conclusions of the study. We apologize for any inconvenience this may havecaused.
文摘目的:总结国内外对DNA依赖性蛋白激酶(DNA-dependent protein ki-nase,DNA-PK)作为放化疗增敏靶点研究的现状。方法:检索Medline及CHKD期刊全文数据库检索系统,以"DNA-PK、放射敏感性和化疗敏感性"为关键词,检索1998-01-2008-03关于DNA-PK研究的文献,共检索到英文文献1660篇,中文文献115篇。纳入标准:1)DNA-PK的功能;2)DNA-PK作为肿瘤治疗靶点的研究;3)DNA-PK抑制剂研究。根据纳入标准,精选82篇文献,最后纳入分析35篇文献。结果:放射线和类放射线药物均可导致肿瘤细胞DNA双链断裂(DSB),从而杀伤肿瘤细胞,DNA-PK具有包括促进DSB修复在内的诸多功能。抑制DNA-PK的功能和活性,就可抑制DSB的修复,提高肿瘤细胞对放化疗的敏感性。结论:DNA-PK可以成为肿瘤治疗的理想靶点,但在临床应用方面仍有很多问题需要解决。
