DNA-Dependent Protein Kinase (DNA-PK)-Dependent Cisplatin-Induced Loss of Nucleolar Facilitator of Chromatin Transcription (FACT) and Regulation of Cisplatin Sensitivity by DNA-PK and FACT
DNA-Dependent Protein Kinase (DNA-PK)-Dependent Cisplatin-Induced Loss of Nucleolar Facilitator of Chromatin Transcription (FACT) and Regulation of Cisplatin Sensitivity by DNA-PK and FACT
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DOI：
10.1158/1541-7786.mcr-08-0049
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发表时间：
2009-04-01
影响因子：
5.2
通讯作者：
Lazaro, Jean-Bernard
中科院分区：
文献类型：
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作者：
Dejmek, Janna;Iglehart, J. Dirk;Lazaro, Jean-Bernard
据报道，DNA依赖性蛋白激酶（DNA-PK）的Ku亚基和染色质转录促进剂（FACT）复合物都能结合顺铂-DNA加合物。在这项研究中，我们开发了一种基于去污剂提取的免疫细胞化学检测方法，可以揭示核质和核仁中存在的蛋白质核仁亚群。在各种人类癌细胞系中的免疫荧光分析和分离的核仁的免疫印迹显示，DNA-PK催化亚基（DNA-PKcs）、Ku 86、Werner综合征蛋白（WRN）和FACT的结构特异性识别蛋白1（SSRP 1）亚基共定位于核仁中，并在顺铂处理后退出核仁。Ku的核仁定位也在γ或UV照射和暴露于DNA损伤药物如放线菌素D、丝裂霉素C、羟基脲和阿霉素后丢失。Ku 86和WRN在暴露于低剂量（>1 μ g/mL）顺铂后离开核仁。相比之下，SSRP 1与核仁的关联仅被高剂量（50-100 μ g/mL）的顺铂破坏。用渥曼青霉素或DNA-PK抑制剂NU 7026预处理细胞可抑制顺铂诱导的核仁SSRP 1和DNA-PK活化的丧失，但磷脂酰肌醇3-激酶抑制剂LY 294002不能抑制顺铂诱导的核仁SSRP 1和DNA-PK活化。在相同的条件下，激酶抑制剂没有改变退出DNA-PKcs和WRN，表明不同的机制调节退出DNA-PK/WRN和FACT从核仁。此外，DNA-PKcs的RNA沉默阻断了顺铂诱导的核仁SSRP 1的退出。最后，通过短发夹RNA沉默DNA-PKcs或SSRP 1显著增加癌细胞对顺铂的敏感性。(Mol Cancer Res 2009;7（4）：581-91）
Both the Ku subunit of the DNA-dependent protein kinase (DNA-PK) and the facilitator of chromatin transcription (FACT) complex reportedly bind cisplatin-DNA adducts. For this study, we developed an immunocytochemical assay based on detergent extraction allowing unveiling nucleolar subpopulations of proteins present in both the nucleoplasm and the nucleolus. Immunofluorescence analysis in various human cancer cell lines and immunoblotting of isolated nucleoli show that DNA-PK catalytic subunit (DNA-PKcs), Ku86, the Werner syndrome protein (WRN), and the structure-specific recognition protein 1 (SSRP1) subunit of FACT colocalize in the nucleolus and exit the nucleolus after cisplatin treatment. Nucleolar localization of Ku is also lost after y or UV irradiation and exposure to DNA-damaging drugs, such as actinomycin D, mitomycin C, hydroxyurea, and doxorubicin. Ku86 and WRN leave the nucleolus after exposure to low (>1 mu g/mL) doses of cisplatin. In contrast, the SSRP1 association with the nucleolus was disrupted only by high (50-100 mu g/mL) doses of cisplatin. Both cisplatin-induced loss of nucleolar SSRP1 and DNA-PK activation are suppressed by pretreatment of the cells with wortmannin or the DNA-PK inhibitor NU7026 but not by the phosphatidylinositol 3-kinase inhibitor LY294002. In the same conditions, kinase inhibitors did not alter the exit of DNA-PKcs and WRN, suggesting that different mechanisms regulate the exit of DNA-PK/WRN and FACT from the nucleolus. Furthermore, RNA silencing of DNA-PKcs blocked the cisplatin-induced exit of nucleolar SSRP1. Finally, silencing of DNA-PKcs or SSRP1 by short hairpin RNA significantly increased the sensitivity of cancer cells to cisplatin. (Mol Cancer Res 2009;7(4):581-91)