Che Yiping, Best O Giles, Zhong Ling, Kaufman Kimberley L, Mactier Swetlana, Raftery Mark, Graves Lee M, Mulligan Stephen P, Christopherson Richard I
Cancer Proteomics Laboratory, School of Molecular Bioscience, University of Sydney , Sydney, NSW 2006, Australia.
J Proteome Res. 2013 Apr 5;12(4):1710-22. doi: 10.1021/pr301055y. Epub 2013 Mar 22.
The proteomic effects of the Hsp90 inhibitor, SNX-7081, have been determined on the p53-mutated B-cell chronic lymphocytic leukemia (CLL) cell line, MEC1. Following SNX-7081 treatment (500 nM, 24 h), 51 proteins changed abundance by more than 2-fold (p < 0.05); 7 proteins increased while 44 proteins decreased. Proteins identified as differentially abundant by LC-MS/MS were validated by Western blotting (DDB1, PCNA, MCM2, Hsp90, Hsp70, GRP78, PDIA6, HLA-DR). RT-PCR showed that SNX-7081 unexpectedly modulates a number of these proteins in MEC1 cells at the mRNA level (PCNA, MCM2, Nup155, Hsp70, GRP78, PDIA6, and HLA-DR). Pathway analysis determined that 3 of the differentially abundant proteins (cyclin D1, c-Myc and pRb) were functionally related. p53 levels did not change upon SNX-7081 treatment of p53 wild-type Raji cells or p53-mutated MEC1 and U266 cells, indicating that SNX-7081 has a p53-independent mechanism. The decreases in DDB1, MCM2, c-Myc, and PCNA and increases of pRb and cyclin D1 were confirmed in MEC1, U266, Raji, and p53 null HL60 cells by Western blotting. These data suggest that SNX-7081 arrests the cell cycle and inhibits DNA replication and r epair and provides evidence for the mechanism of the observed synergy between Hsp90 inhibitors and drugs that induce DNA strand breaks.
已确定热休克蛋白90（Hsp90）抑制剂SNX - 7081对p53突变的B细胞慢性淋巴细胞白血病（CLL）细胞系MEC1的蛋白质组学效应。用SNX - 7081处理（500 nM，24小时）后，51种蛋白质的丰度变化超过2倍（p < 0.05）；7种蛋白质增加，44种蛋白质减少。通过液相色谱 - 串联质谱法（LC - MS/MS）鉴定为差异丰度的蛋白质经蛋白质免疫印迹法（DDB1、增殖细胞核抗原（PCNA）、微小染色体维持蛋白2（MCM2）、Hsp90、Hsp70、葡萄糖调节蛋白78（GRP78）、蛋白二硫键异构酶A6（PDIA6）、人类白细胞抗原DR（HLA - DR））验证。逆转录 - 聚合酶链反应（RT - PCR）表明，SNX - 7081意外地在mRNA水平调节MEC1细胞中许多此类蛋白质（PCNA、MCM2、核孔蛋白155（Nup155）、Hsp70、GRP78、PDIA6和HLA - DR）。通路分析确定差异丰度的3种蛋白质（细胞周期蛋白D1、c - Myc和视网膜母细胞瘤蛋白（pRb））在功能上相关。用SNX - 7081处理p53野生型Raji细胞或p53突变的MEC1和U266细胞后，p53水平未发生变化，表明SNX - 7081具有不依赖p53的机制。通过蛋白质免疫印迹法在MEC1、U266、Raji和p53缺失的HL60细胞中证实了DDB1、MCM2、c - Myc和PCNA的减少以及pRb和细胞周期蛋白D1的增加。这些数据表明，SNX - 7081使细胞周期停滞并抑制DNA复制和修复，并为观察到的Hsp90抑制剂与诱导DNA链断裂的药物之间协同作用的机制提供了证据。
