Xu Jian-Ping, Hu Jian-Da, Lin Min-Hui, Li Jing, Liu Ting-Bo
Fujian Institute of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2011 Dec;19(6):1383-7.
The purpose of this study was to compare the differences of the protein expression profiles between human myeloid leukemia K562 cells and adriamycin-resistant K562/A02 cells, as well as to select novel resistance-related proteins in myeloid leukemia by means of proteomics. The total cellular proteins were separated from K562 and adriamycin-resistant K562/A02 cells by using technique of two dimensional difference in gel electrophoresis (2D-DIGE). Differentially expressed proteins were analyzed by matrix-assisted laser desorption ionization/time of flight-mass spectrometry (MALDI-TOF/MS), and by protein database searching. Moreover, the differentially expressed proteins were verified at protein and mRNA levels by Western blot assay and quantitative real time PCR. The results showed that 8 proteins differentially expressed in adriamycin-resistant K562/A02 cells, among them 2 proteins were identified to be down-regulated and 6 to be up-regulated. These identified proteins involved in the cell energy metabolism, cell proliferation, cell apoptosis, signal transduction, gene transcription and translation respectively. The results assayed by Western blot were similar to those detected by 2D-PAGE. Two up-regulated proteins Stathmin and CrkL were selected for verification in K562 and K562/A02 cells. As a result, the results detected by Western blot were identical with results from 2D-DIGE; real time quantitative PCR assay showed that the changes of CrkL at mRNA level were identical with changes at protein level, but no complete identity of Stathmin changes at mRNA level and protein level was observed. It is concluded that the difference of protein expression profile exists in K562 and K562/A02 cells. Stathmin and CrkL proteins may be involved in the drug resistance and suggest a novel clue for the resistant mechanisms in myeloid leukemia, which is worth further to explore.
本研究旨在比较人髓系白血病K562细胞与阿霉素耐药K562/A02细胞之间蛋白质表达谱的差异，并通过蛋白质组学方法筛选髓系白血病中与耐药相关的新蛋白。采用二维差异凝胶电泳（2D-DIGE）技术分离K562细胞和阿霉素耐药K562/A02细胞的总细胞蛋白。通过基质辅助激光解吸电离/飞行时间质谱（MALDI-TOF/MS）和蛋白质数据库搜索分析差异表达蛋白。此外，通过蛋白质免疫印迹法和实时定量PCR在蛋白质和mRNA水平验证差异表达蛋白。结果显示，阿霉素耐药K562/A02细胞中有8种蛋白质差异表达，其中2种蛋白质下调，6种蛋白质上调。这些鉴定出的蛋白质分别参与细胞能量代谢、细胞增殖、细胞凋亡、信号转导、基因转录和翻译。蛋白质免疫印迹法检测结果与二维聚丙烯酰胺凝胶电泳（2D-PAGE）检测结果相似。选择两种上调蛋白Stathmin和CrkL在K562和K562/A02细胞中进行验证。结果，蛋白质免疫印迹法检测结果与2D-DIGE结果一致；实时定量PCR分析显示，CrkL在mRNA水平的变化与蛋白质水平的变化一致，但未观察到Stathmin在mRNA水平和蛋白质水平的变化完全一致。结论是，K562和K562/A02细胞存在蛋白质表达谱差异。Stathmin和CrkL蛋白可能参与耐药过程，为髓系白血病的耐药机制提供了新线索，值得进一步探索。
