Qin Hui, Liu Ting, Yang Jin-liang, Huang Xin, Liu Bin, Song Xin, Zhao Xia, Wei Yu-quan
Department of Hematology, West China Hospital, Sichuan University, Chengdu 610041, China.
Zhonghua Yi Xue Za Zhi. 2007 Feb 27;87(8):520-5.
To compare the expression profiles of differential proteins between retinoic acid resistant and sensitive cells and screen the proteins related to retinoic acid (RA) resistance by proteomic analysis.
The total cellular proteins from the RA sensitive cells of the line NB4 and the RA resistant cells of the line MR2 obtained from a patient with acute promyelocytic leukemia (APL) were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and analyzed by PDQuest v7.1 analysis software to screen the differential protein spots. Differentially expressed spots were analyzed by mass spectrometry for peptide mass finger (PMF) data and identified by Mascot software and SWISS-PROT protein database. The differentially expressed proteins were verified by Western blotting assay and semi-quantification RT-PCR.
2-DE patterns of APL cell lines with high-resolution and reproducibility were obtained. The average spots of the RA resistant cell line MR2 and RA sensitive cell line NB4 were 890 +/- 45 and 912 +/- 56 respectively. 57 significantly differentially expressed protein spots were screened, among which 23 protein spots were founded to be upregulated and 34 protein spots down regulated in the RA resistant cell line MR2. 25 differential protein spots were identified by mass spectrometry and 17 proteins were successfully assigned to 13 gene-reading frames, of which one was unknown function protein (FLJ00279) and the others were included in the categories of oncoprotein (DJ-1), transcription factor (MYC promoter-binding protein 1), molecule chaperone (HSP70, HSP60 and protein disulfide isomerase), metabolism protein (prohibitin, triosephosphate isomerase 1, and calreticulin), signal transduction (Rho GDP dissociation inhibitor), and cytoskeleton (ACTG1 protein, Beta 5-tubulin, and keratin 10). The results of Western blotting were similar to those of 2D-PAGE and showed differential expression of DJ-1 and calreticulin in several isoforms. Semi-quantification RT-PCR analysis showed that there was no correlation between the protein expression changes and mRNA levels of HSP70 or HSP60. Those results indicated that post-translational events might modify or shear the protein content of the specific spots.
The utilization of two-dimensional polyacrylamide gel electrophoresis coupled with mass spectrometry is effective in screening the all-trans RA resistance-associated proteins and could provide novel clue for study of elucidating all-trans RA resistance mechanisms.
通过蛋白质组学分析比较维甲酸耐药和敏感细胞之间差异蛋白质的表达谱，筛选与维甲酸（RA）耐药相关的蛋白质。
从一名急性早幼粒细胞白血病（APL）患者获得的NB4细胞系的RA敏感细胞和MR2细胞系的RA耐药细胞中提取总细胞蛋白，通过二维聚丙烯酰胺凝胶电泳（2D-PAGE）进行分离，并用PDQuest v7.1分析软件进行分析，以筛选差异蛋白点。对差异表达的点进行质谱分析以获取肽质量指纹（PMF）数据，并通过Mascot软件和SWISS-PROT蛋白质数据库进行鉴定。通过蛋白质免疫印迹法和半定量RT-PCR对差异表达的蛋白质进行验证。
获得了具有高分辨率和可重复性的APL细胞系的2-DE图谱。RA耐药细胞系MR2和RA敏感细胞系NB4的平均点数分别为890±45和912±56。筛选出57个差异显著的表达蛋白点，其中在RA耐药细胞系MR2中有23个蛋白点上调，34个蛋白点下调。通过质谱鉴定了25个差异蛋白点，17种蛋白质成功匹配到13个基因阅读框，其中一个是功能未知蛋白（FLJ00279），其他包括癌蛋白（DJ-1）、转录因子（MYC启动子结合蛋白1）、分子伴侣（HSP70、HSP60和蛋白二硫键异构酶）、代谢蛋白（抑制素、磷酸丙糖异构酶1和钙网蛋白）、信号转导（Rho GDP解离抑制剂）和细胞骨架（ACTG1蛋白、β5-微管蛋白和角蛋白10）等类别。蛋白质免疫印迹结果与2D-PAGE结果相似，显示DJ-1和钙网蛋白在几种异构体中存在差异表达。半定量RT-PCR分析表明，HSP70或HSP60的蛋白质表达变化与mRNA水平之间没有相关性。这些结果表明翻译后事件可能会修饰或剪切特定斑点的蛋白质含量。
二维聚丙烯酰胺凝胶电泳结合质谱法可有效筛选全反式维甲酸耐药相关蛋白，为阐明全反式维甲酸耐药机制的研究提供新线索。
