Sinha P, Hütter G, Köttgen E, Dietel M, Schadendorf D, Lage H
Institut für Laboratoriumsmedizin und Pathobiochemie, Universitätsklinikum Charité, Berlin, Germany.
J Biochem Biophys Methods. 1998 Nov 18;37(3):105-16. doi: 10.1016/s0165-022x(98)00020-7.
The therapy of advanced cancer using chemotherapy alone or in combination with radiation or hyperthermia yields an overall response rate of about 20-50%. This success is often marred by the development of resistance to cytostatic drugs. Our aim was to study the global analysis of protein expression in the development of chemoresistance in vitro. We therefore used a cell culture model derived from the gastric carcinoma cell line EPG 85-257P. A classical multidrug-resistant subline EPG85-257RDB selected to daunorubicin and an atypical multidrug-resistant cell variant EPG85-257RNOV selected to mitoxantrone, were analysed using two-dimensional electrophoresis in immobilized pH-gradients (pH 4.0-8.0) in the first dimension and linear polyacrylamide gels (12%) in the second dimension. After staining with coomassie brilliant blue, image analysis was performed using the PDQuest system. Spots of interest were isolated using preparative two-dimensional electrophoresis and subjected to microsequencing. A total of 241 spots from the EPG85-257RDB-standard and 289 spots from the EPG85-257RNOV-standard could be matched to the EPG85-257P-standard. Microsequencing after enzymatic hydrolysis in gel, mass spectrometric data and sequencing of the peptides after their fractionation using microbore HPLC identified that two proteins annexin I and thioredoxin were overexpressed in chemoresistant cell lines. Annexin I was present in both the classical and the atypical multidrug-resistant cells. Thioredoxin was found to be overexpressed only in the atypical multidrug-resistant cell line.
单独使用化疗或联合放疗或热疗治疗晚期癌症的总体缓解率约为20%-50%。但这种成功常常因对细胞生长抑制剂产生耐药性而受到影响。我们的目的是研究体外化疗耐药性发展过程中蛋白质表达的全局分析。因此，我们使用了源自胃癌细胞系EPG 85-257P的细胞培养模型。对选择柔红霉素的经典多药耐药亚系EPG85-257RDB和选择米托蒽醌的非典型多药耐药细胞变体EPG85-257RNOV，在第一维使用固定pH梯度（pH 4.0-8.0）的二维电泳，在第二维使用线性聚丙烯酰胺凝胶（12%）进行分析。用考马斯亮蓝染色后，使用PDQuest系统进行图像分析。通过制备性二维电泳分离感兴趣的斑点并进行微量测序。来自EPG85-257RDB标准品的总共241个斑点和来自EPG85-257RNOV标准品的289个斑点可以与EPG85-257P标准品匹配。凝胶内酶解后的微量测序、质谱数据以及使用微径HPLC对肽段进行分级分离后的测序鉴定出，两种蛋白质膜联蛋白I和硫氧还蛋白在耐药细胞系中过表达。膜联蛋白I存在于经典和非典型多药耐药细胞中。硫氧还蛋白仅在非典型多药耐药细胞系中过表达。
