Aboagye-Mathiesen G, Ebbesen P, von der Maase H, Celis J E
Institute for Medical Biochemistry and Danish Center for Human Genome Research, Aarhus University, Aarhus C.
Electrophoresis. 1999 Feb;20(2):344-8. doi: 10.1002/(SICI)1522-2683(19990201)20:2<344::AID-ELPS344>3.0.CO;2-V.
Poly(A) mRNA was isolated from human placental trophoblast cells stimulated with 100 U/mL of interleukin-2 and 5 microg/mL of phytohemagglutinin and reverse-transcribed. The cDNA coding for the mature interferon-gamma (IFN-gamma) protein was amplified using specific primers, cloned into the pGEX-4T2 vector, and expressed in Escherichia coli. Treatment of four fresh bladder transitional cell carcinoma (TCC) biopsies (TCCs 845-1, grade II, Ta; TCC 925-1, grade II, Ta; TCC 919-1, grade III, T1; TCC 950-1, grade III, T1) with the purified recombinant trophoblast IFN-gamma (50 U/mL, 20 h), followed by proteome analysis using two-dimensional gel electrophoresis, revealed several major proteins whose level of expression were affected by this cytokine. Of these, five (tryptophanyl-tRNA synthetase, the interferon gamma-inducible protein gamma3, mangase superoxide dismutase, and two unknown proteins of apparent molecular masses of 35.8 and 11.2 kDa, respectively) were upregulated in at least 75% of the tumors analyzed while one was downregulated (aldose reductase). Proteins were identified using a combination of techniques that included microsequencing, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) immunoblotting and comparison with the transitional cell carcinoma isoelectric focusing (IEF) database (http://biobase.dk/cgi-bin/celis). Proteome profile analysis of primary cultures from a low-grade lesion (TCC 846-1, Grade II, Ta) labeled in the presence and absence of IFN-gamma showed that all of the proteins disregulated in vivo were also affected in the cultures. The cultured cells, on the other hand, exhibited additional changes that were not detected in vivo and that may reflect adaptation to the culturing conditions. Taken together, the results provide a first glance at the effect of IFN-gamma on the protein expression profiles of TCCs, and in due course may form the basis for more comprehensive studies aimed at evaluating the usefulness of this cytokine in bladder cancer management.
从用100 U/mL白细胞介素-2和5 μg/mL植物血凝素刺激的人胎盘滋养层细胞中分离出聚腺苷酸（Poly(A)）mRNA并进行逆转录。使用特异性引物扩增编码成熟干扰素-γ（IFN-γ）蛋白的cDNA，将其克隆到pGEX-4T2载体中，并在大肠杆菌中表达。用纯化的重组滋养层IFN-γ（50 U/mL，20小时）处理四份新鲜膀胱移行细胞癌（TCC）活检样本（TCC 845-1，II级，Ta期；TCC 925-1，II级，Ta期；TCC 919-1，III级，T1期；TCC 950-1，III级，T1期），然后使用二维凝胶电泳进行蛋白质组分析，结果显示几种主要蛋白质的表达水平受到这种细胞因子的影响。其中，五种蛋白质（色氨酰-tRNA合成酶、干扰素γ诱导蛋白γ3、锰超氧化物歧化酶以及两种表观分子量分别为35.8 kDa和11.2 kDa的未知蛋白质）在至少75%的分析肿瘤中上调，而一种蛋白质（醛糖还原酶）下调。使用包括微量测序、二维聚丙烯酰胺凝胶电泳（2-D PAGE）免疫印迹以及与移行细胞癌等电聚焦（IEF）数据库（http://biobase.dk/cgi-bin/celis）比较在内的多种技术组合来鉴定蛋白质。对在有和没有IFN-γ存在的情况下标记的低级别病变（TCC 846-1，II级，Ta期）的原代培养物进行蛋白质组图谱分析表明，所有在体内失调的蛋白质在培养物中也受到影响。另一方面，培养的细胞表现出在体内未检测到的其他变化，这可能反映了对培养条件的适应。综上所述，这些结果初步展示了IFN-γ对TCC蛋白质表达谱的影响，并且在适当的时候可能为旨在评估这种细胞因子在膀胱癌治疗中效用的更全面研究奠定基础。
