Celis A, Rasmussen H H, Celis P, Basse B, Lauridsen J B, Ratz G, Hein B, Ostergaard M, Wolf H, Orntoft T, Celis J E
Department of Medical Biochemistry and Danish Centre for Human Genome Research, The University of Aarhus.
Electrophoresis. 1999 Feb;20(2):355-61. doi: 10.1002/(SICI)1522-2683(19990201)20:2<355::AID-ELPS355>3.0.CO;2-N.
Fresh, superficial transitional cell carcinomas (TCCs) of low-grade atypia (3 grade I, Ta; 6 grade II, Ta), as well as primary cultures derived from them were labeled with [35S]methionine for 16 h, between 2 and 6 days after inoculation. Whole protein extracts were subjected to IEF (isoelectric focusing) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by autoradiography. Proteins were identified by a combination of proteomic technologies that included microsequencing, mass spectrometry, 2-D PAGE immunoblotting and comparison with the bladder TCC protein database available on the internet (http://biobase.dk/cgi-bin/celis). Comparison of the IEF 2-D gel protein profiles of fresh tumors and their primary cultures showed that the overall expression profiles were strikingly similar, although differing significantly in the levels of several proteins whose rate of synthesis was differentially regulated in at least 85% of the tumor/culture pairs as a result of the short-term culturing. Most of the proteins affected by culturing were upregulated and among them we identified components of the cytoskeleton (keratin 18, gelsolin and tropomyosin 3), a molecular chaperone (hsp 28), aldose reductase, GST pi, metastasin, synuclein, the calreticulin precursor and three polypeptides of unknown identity. Only four major proteins were downregulated, and these included two fatty acid-binding proteins (FABP:FABP5 and A-FABP) which are thought to play a role in growth control, the differentiation-associated keratin 20, and the calcium-binding protein annexin V. Proteins that were differentially regulated in only some of the cultured tumors included alpha-enolase, triosphosphate isomerase, members of the 14-3-3 family, hnRNPs F and H, PGDH, hsp (heat-shock protein) 60, BIP, the interleukin-1 receptor antagonist, the nucleolar protein B23, as well as several proteins of yet unknown identity. The suitability of in vitro bladder tumor culture models to study complex biological phenomena such as malignancy and invasion is discussed.
对新鲜的、低级别异型性的浅表移行细胞癌（TCCs）（3例I级，Ta期；6例II级，Ta期）及其原代培养物在接种后2至6天用[35S]甲硫氨酸标记16小时。将全蛋白提取物进行等电聚焦（IEF）二维聚丙烯酰胺凝胶电泳（2-D PAGE），然后进行放射自显影。通过包括微量测序、质谱分析、2-D PAGE免疫印迹以及与互联网上可用的膀胱TCC蛋白质数据库（http://biobase.dk/cgi-bin/celis）进行比较等蛋白质组学技术的组合来鉴定蛋白质。新鲜肿瘤及其原代培养物的IEF 2-D凝胶蛋白质谱比较表明，尽管由于短期培养，在至少85%的肿瘤/培养物对中，几种蛋白质的合成速率受到差异调节，导致其水平有显著差异，但总体表达谱惊人地相似。大多数受培养影响的蛋白质被上调，其中我们鉴定出细胞骨架成分（角蛋白18、凝溶胶蛋白和原肌球蛋白3）、一种分子伴侣（hsp 28）、醛糖还原酶、GST pi、转移抑制蛋白、突触核蛋白、钙网蛋白前体以及三种身份不明的多肽。只有四种主要蛋白质被下调，其中包括两种被认为在生长控制中起作用的脂肪酸结合蛋白（FABP：FABP5和A-FABP）、与分化相关的角蛋白20以及钙结合蛋白膜联蛋白V。仅在一些培养肿瘤中差异调节的蛋白质包括α-烯醇化酶、磷酸丙糖异构酶、14-3-3家族成员、hnRNPs F和H、PGDH、hsp（热休克蛋白）60、BIP、白细胞介素-1受体拮抗剂、核仁蛋白B23以及几种身份不明的蛋白质。讨论了体外膀胱肿瘤培养模型用于研究诸如恶性肿瘤和侵袭等复杂生物学现象的适用性。
