Stierum Rob, Gaspari Marco, Dommels Yvonne, Ouatas Taoufik, Pluk Helma, Jespersen Sonja, Vogels Jack, Verhoeckx Kitty, Groten John, van Ommen Ben
TNO Nutrition and Food Research, Department of Biomolecular Sciences, P.O. Box 360, 3700 AJ, Zeist, The Netherlands.
Biochim Biophys Acta. 2003 Aug 21;1650(1-2):73-91. doi: 10.1016/s1570-9639(03)00204-8.
Here, we describe a proteomics approach to study protein expression changes in differentiating Caco-2 cells. Caco-2 is a colorectal carcinoma cell line, which upon differentiation loses its tumorigenic phenotype and displays characteristics of mature enterocytes, including brush borders with microvilli. Cells were grown in culture flasks and harvested at different stages of differentiation (days post-confluence: -3, 0, 3, 7, 10, 14, and 18). Two-dimensional gel electrophoresis was used to analyse proteome changes. Approximately 1400 protein spots were detected within the Caco-2 proteome, within the pH 4-7 range. Two-dimensional gel electrophoresis allowed for the detection of 18 proteins from which the levels of expression were found to be associated with differentiation. Of these proteins, 11 were identified by means of MALDI-TOF or NANO-ESI-MS/MS mass spectrometry and include liver fatty acid binding protein (FABL), three forms of alpha-enolase (ENOA), nucleoside diphosphate kinase A (NDKA), cofilin-1 (COF1), translationally controlled tumour protein (TCTP), mitochondrial 60-kDa heat shock protein (CH60), probable protein disulfide isomerase (ER60), creatine kinase B (KCRB), and glutathione S-transferase alpha (GTA1). Thus, proteomics revealed that the differentiation-related change in phenotype of Caco-2 involves changes in a variety of distinct biochemical pathways. Some of these proteins have not been shown before to be associated with Caco-2 differentiation (ER60; COF1; CH60; NDKA; TCTP and ENOA). Therefore, processes related to protein folding and disulfide bridge formation, cytoskeleton formation and maintenance, nucleotide metabolism, glycolysis as well as tumorigenesis-associated proteins may be involved in Caco-2 differentiation. Changes in the expression of CH60, TCTP, GTA1, NDKA, and FABL have also been reported to be associated with in vivo colon carcinogenesis. These findings illustrate that a combination of proteomics and cell culture is a useful approach to find markers for Caco-2 differentiation, which could contribute to the comprehension of the process of colon carcinogenesis.
在此，我们描述了一种蛋白质组学方法，用于研究分化中的Caco-2细胞的蛋白质表达变化。Caco-2是一种结肠癌细胞系，分化后会失去其致瘤表型，并呈现成熟肠上皮细胞的特征，包括带有微绒毛的刷状缘。细胞在培养瓶中生长，并在分化的不同阶段（汇合后天数：-3、0、3、7、10、14和18）收获。二维凝胶电泳用于分析蛋白质组变化。在pH 4-7范围内，在Caco-2蛋白质组中检测到约1400个蛋白质斑点。二维凝胶电泳检测到18种蛋白质，其表达水平与分化相关。在这些蛋白质中，11种通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）或纳升电喷雾串联质谱（NANO-ESI-MS/MS）鉴定，包括肝脂肪酸结合蛋白（FABL）、三种形式的α-烯醇化酶（ENOA）、核苷二磷酸激酶A（NDKA）、丝切蛋白-1（COF1）、翻译控制肿瘤蛋白（TCTP）、线粒体60 kDa热休克蛋白（CH60）、可能的蛋白质二硫键异构酶（ER60）、肌酸激酶B（KCRB）和谷胱甘肽S-转移酶α（GTA1）。因此，蛋白质组学显示Caco-2表型的分化相关变化涉及多种不同生化途径的变化。其中一些蛋白质以前未被证明与Caco-2分化相关（ER60；COF1；CH60；NDKA；TCTP和ENOA）。因此，与蛋白质折叠和二硫键形成、细胞骨架形成和维持、核苷酸代谢、糖酵解以及肿瘤发生相关蛋白的过程可能参与Caco-2分化。据报道，CH60、TCTP、GTA1、NDKA和FABL表达的变化也与体内结肠癌发生相关。这些发现表明，蛋白质组学和细胞培养相结合是寻找Caco-2分化标志物的有用方法，这有助于理解结肠癌发生过程。
