de Albuquerque Garcia Redondo Patricia, Nakamura Celso Vataru, de Souza Wanderley, Morgado-Díaz José Andrés
Divisão de Biologia Celular, Grupo de Biologia Estrutural, Centro de Pesquisa, Instituto Nacional de Câncer, Rio de Janeiro, Brasil.
J Histochem Cytochem. 2004 May;52(5):629-40. doi: 10.1177/002215540405200507.
In this study we investigated the levels of expression of sialic acid and N-acetylgalactosamine residues on the cell surface of a normal intestinal epithelium cell line, IEC-6, and in two colon adenocarcinoma cell lines with different metastatic potential, Caco-2 and HCT-116. Glycoprotein expression was estimated initially by cytochemistry with WGA and HPA lectins and biochemistry with isolated plasma membrane fractions of the cells. Fluorescence and electron microscopic analyses revealed differences in the expression profile of carbohydrates recognized by the lectins used on the cell surface of IEC-6, Caco-2, and HCT-116 cells. Lectin blotting identified a range of eight HPA-binding glycoprotein bands with molecular weights of 16-66 kD in Caco-2 cells, six glycoproteins of 16-36 kD, and three protein bands of 35, 24, and 21 kD in IEC-6 cells. A minor band of 66 kD and a major one of 50 kD for WGA-binding glycoproteins were observed in IEC-6 cells and seven glycoproteins of 18-97 kD in Caco-2 and HCT-116 cells but with a visible higher expression of these glycoproteins in the latter. Furthermore, significant quantitative difference in levels of expression of WGA- but not of HPA-binding glycoconjugates was noted, as analyzed by high-resolution scanning electron microscopy using backscattered electron images of cells incubated with gold-labeled lectins.
在本研究中,我们调查了正常肠上皮细胞系IEC-6以及两种具有不同转移潜能的结肠腺癌细胞系Caco-2和HCT-116细胞表面唾液酸和N-乙酰半乳糖胺残基的表达水平。最初通过用WGA和HPA凝集素进行细胞化学以及用细胞分离的质膜组分进行生物化学分析来评估糖蛋白表达。荧光和电子显微镜分析揭示了IEC-6、Caco-2和HCT-116细胞表面上所用凝集素识别的碳水化合物表达谱的差异。凝集素印迹法在Caco-2细胞中鉴定出一系列分子量为16 - 66 kD的八条HPA结合糖蛋白带,在IEC-6细胞中鉴定出六条分子量为16 - 36 kD的糖蛋白带以及三条分子量为35、24和21 kD的蛋白带。在IEC-6细胞中观察到一条66 kD的次要WGA结合糖蛋白带和一条50 kD的主要带,在Caco-2和HCT-116细胞中观察到七条分子量为18 - 97 kD的糖蛋白带,但后者中这些糖蛋白的表达明显更高。此外,通过使用与金标记凝集素孵育的细胞的背散射电子图像的高分辨率扫描电子显微镜分析,发现WGA结合糖缀合物而非HPA结合糖缀合物的表达水平存在显著的定量差异。