Subramaniam Venkateswaran, Vincent Isabella R, Jothy Serge
Department of Laboratory Medicine, St. Michael's Hospital, 30 Bond Street, Toronto, ON, Canada.
Exp Mol Pathol. 2005 Dec;79(3):187-93. doi: 10.1016/j.yexmp.2005.08.004. Epub 2005 Oct 14.
Colon cancer is among the leading causes of cancer death in North America. Dysregulation of the crypt homeostasis is evident in the early stage of colon cancer. Moreover, cytoskeletal rearrangement of actin also plays a crucial role in morphological changes during apoptosis. CD44, an adhesion and anti-apoptotic molecule is overexpressed in colon cancer and is known to interact with certain cytoskeletal proteins. In this study, we used the human colon cancer cell line SW620, which does not express CD44 but stably transfected with standard, 3-10v and 8-10v variant isoforms of CD44. By two-dimensional isoelectric focusing (2DIEF), we found an increasing concentration of a 21-kDa protein in SW620 colon cancer cells transfected with CD44 3-10v, as compared to cells transfected with an empty vector. Mass spectrometry (MS) and proteomic analysis of this protein identified the peptide fragment (YALYDATYETK) of 11 amino acids in length spanning residue 82 to 92 of cofilin, a widely distributed 21-kDa actin-modulating protein. Western blot analysis of lysates from cells expressing CD44 variant isoforms 3-10v had increased level of expression of cofilin compared to vector control consistent with our finding by 2DIEF. Also, immunocytochemistry showed that cofilin expression in colonic epithelial cells was greater in cells transfected with CD44 3-10v, as compared to vector controls. We observed that the phosphorylated form of cofilin is downregulated in cells expressing the 3-10v isoform of CD44 both by Western blot and immunocytochemistry. Cofilin expression is thus mechanistically associated with CD44 expression and its 3-10v isoform. Dephosphorylation of cofilin could bring about directional motility of cells that could have important implications to the proliferation and motility of colonic epithelial cells in cancer.
结肠癌是北美癌症死亡的主要原因之一。隐窝稳态失调在结肠癌早期很明显。此外，肌动蛋白的细胞骨架重排在细胞凋亡过程中的形态变化中也起着关键作用。CD44是一种黏附分子和抗凋亡分子，在结肠癌中过度表达，并且已知与某些细胞骨架蛋白相互作用。在本研究中，我们使用了人结肠癌细胞系SW620，该细胞系不表达CD44，但稳定转染了CD44的标准、3-10v和8-10v变异亚型。通过二维等电聚焦（2DIEF），我们发现与转染空载体的细胞相比，转染CD44 3-10v的SW620结肠癌细胞中一种21 kDa蛋白的浓度增加。对该蛋白的质谱（MS）和蛋白质组学分析确定了11个氨基酸长度的肽片段（YALYDATYETK），其跨越广泛分布的21 kDa肌动蛋白调节蛋白丝切蛋白的82至92位残基。与载体对照相比，对表达CD44变异亚型3-10v的细胞裂解物进行的蛋白质印迹分析显示丝切蛋白的表达水平增加，这与我们通过2DIEF得到的结果一致。此外，免疫细胞化学显示，与载体对照相比，转染CD44 3-10v的细胞中结肠上皮细胞中的丝切蛋白表达更高。我们通过蛋白质印迹和免疫细胞化学观察到，在表达CD44 3-10v亚型的细胞中，丝切蛋白的磷酸化形式下调。因此，丝切蛋白的表达在机制上与CD44表达及其3-10v亚型相关。丝切蛋白的去磷酸化可能导致细胞的定向运动，这可能对结肠癌细胞中结肠上皮细胞的增殖和运动具有重要意义。
