Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cells

Abstract

Self-renewing tissues such as the intestine contain progenitor proliferating cells which subsequently differentiate. Cell proliferation and differentiation involve gene regulation processes which take place in the nucleus. A human intestinal epithelial cell line model (Caco2/TC7) which reproduces these dynamic processes has been used to perform proteomic studies on nuclear proteins. Nuclei from Caco2/TC7 cells at proliferative and differentiated stages were purified by subcellular fractionation. After two-dimensional gel electrophoresis separation and ruthenium staining, 400 protein spots were detected by image analysis. Eighty-five spots corresponding to 60 different proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry in nuclei from proliferative cells. Comparison of nuclear proteomes from proliferative or differentiated cells by differential display resulted in the identification of differentially expressed proteins such as nucleolin, hnRNP A2/B1 and hnRNP A1. By using Western blot analysis, we found that the expression and number of specific isoforms of these nuclear proteins decreased in differentiated cells. Immunocytochemistry experiments also showed that in proliferative cells nucleolin was distributed in nucleoli-like bodies. In contrast, hnRNPs A2/B1 and A1 were dispersed throughout the nucleus. This study of the nuclear proteome from intestinal epithelial cells represents the first step towards the establishment of a protein database which will be a valuable resource in future studies on the differential expression of nuclear proteins in response to physiological, pharmacological and pathological modulations.

Proteomic Analysis Reveals Hyperactivation of the Mammalian Target of Rapamycin Pathway in Neurofibromatosis 1–Associated Human and Mouse Brain Tumors

“…However, fibrillarin, a nucleolar protein responsible for early processing of 47S rRNA molecules, was unchanged in Nf1À/À astrocytes, suggesting that increases in nucleolar ribosome components was selective following loss of neurofibromin expression. Consistent with this notion, both NPM and nucleolin are positive regulators of cell growth, whereas other nucleolar ribosome components, such as fibrillarin and L11, either exhibit little influence on cell cycle progression or act as putative tumor suppressors (25)(26)(27). Notably, NPM is a potent oncogene and its continued expression is required for efficient ribosome export and cell cycle progression (28), making it an attractive downstream target for increased proliferation in Nf1À/À astrocytes.…”

Gene expression analysis of a human enterocyte cell line reveals downregulation of cholesterol biosynthesis in response to short‐chain fatty acids

“…Caco2/TC-7, which is a clonal cell population isolated from late passages of the Caco2 cell line, exhibits more rapid growth and a higher differentiation potential (28).…”

Characteristic Changes in Cell Surface Glycosylation Accompany Intestinal Epithelial Cell (IEC) Differentiation: High Mannose Structures Dominate the Cell Surface Glycome of Undifferentiated Enterocytes

“…A characteristic feature of Caco-2 cells is their spontaneous enterocyte-like differentiation in culture after cells reach confluence (21). Although proliferation and differentiation of Caco-2 has been studied extensively, including quantitative proteomic and transcriptomic analyses (22)(23)(24)(25)(26), the associated changes in glycosylation that accompany Caco-2 cell differentiation have yet to be comprehensively characterized. More specifically, there is only modest knowledge about its cell surface glycome, despite the importance of the plasma membrane in many key biological functions.…”