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. 2003 Jan;162(1):69-80.
doi: 10.1016/S0002-9440(10)63799-6.

Fascin, an Actin-Bundling Protein, Modulates Colonic Epithelial Cell Invasiveness and Differentiation in Vitro

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Fascin, an Actin-Bundling Protein, Modulates Colonic Epithelial Cell Invasiveness and Differentiation in Vitro

Aida U Jawhari et al. Am J Pathol. .
Free PMC article

Abstract

In epithelial tissue, cell-matrix and cell-cell adhesive interactions have important roles in the normal organization and stabilization of the cell layer. The malignant conversion of epithelial cells involves alterations in the expression and function of these adhesion systems that enable a switch to a migratory phenotype in tumor invasion and metastasis. Fascin is an actin-crosslinking protein that is found in the core actin bundles of cell-surface spikes and projections that are implicated in cell motility. We demonstrate that fascin is not detectable in normal colonic epithelium, but is dramatically up-regulated in colorectal adenocarcinoma. To test the hypothesis that fascin could participate in tumor invasive behavior, we developed a cell culture model to examine the effect of fascin expression on the adhesive interactions, invasiveness, and differentiation of colonic epithelial cells. We report marked effects on the organization of cell-surface protrusions, actin cytoskeleton, and focal adhesions in the absence of alterations in the protein levels of the major components of these structures. These effects correlate with alterations in cell movements on two-dimensional matrix, and increased invasiveness in three-dimensional matrix. The cells also show increased proliferation and decreased capacity for normal glandular differentiation in collagen gels. We propose that up-regulation of fascin, by promoting the formation of protrusive, actin-based, cell-motility structures, could be a significant component in the acquisition of invasive phenotype in colonic carcinoma.

Figures

Figure 1.
Figure 1.
Fascin immunoreactivity in normal colonic mucosa (A) and colonic adenocarcinoma (B). Overexpression of fascin is detected in colonic carcinoma cells.
Figure 2.
Figure 2.
A: Western blot for fascin expression in a panel of gastric, colonic, and pancreatic cell lines. Kato III, HSC39, and SW1222 had low or undetectable fascin. B: Western blot of fascin expression in SW1222 transfectant clonal lines. Clones C9 and C11 were chosen for further examination.
Figure 3.
Figure 3.
Analysis of Ki67-expressing cells in three-dimensional colonies. Sections were stained with MIB I antibody to Ki67: VC1 (A), C9 (B), and C11 (C). Scale bar, 100 μm.
Figure 4.
Figure 4.
Phase-contrast images of VC1 (A), C9 (B), and C11 (C) cell lines. With reference to VC1, cells in B and C show typical changes in colony morphology including increased spreading of marginal cells (arrows in B) and cellular projections (arrows in C). Scale bar, 100 μm.
Figure 5.
Figure 5.
Analysis of β-catenin cellular localization in laminin-adherent fascin transfectants. VC1 (A, B), C9 (C), C11 (D). Scale bar, 10 μm.
Figure 6.
Figure 6.
Analysis of β1-integrin cellular localization in laminin-adherent fascin transfectants. VC1 (A), C9 (B), C11 (C, D). Small arrows indicate concentration of β1 integrin at cell-cell borders. Scale bar, 10 μm.
Figure 7.
Figure 7.
Analysis of cytoskeletal organization in laminin-adherent fascin transfectants. Small colonies of VC1 (A) and C11 (B) were stained with tetramethyl-rhodamine isothiocyanate-phalloidin. Small arrows in B indicate presence of F-actin at the free edges of adherent cells at the margins of colonies. Scale bar, 10 μm.
Figure 8.
Figure 8.
Analysis of vinculin and fascin cellular localization in laminin adherent fascin transfectants. Small colonies of VC1 (A), C9 (B, E), and C11 (C, D, F) were stained for vinculin (A–D) and fascin (E, F). Arrows in B, C, D, and F indicate the increased assembly of focal contacts by single cells or cells in colonies. Large arrow in F indicates apical cluster of fascin projections. Scale bar, 10 μm.
Figure 9.
Figure 9.
Cell-matrix adhesion and migration on different extracellular matrix proteins of fascin transfectants (VC1, C9, and C11 cell lines). Quantitation of attachment to collagen IV (A) and laminin (B) under serum-free conditions. Values are means ± SE. No significant difference in adhesion was observed. C: Cell migration on different extracellular matrix proteins of fascin transfectants (VC1, C9, and C11 cell lines). Quantitation of migration across collagen IV (C)-coated filters. Values are means ± SE.
Figure 10.
Figure 10.
Organization of cell colonies in three-dimensional collagen gels. VC1 (A, D), C9 (B, E), and C11 (C, F) were grown in collagen gels for 3 weeks, fixed, sectioned, and stained with H&E (A–C) or Alcian Blue/periodic acid-Schiff (D–F) stain to visualize mucins. C9 and C11 showed increased production of both acidic (blue stain, arrows in E) and neutral mucin (magenta stain, arrows in F). Scale bar, 100 μm.
Figure 11.
Figure 11.
Analysis of colony organization by transmission electron microscopy. Ultra-thin sections of C11 cells counterstained with uranyl acetate. A: Section at cell-cell junction. B: Section across glandular lumen (L). AJ, adherens junction; MV, microvilli; MF, actin microfilament. Original magnifications: ×35,625 (A); ×14,375 (B).

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