CEACAM1-4S, a cell-cell adhesion molecule, mediates apoptosis and reverts mammary carcinoma cells to a normal morphogenic phenotype in a 3D culture - PubMed (original) (raw)

CEACAM1-4S, a cell-cell adhesion molecule, mediates apoptosis and reverts mammary carcinoma cells to a normal morphogenic phenotype in a 3D culture

Julia Kirshner et al. Proc Natl Acad Sci U S A. 2003.

Abstract

In a 3D model of breast morphogenesis, CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) plays an essential role in lumen formation in a subline of the nonmalignant human breast cell line (MCF10A). We show that mammary carcinoma cells (MCF7), which do not express CEACAM1 or form lumena when grown in Matrigel, are restored to a normal morphogenic program when transfected with CEACAM1-4S, the short cytoplasmic isoform of CEACAM1 that predominates in breast epithelia. During the time course of lumen formation, CEACAM1-4S was found initially between the cells, and in mature acini, it was found exclusively in an apical location, identical to its expression pattern in normal breast. Lumena were formed by apoptosis as opposed to necrosis of the central cells within the alveolar structures, and apoptotic cells within the lumena expressed CEACAM1-4S. Dying cells exhibited classical hallmarks of apoptosis, including nuclear condensation, membrane blebbing, caspase activation, and DNA laddering. Apoptosis was mediated by Bax translocation to the mitochondria and release of cytochrome c into the cytoplasm, and was partially inhibited by culturing cells with caspase inhibitors. The dynamic changes in CEACAM1 expression during morphogenesis, together with studies implicating extracellular matrix and integrin signaling, suggest that a morphogenic program integrates cell-cell and cell-extracellular matrix signaling to produce the lumena in mammary glands. This report reveals a function of CEACAM1-4S relevant to cellular physiology that distinguishes it from its related long cytoplasmic domain isoform.

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Figures

Figure 1

Figure 1

Time course of apoptosis and lumen formation in normal and carcinoma cells. MCF10F and MCF7/CEACAM1-4S cells (2.5 × 105 per well) were grown in Matrigel for the indicated number of days, fixed, embedded in paraffin, and stained with caspase-cleaved cytokeratin 18 detecting mAb (M30) and anti-CEACAM1 mAb (4D1C2). For annexin V expression, cells were grown in Matrigel for 8 days, stained with annexin V-FITC, and visualized by confocal microscopy. For MCF7/CEACAM1-4S cells, the annexin V-FITC staining image was superimposed on the transmission image for easier visualization. (Magnification, ×600.)

Figure 2

Figure 2

CEACAM1-4S mediated reversion of MCF7 cells to normal phenotype. (A) Cell lysate preparations (60 μg of total protein) from cells grown on plastic were separated by 8% reducing SDS/PAGE gel and immunoblotted with anti-CEACAM1 mAb (4D1C2). Lane 1, positive control HT29 colon carcinoma cells; lane 2, parental MCF7 cells; lane 3, MCF7/pHβ (vector); lane 4, MCF7/CEACAM1-4S; lane 5, MCF7/CEACAM1-4L. (B_–_E) Cells (2.5 × 105 per well) were grown in Matrigel for 12 days, fixed, and embedded in paraffin, and sections were stained with anti-CEACAM1 mAb (4D1C2). (B) MCF7 cells exhibiting no lumen formation. (C) Vector-transfected MCF7 cells exhibiting no lumen formation. (D) Nonmalignant mammary epithelial cells MCF10F exhibiting lumen formation. (E) MCF7/CEACAM1-4S-transfected cells exhibiting lumen formation. (Magnification, ×600.)

Figure 3

Figure 3

Morphologic analysis of apoptotic cells during lumen formation. MCF7/CEACAM1-4S cells (2.5 × 105 per well) were grown in Matrigel for 5 (A and B) or 12 (C) days, and transmission electron microscopy was performed. (A) An acinus undergoing apoptosis of the central cell (box). (Magnification, ×5,500.) (B) Nuclear condensation occurring during apoptosis of the central cell seen in A. (Magnification, ×7,375.) (C) An apoptotic body in the lumen of the acinus. (Magnification, ×16,525.) Arrows point to the vacuoles seen in the cytoplasm of the apoptotic cell.

Figure 4

Figure 4

Mitochondrial potential and CEACAM1-4S expression in apoptotic cells. Cells (2.5 × 105 per well) were grown in Matrigel for the indicated number of days, stained with JC-1 dye, and visualized by confocal microscopy. (A) MCF7/pHβ (vector) cells stained with JC-1 dye. (B) MCF7/CEACAM1-4S cells stained with JC-1 dye. (C) MCF7/CEACAM1-4S-ectoGFP cells. (D_–_F) MCF7/CEACAM1-4S-ectoGFP cells stained with JC-1 dye. (Magnification, ×600.)

Figure 5

Figure 5

Bax expression and cytochrome c release during lumen formation. (A) MCF7/pHβ (vector) or MCF7/CEACAM1-4S cells (2.5 × 105 per well) were grown in Matrigel for 5 days, lysed, and fractionated into mitochondrial (M) and cytosolic (C) fractions. Total lysates (T) and the fractions of vector and CEACAM1-4S-transfected MCF7 cells were separated on an SDS/PAGE gel and immunoblotted with anti-Bax antibody. (B and C) Cells (2.5 × 105 per well) were grown in Matrigel for 12 days, fixed, and embedded in paraffin, and sections were stained with anti-cytochrome c antibody. (B) MCF7/pHβ (vector). (C) MCF7/CEACAM1-4S. (Magnification, ×600.)

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