Cells Expressing the C/EBPbeta Isoform, LIP, Engulf Their Neighbors

<div><p>Descriptions of various processes that lead to cell-in-cell structures have been reported for decades. The exact molecular mechanism(s) of their formation and the physiological significance of cell-in-cell structures remain poorly understood. We had previously shown that an isoform of the CCAAT/enhancer-binding protein beta (C/EBPbeta) transcription factor, liver-enriched inhibitory protein (LIP), induces cell death in human breast cancer cells and stimulates autophagy. Here we describe a non-apoptotic cell death process where LIP mediates the engulfment of neighboring cells. We provide evidence of LIP-mediated engulfment via DNA profiling, fluorescent imaging and cell sorting studies, as well as ultrastructure analysis of LIP-expressing MDA-MB-468 breast cancer cells. Our work illustrates that expression of a specific transcription factor, LIP, can mediate cell engulfment.</p> </div

DNA content of LIP-expressing MDA-MB-468 cells.

<p>A, Representative cell cycle profiles of control uninfected: no virus (NV) (panel a), Ad-GFP (panel b), and Ad-LIP (panel c) MDA-MB-468 cells are shown. B, Quantification of the percent of cells with greater than 4n DNA content as measured by the area marked as R2 in the graphs is presented. Data shown represents the mean + SD of three separate experiments. *p-value <0.05 as determined by ANOVA followed by Friedman test.</p

LIP-mediated cell engulfment is not dependent on adenoviral infection.

<p>MDA-MB-468 cells cotransfected with LIP and GFP expression vectors were stained with anti-beta-catenin antibody (red) and Hoechst nuclear stain (blue). GFP positive cells in panels a–f are LIP expressing cells. Panels a–f are fluorescent micrographs of separate, randomly chosen fields; cells were imaged 4–6 days post transfection.</p

Ultrastructure analysis of LIP-mediated cell engulfment in MDA-MB-468 cells.

<p>Panels a–c, ultrastructure of engulfing intermediates at 48 hrs post infection; scale bar is 2µm. 1 and 2 indicate higher magnification of cell contacts present in panel c. Arrows indicate formation of cell contacts. Scale bar is 100 nm and 500 nm for panel 1 and 2, respectively. Panels d and e show LIP-expressing cells that have enormous vacuoles; scale bar is 2 µm. Panels f–h illustrate an internalized cell at 72 hrs post infection; scale bar is 2 µm. Panel g is a higher magnification to show the nucleus of engulfing (host) cell and the various projections typical of actively phagocytic cells. Panel h depicts the engulfed cell inside of the vacuole of the host cell.</p

Quantification of LIP-mediated cell engulfment.

<p>A, Panel a shows the percent of Ad-LIP MDA-MB-468 cells that are double positive for GFP and CellTracker label. Panel b shows the percent of control Ad-GFP MDA-MB-468 cells that are double positive for GFP and CellTracker label. B, Quantification of the percent of GFP positive cells that have engulfed uninfected CellTracker labeled cells. Results are shown for 16 different experiments with a p-value of <0.0001 using paired t-test and Wilcoxon matched pairs test for statistical analysis. C, Quantification of the percent of GFP positive cells that have engulfed uninfected CellTracker labeled cells after treatment with 40 µM of the ROCK inhibitor, Y-27632. Results are shown for 5 different experiments with a p-value of <0.0001 using ANOVA followed by the Student-Newman-Keuls multiple comparisons test.</p

LIP-expressing MDA-MB-468 cells engulf neighboring cells.

<p>GFP positive LIP-expressing MDA-MB-468 cells were mixed with uninfected CellTracker orange labeled MDA-MB-468 cells. Hoechst dye was used to stain DNA. Cells were imaged at 36–48 hrs post-mix.</p

Cell disintegration following exogenous expression of LIP.

<p>Microscopic examination of cell breakdown. Equal numbers of MDA-MB-468 cells were plated for adenoviral infection with either control Ad-GFP or Ad-LIP. Representative fluorescent photomicrographs of cells imaged at 72 hrs post infection are shown. Panel a represents Ad-GFP MDA-MB-468 cells. Panels b-i represent Ad-LIP MDA-MB-468 cells.</p