Reduced surface area in apoptotic rounding of human Chang liver cells from serum deprivation.

BACKGROUND

The early stages of apoptosis (programmed cell death) are said to be characterized by internucleosomal DNA fragmentation and "condensation of the cytoplasm" in which cells round up, detach, and increase in density. We studied the causation of apoptotic rounding.

METHODS

Human Chang liver cells in normal monolayer culture were compared with apoptotic counterparts derived from serum growth factor deprivation. Cell-by-cell analysis using the Coulter EPICS PROFILE II flow cytometer studied 1) the cell cycle from propidium iodide-DNA bindings, 2) uptake of neutral red (NR) dye, a viable cell marker, and 3) cytosolic pH (pHi) modulations from 2',7'-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) fluorescence ratios with NH4Cl prepulsing and forward scatter bitmapping of cell surface area. Morphometric studies were done in the Quantimet 570 image analyser. Uptake of trypan blue, neutral red, and 2 million mol.wt fluoresceinated dextrans was studied by light microscopy. Cytological profiles were examined in light microscopy and transmission and scanning electron microscopy.

RESULTS

Three days of serum growth factor deprivation caused confluent flat substrate-attached cells to retract and round up, tethering tenuously to the substrate via thin microvillus attachments only. Ninety percent of cell surface area was lost with this flat-to-round change. There was high trypan blue staining with total loss of proliferative potential, and the entire genome was just fragmented DNA making up the solitary Ao (apoptotic) peak in cell cycle profiles. However, these rounded apoptotic cells also internalized huge 2 million mol.wt dextran particles and impermeant neutral red which is an established viable cell marker. The rounded apoptotic cells had an intensely acidic (pH 5.6) cytosol and therefore a steep [H+]i/[H+]o gradient promoting proton extrusion. The pHi upshifted dynamically upon acidification, recovering and even exceeding resting level by a whole pH unit. Surface area reduction occurred concomitantly in real time with pHi upshifts in these apoptotic cells. Acidification and recovery in apoptotic cells also produced enhanced uptake of neutral red. Cytological profiles showed abundant large endocytic channels and endosomes in the rounded apoptotic cells.

CONCLUSIONS

Gross surface area reduction with evidence of distinctive endocytic activity including uptake of huge 2 million mol.wt dextran particles suggested large channel endocytic internalization as a causal factor in apoptotic rounding, in common with rounding in M-phase and interphase cells with pHi upshifting where concomitant surface area reduction and uptake of impermeant particles were similarly demonstrable. The reduction in size of the cell envelope, together with consequential concentration pressures, could account for the observed rise in cell density and shrinkage in cell size. As a symptom of continual pHi upshifting, apoptotic rounding appears to be a recovery-associated response rather than a direct consequence of the disruptive forces causing its death.