Normotonic cell shrinkage induces apoptosis under extracellular low Cl − conditions in human lymphoid and epithelial cells

Aim:  Sustained cell shrinkage is associated with apoptosis. Apoptotic volume decrease, which is known to be induced by release of osmolytes including Cl − ions, may be an essential event for apoptosis induction. Provided any anion channels and/or anion transporters are basally functioning, there is a possibility that imposition of a driving force for Cl − efflux per se results in sustained cell shrinkage and thereby induces apoptotic death. Here, this possibility was tested by reducing the extracellular Cl − concentration. Methods:  Human lymphoid U937 and epithelial HeLa cells were provided for experiments after exposing to isotonic electrolyte solution which contains 146 or 1 m m Cl − . Measurements of mean cell volume, caspase‐3 activity and cell viability were performed by a Coulter‐type cell size analyzer, a fluorometric assay and a colorimetric assay, respectively. Results:  After exposure to low Cl − solution in which most chloride was replaced with aspartate, gluconate, phosphate or methanesulphonate, both U937 and HeLa cells exhibited, for up to 60 min, shrinkage to a level (90–80%) significantly lower than that in control high Cl − solution. Reduction in cell viability started within 2 h and reached below 20% within 8 h after exposure to low Cl − solution. The cell death was found to be associated with caspase‐3 activation and DNA fragmentation. Conclusions:  Exposure to isotonic low Cl − solution induced sustained shrinkage and thereafter apoptotic death in U937 and HeLa cells. Thus, it is suggested that sustained cell shrinkage per se provides a sufficient condition for apoptosis induction.