Dual response of human leukemia U937 cells to hypertonic shrinkage: initial regulatory volume increase (RVI) and delayed apoptotic volume decrease (AVD).

BACKGROUND/AIMS: Osmotic cell shrinkage is a powerful trigger of suicidal cell death or apoptosis, which is paralleled and enforced by apoptotic volume decrease (AVD). Cells counteract cell shrinkage by volume regulatory increase (RVI). The present study explored the response of human U937 cells to hypertonic solution thus elucidating the relationship between RVI and AVD.

METHODS

Cell water, concentration of monovalent ions and the appearance of apoptotic markers were followed for 0.5-4 h after the cells were transferred to a hypertonic medium. Intracellular water, K+, Na+, and Cl- content, ouabain-sensitive and -resistant Rb+ influxes were determined by measurement of the cell buoyant density in Percoll density gradient, flame emission analysis and 36Cl- assay, respectively. Fluorescent microscopy of live cells stained by acridine orange and ethidium bromide was used to verify apoptosis.

RESULTS

After 2-4 h incubation in hypertonic media the cell population was split into light (L) and heavy (H) fractions. According to microscopy and analysis of monovalent ions the majority of cells in the L population were healthy, while the H fractions were enriched with apoptotic cells. The density of L cells was decreasing with time, while the density of H cells was increasing, thus reflecting the opposite effects of RVI and AVD. At the same time, some of the cells were shifting from L to H fractions, indicating that apoptosis was gradually extending to cells that were previously displaying normal RVI.

CONCLUSION

The findings suggest that apoptosis can develop in cells capable of RVI.