ABSENCE OF VOLUME REGULATORY MECHANISMS CONTRIBUTES TO THE RAPID ACTIVATION OF APOPTOSIS IN THYMOCYTES

A common event that occurs during apoptosis is a loss of cell volume, but little information is available on its role in the cell death proc ess. Lymphocytes undergo apoptosis in response to glucocorticoids and exhibit cell shrinkage, nuclear condensation, internucleosomal DNA fra gmentation, and apoptotic body formation. Interestingly, only cells th at exhibit a loss in cell volume degrade their DNA. To determine if ph ysical shrinkage was sufficient to initiate apoptosis, S49 Neo lymphoc ytes were cultured in hypertonic medium. The normal osmolarity (simila r to 300 mosM) of tissue culture medium was increased to either 550 or 800 mosM, using impermeant sugars such as mannitol and sucrose or NaC l. These hypertonic conditions led to a rapid killing of S49 Neo cells . Evaluation of the mode of cell death revealed that these hypertonic conditions resulted in apoptosis. Unlike glucocorticoid-induced cell d eath, hypertonically induced apoptosis did not require protein synthes is. When S49 Neo cells were cultured under hypotonic conditions, the c ells swelled but apoptosis did not occur. Analysis of several cell typ es revealed that all lymphoid cells examined (S49 Neo, CEM-C7, primary thymocytes) undergo apoptosis in response to hypertonic conditions, w hereas several other cell types (L cells, COS, HeLa, GH(3)) did not. A lthough these nonlymphoid cells showed a similar initial reduction in cell volume in response to hypertonic conditions, they subsequently ma intained volume or regulated back to a near normal cell volume. These data indicate that thymic lymphoid cells have the machinery in place f or rapid induction of apoptosis in response to physical shrinkage, whe reas other cell types resist shrinkage-induced apoptosis by the activa tion of cell volume regulatory mechanisms.