Effect of overexpression of gelsolin on apoptosis of Jurkat T cells in response to ceramide treatment or to Fas ligation.

Overexpression of gelsolin did not prevent apoptosis of Jurkat T cells in response to ceramide treatment or to Fas ligation, although gelsolin was cleaved during apoptosis. (A) Both endogenous and overexpressed gelsolin bound actin. Human gelsolin was immunoprecipitated from lysates of transfected Jurkat cells (2 × 10⁷cells/sample). Precipitated proteins were separated by 10% SDS-PAGE on a 10% gel, transferred to PVDF membrane, and immunoblotted for gelsolin and actin as described. These results are representative of 2 independent experiments. (B) Transfected Jurkat T cells (solid bars represent those that overexpress gelsolin, hatched bars represent the vector control) were incubated with 50 μmol/L ceramide (closed or gray bar) or its vehicle, 0.025% Me₂SO (open bars), for 12 hours. The percentage of apoptosis was determined by trypan blue exclusion. Level of expression of gelsolin during this experiment was determined by immunoblot (inset). (C) Transfected Jurkat cells (solid bars represent those that overexpress gelsolin, hatched bars represent the vector control) were incubated for 4 hours with anti-Fas mAb (1:500 dilution of 7C11) (closed or gray bars) or with medium alone (open bars), fixed, and stained with propidium iodide for quantification of apoptosis by nuclear morphology. Immunoblot confirmed the overexpression of gelsolin at the time of the experiment (inset). Results are representative of 4 independent experiments.