Figure 2.
Apoptosis induced by overexpression of c-Kit. U87 cells (5 × 105 cells) were transfected with 2.5 μg or the indicated amount of pcKit (○) or the pcDNA3 vector (•) by lipofection with N-[1-(2,3-dioleoyl)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP; Boehringer Mannheim). The transfection efficiency was 80–90% as determined by transfection with pCMV-βgal. (a) Cell survival was determined by counting the number of cells that excluded trypan blue. (b) The percentage of apoptotic cells was determined by analysis of nuclear morphology after staining with propidium iodide. (c and d) Analysis of DNA fragmentation on agarose gels. Soluble cytoplasmic DNA was isolated from cells transfected with pcKit at the indicated time points (c) or 48 h (d) after transfection. Apoptosis induced by staurosporine (0.1 mM for 16 h) gave the same pattern of DNA fragmentation as that shown in c and d, lanes 4–6 (data not shown). (e) Hoechst 33342 staining demonstrates apoptotic nuclei (arrows) in cells transfected with pcKit but not the control vector pCDNA3. (f) Apoptotic nuclei in c-Kit-positive cells (solid arrows) demonstrated by Hoechst 33342 staining. A normal nucleus is indicated by the open arrow. For e and f, cells were fixed at 48 h after transfection. Data in a and b are the means ± SD for duplicate determinations. Results shown are representative of at least two experiments.