Adaphostin and STI571 induce apoptotic biochemical changes at different rates.

(A) Identification of caspase-9 as the initiating caspase for adaphostin and STI571. K562 cells were transiently transfected with plasmids encoding EGFP, dominant-negative procaspase-9 fused to EGFP, or a 5:1 ratio of plasmids encoding crmA and EGFP as described in “Materials and methods.” After 24-hour incubation, cells expressing EGFP were isolated by FACS, suspended in medium A, treated for 48 hours with 2.5 μM adaphostin or 625 nM STI571, fixed, and examined for apoptotic morphologic changes. As a control, Jurkat cells transfected with EGFP or a 5:1 ratio of crmA and EGFP were isolated by FACS, treated for 24 hours with 20 ng/mL CH-11 agonistic anti-Fas antibody, fixed, and examined for apoptotic morphologic changes. (B-K) Changes in polypeptide levels during adaphostin- or STI571-induced apoptosis. Cells were treated with 10 μM adaphostin (left) or 20 μM STI571 (right) for the indicated length of time. Lysates containing 50 μg total cellular protein were subjected to SDS-PAGE followed by blotting with reagents that recognize XIAP (B), Bcl-x_L (C), Mcl-1 (D), Bax (E), procaspase-9 (F), procaspase-3 (G), cleaved caspase-3 (H), poly(ADP-ribose) polymerase (I, PARP), lamins A and C (J) or, as a loading control, FADD (K). In each panel, lanes 1 to 17 correspond to nonadjacent lanes from a single blot. The same blots used in Figure 3were probed in this figure. Arrows, cleaved fragments corresponding to previously described43 caspase cleavage products. Percentages of cells that were apoptotic at each time point of this experiment are indicated in Figure 1B. Results shown are representative of at least 3 independent experiments.