Activation of caspases in vitro and in intact cells. (A) Activation of caspases in vitro. Aliquots containing 50 μg of cytosolic protein from untreated HL-60 or K562 cells were incubated with or without dATP and cytochrome C for the indicated length of time, subjected to SDS-PAGE, transferred to PVDF membrane, and probed with the indicated antibody. Arrow indicates 17-kD large subunit of active caspase-3. (B) Activation of caspases in vivo. HL-60 and K562 cells were treated with 68 μmol/L etoposide for the indicated length of time. Samples containing total cellular polypeptides from 3 × 10⁵ cells were subjected to SDS-PAGE followed by immunoblotting with antibodies to the indicated caspase precursor. The lower panel was derived from a single x-ray film, permitting direct comparison of polypeptide levels. Because procaspase-3 levels were roughly fivefold higher in K562 cells than in HL-60 cells, the exposure of the blot shown in lanes 7 through 12 was adjusted to give a signal comparable to that shown in lanes 1 through 6 of the top panel. (C and D) Detection of peptidase activity in cytosol from etoposide-treated HL-60 or K562 cells. Cytosol was simultaneously prepared from HL-60 (•) or K562 cells (○) treated with 68 μmol/L etoposide for the indicated length of time. Aliquots (50 μg protein) from the same set of extracts were incubated with DEVD-AFC (C) or VEID-AMC (D). The amount of fluorochrome released was determined by comparison to an AFC or AMC standard curve. (E) Detection of DEVD-AFC cleavage activity in nuclei prepared from HL-60 or K562 cells after treatment with 68 μmol/L etoposide for the indicated length of time. (F) Effect of protein synthesis inhibitors on etoposide-induced activation of DEVD-AFC cleavage activity. K562 cells were treated for 24 hours with 70 μmol/L cycloheximide or 100 μmol/L puromycin alone or in combination with 68 μmol/L etoposide. At the completion of the incubation, cytosolic extracts were prepared and assayed for DEVD-AFC cleavage activity. (G) Time course of appearance of cytochrome c in cytosol. Aliquots containing 50 μg of cytosol protein prepared from HL-60 or K562 cells treated with 68 μmol/L etoposide for the indicated length of time were subjected to SDS-PAGE and blotting with anti-cytochrome c. Lanes 10 through 12, 270, 27, and 2.7 ng of cytochrome c, respectively. In contrast to HL-60 cells, where cytochrome c release to cytosol is evident within 1.5 hours (lane 2 and Yang et al36 ), cytochrome c release to cytosol of K562 cells was not evident until 24 hours. Results are representative of three to six independent experiments.