DNA fragmentation, caspase-3–like activity, and caspase-9–like activity in sensitive and resistant cell lines treated with CdA.

All cells were treated in the absence or presence of 100 nM or 1 μM CdA. (A) DNA fragmentation. Cells were incubated in the presence or absence of drug for 48 hours. Subsequent propidium iodide staining and FACS analysis (as described in “Materials and methods”) was conducted to quantitate the number of cells with subdiploid amounts of DNA. This subdiploid population was indicative of cells with fragmented DNA and is depicted graphically. Open bars depict cells treated with diluent alone, gray bars represent cells treated with 100 nM CdA, and black bars show cells treated with 1 μM CdA. (B) Caspase-3–like activity. Cells were incubated in the presence or absence of CdA for 12 hours. DEVDase activity was measured using a commercially available labeled peptide, DEVD-AMC. Cleavage of this fluorescently labeled peptide and consequent release of free AMC, which was measurable with the use of a spectrofluorimeter, indicated the presence of caspase-3–like proteases in cell extracts. Color designation of bars is identical to that for panel A. (C) Caspase-9–like activity. Cells were incubated in the presence or absence of CdA for 12 hours. LEHDase activity was quantitated using the caspase-9 substrate, LEHD-AMC. Cleavage of this fluorescently labeled peptide and consequent release of free AMC, which was measurable with the use of a spectrofluorimeter, indicated the presence of caspase-9–like proteases in cell extracts. Results shown for panels A-C are representative of 4 separate experiments.