Figure 6.

Bacterially expressed CAD is capable of inducing apoptotic morphology in added nuclei in the absence of apoptotic extract. (A) Diagram of the experimental protocol. After CAD was activated during a preincubation with purified caspase-3 at 25°C, substrates were added and incubated for various times with the active enzyme at 37°C. (B) Effects of various incubations on CAD nuclease activity using either a purified plasmid substrate (pBluescript, upper panels) or added nuclei (lower panels). The morphology of the nuclei in these same incubations is shown in C, where the panel numbers refer to lanes in B. Buffer and caspase-3 do not possess nuclease activity or induce apoptotic morphology in added nuclei (lanes and panels 2 and 3). Preincubation of CAD alone induces nuclease activity and apoptotic morphology (lane and panel 4), presumably because of the presence of a contaminating protease. ICAD is cleaved during this preincubation (data not shown). This spontaneous activation of CAD is not observed if noncleavable ICAD is present from the start of the preincubation (lane and panel 5). Preincubation of CAD with caspase-3 induces nuclease activity and apoptotic morphology (lane and panel 6). This activity is not observed if noncleavable ICAD is present either from the start of the preincubation, or if it is added at the end of the preincubation together with the substrate nuclei (lanes and panels 7 and 8). Addition of DEVD-fmk to the preincubation blocks CAD activation but has no effect on nuclease activity or induction of apoptotic morphology if added once the preincubation is complete (lanes and panels 9 and 10). The induction of apoptotic morphology by CAD in the presence of DEVD-fmk was slightly, but reproducibly, reduced (panel 10). These results demonstrate that CAD can induce nuclear apoptosis in the absence of caspase-3 activity. I and II at the right of B indicate the migration of form I (supercoiled) and form II (nicked circular) plasmid DNA.