Cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway were prepared to analyze changes in nuclear morphology during apoptotic execution. When isolated interphase nuclei were exposed to S/M extracts prepared from morphologically normal cells in the "committed stage" stage of the apoptotic pathway, they became condensed and the DNA was cleaved. These changes were dependent on caspase activity. In contrast, when the same nuclei were exposed to E/X extracts, prepared from morphologically apoptotic cells, they underwent apoptotic changes in a caspase-independent manner. These results suggest that nuclear disassembly is driven largely by factors activated downstream of the caspases at the onset of apoptotic execution. One such factor, the caspase-activated DNase, CAD/CPAN/DFF40, can induce apoptotic chromatin condensation in isolated HeLa cell nuclei in the absence of other cytosolic factors. However, as morphological changes occur even when CAD activity is inhibited, CAD cannot be the sole factor triggered by caspases. Indeed, in this study it was found that DNA topoisomerase IIa (topoIIa), which is essential both for chromosome condensation and segregation in mitosis, also functions during apoptotic execution. Simultaneous inhibition of topo IIa plus either caspases or CAD completely abolished apoptotic chromatin condensation. Furthermore, in vitro experiments demonstrated that CAD binds to topo IIa. This study presented the first evidence that activities such as CAD and topo II that function downstream of caspases in apoptosis are largely responsible for nuclear disassembly. Furthermore, it is demonstrated that CAD and topo II which are located in the nucleus in healthy cells, have interactions but work in parallel during chromatin condensation.