Selective inhibition of topoisomerase II by ICRF-193 does not support a role for topoisomerase II activity in the fragmentation of chromatin during apoptosis of human leukemia cells

Specific inhibitors of topoisomerase II (e.g., ICRF-193, an inhibitor of the catalytic activity of topoisomerase II and etoposide that stabilizes enzyme/DNA cleavable complexes) have been used to probe the role of topoisomerase II in the fragmentation of DNA during drug-induced apoptosis of human HL-60 leukemia cells. Topoisomerase II plays a role in the attachment of 50-kilobase domains of DNA to the nuclear matrix; fragments of this size are cleaved during apoptosis. Apoptosis was induced by 50 mu M etoposide or 300 mM N-methylformamide (NMF), a nongenotoxic agent. Treatment with etoposide or NMF induced the morphology of apoptosis within 4 hr. Analysis of DNA integrity by electrophoresis showed coincident fragmentation from 50 kb and to integers of 200 bp. Transient protein-associated DNA strand breaks, characteristic of etoposide-induced damage, were visualized as DNA fragments of >600 kb, Preincubation with ICRF-193 (100 mu M) reduced the number of etoposide-induced DNA strand breaks by 50% and delayed the appearance of DNA fragmentation by similar to 18 hr. However, ICRF-193 had no effect on either NMF- or camptothecin-induced DNA fragmentation. The induction of apoptosis by both etoposide and NMF was associated with a reduction in the cellular levels of topoisomerases II alpha and II beta. ICRF-193 inhibited proteolytic cleavage of topoisomerase II induced by etoposide but not by NMF. The data suggest that the activity of topoisomerase II is not required for the cleavage of DNA to 50-kb fragments but that proteolysis of topoisomerase II represents a conserved event of apoptosis.