Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Apoptosis is characterized by the nonrandom cleavage of DNA. After continuous treatment of MOLT-4 human T lymphoblastoid cells with the topoisomerase II inhibitor etoposide (50 microM) and the nongenotoxic agent N-methylformamide (300 mM), apoptosis was confirmed by electron microscopy. Analysis of DNA integrity by conventional gel electrophoresis failed to detect internucleosomal DNA cleavage. Resolution of DNA by field inversion gel electrophoresis showed fragments of 50 kilobases (kb). Etoposide induced the transient appearance of an additional DNA band of > 600 kb, which was temporally coincident with DNA-protein complex formation and was rapidly reversible upon drug removal. This DNA band was not observed after N-methylformamide treatment. In situ DNA end-labeling showed the incorporation of biotinylated dUTP into 50-kb DNA fragments but not etoposide-induced DNA fragments of > 600 kb. DNA end-labelling with terminal deoxynucleotidyltransferase was therefore not dependent upon intenucleosomal DNA cleavage, and fragments of approximately 50 kb were characterized by free 3'-OH termini that were not occluded by topoisomerase II protein. Although we considered that topoisomerase II potentially played an active role in the fragmentation of higher order chromatin during apoptosis, the results showed that DNA cleavage by topoisomerase II induced reversible, protein-associated fragments of > 600 kb and not irreversible cleavage to 50-kb fragments. The reversible cleavage of DNA to fragments of > 600 kb appears to be a signal for the engagement of apoptosis and is not an initial step in the sequential unwinding of chromatin.
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PMID:Investigation of the mechanism of higher order chromatin fragmentation observed in drug-induced apoptosis. 774 85

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 microM 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 microM) reduced the number of etoposide-induced DNA strand breaks by 50% and delayed the appearance of DNA fragmentation by approximately 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.
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PMID: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. 862 34