Isolation of intercalator-dependent protein-linked DNA strand cleavage activity from cell nuclei and identification as topoisomerase II

Abstract

DNA intercalating agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) have previously been found to induce in mammalian cells the formation of protein-associated DNA single- and double-strand breaks. In the current work, an activity characterized by the production of DNA-protein links associated with DNA strand breaks and by stimulation by m-AMSA was isolated from L1210 cell nuclei and was shown to be due to topoisomerase II. Nuclei were extracted with 0.35 M NaCl, and the extract was fractionated by gel filtration, DNA-cellulose chromatography, and glycerol gradient centrifugation. A rapid filter binding assay was devised to monitor the fractionation procedure on the basis of DNA-protein linking activity. The active DNA-cellulose fraction contained both topoisomerase I and topoisomerase II whereas the glycerol gradient purified material contained only topoisomerase II activity. The properties of the active material were studied at both stages of purification. m-AMSA enhanced the formation of complexes between purified topoisomerase II and SV40 DNA in which the DNA sustained a single- or double-strand cut and the enzyme was covalently linked to the 5' terminus of the DNA. This action was further enhanced by ATP, as well as by nonhydrolyzable ATP analogues. m-AMSA inhibited the topoisomerization and catenation reactions of topoisomerase II, probably because of trapping of the enzyme-DNA complexes. The activity showed a dependence on the type of DNA intercalators used, analogous to what was previously observed in intact cells. m-AMSA had no effect on topoisomerase I.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered stability of etoposide-induced topoisomerase II-DNA complexes in resistant human leukaemia K562 cells

“…Topoisomerase II also allows for separation of daughter DNA strands during mitosis and is thought to play a role in recombinational events (Wang, 1985). Topoisomerase II is a target for a number of clinically effective antineoplastic agents including m-AMSA, doxorubicin, mitoxantrone, VM-26 and VP-16 (Chen et al, 1984; Tewey et al, 1984a,b;Zwelling, 1985;Minford et al, 1986;Zhang, 1990). These drugs interfere with topoisomerase II activity by stabilising topoisomerase II/DNA binding and strand breakage, a result of blockade of the religation/ resealing reaction which follows topoisomerase 1I-mediated strand breakage (Chen et al, 1984;Nelson et al, 1984).…”

Expression of a mutant DNA topoisomerase II in CCRF-CEM human leukemic cells selected for resistance to teniposide.

“…The 170-kDa form is more sensitive to the topoisomerase II inhibitors teniposide and merbarone than the 180-kDa form and the two forms differ in their cleavage site, thermal stability, and inhibition by A+T-rich oligonucleotides (7). Chung et al (8) Several classes of antitumor drugs, including the anthracyclines, epipodophyllotoxins, and aminoacridines, inhibit the catalytic activity of topoisomerase 11 (9)(10)(11)(12)(13)(14), and both rodent and human cell lines have been selected for resistance to these drugs (15)(16)(17)(18)(19)(20)(21). In most cases cells that have been selected for resistance to a single topoisomerase II-inhibiting drug are cross-resistant to drugs of the other classes.…”

Inhibitory effects of quinolones on pro- and eucaryotic DNA topoisomerases I and II

“…These enzymes are the targets of numerous antibacterial and antitumor agents (4,15). While the details of the drug-DNA-topoisomerase interaction are not yet fully elucidated (36), it seems clear that, in the presence of the drug, a complex is formed between enzyme and DNA which, upon exposure to a denaturing agent, results in cleavage of one or both DNA strands (4,26,30,32,33,37). This irreversible formation of DNA strand breakage induces a sequence of events that leads ultimately to cell death.…”