Increased ATP requirement for activity of and complex formation by DNA topoisomerase II from human leukemic CCRF-CEM cells selected for resistance to teniposide.

We showed previously that nuclear extracts from teniposide (VM-26)-resistant sublines of the human leukemic cell line, CCRF-CEM, exhibited decreased DNA topoisomerase II activity and ability to form drug-stabilized covalent protein-DNA complexes (Danks et al., Biochemistry 27:8861-8869; 1988). In the present study, we found that nuclear extracts of these sublines (approximately 50- and approximately 140-fold resistant to VM-26) required 2 and 8 times more adenosine 5'-triphosphate (ATP), respectively, to achieve half-maximal stimulation of unknotting activity compared to extracts from the sensitive cells. When novobiocin, a competitive inhibitor of ATP binding to topoisomerase II, was included in the reaction, this ATP requirement increased 2.5- to 4-fold with the CEM cell extracts and 3.5- to 12-fold with the resistant cell extracts. ATP produced a dose-dependent increase in VM-26-stabilized topoisomerase II-DNA covalent complexes with nuclear extracts from all three cell lines. Extracts from resistant cells, however, formed 40-80% fewer complexes than those from sensitive cells. A similar decrease was seen with 4'-[(9-acridinyl)amino]methanesulphon-m-anisidide, to which the cells are cross-resistant. With nuclear extracts from sensitive cells, the tetralithium salt of 5'-adenylylimidodiphosphate (AMP-PNP), a nonhydrolyzable analog of ATP, was as effective as ATP in promoting the formation of drug-stabilized enzyme-DNA complexes. With extracts from the resistant cell nuclei, however, AMP-PNP was about half as effective as ATP in promoting complex formation. Our results demonstrate that the effect of ATP on strand passing activity of and drug-stabilized complex formation by topoisomerase II is decreased in the nuclear extracts from the drug-resistant cells and suggest a possible basis for this form of drug resistance.