Down-regulation of DNA topoisomerase IIalpha leads to prolonged cell cycle transit in G2 and early M phases and increased survival to microtubule-interacting agents.
Microtubule binders are cell cycle-specific agents with preferential cytotoxicity toward mitotic cells. We have characterized vincristine-selected human leukemia cells to establish whether development of vincristine resistance was accompanied by changes in cell cycle kinetics and distribution. Our results indicate that vincristine resistance is accompanied by delayed G2 transit and prolonged early mitosis in both the absence and the presence of the microtubule binder nocodazole. The altered G2/M regulation is accompanied by resistance to short-term (12 h) but not continuous nocodazole exposure in agreement with the transient nature of the observed cell cycle alterations. Western blot analysis indicates that vincristine-selection is accompanied by down-regulation of topoisomerase IIalpha without detectable alterations of the other mitotic regulators studied, including Cdk1, p21, 14-3-3sigma, and 14-3-3epsilon. This was associated with at least 7-fold less chromosome-associated topoisomerase IIalpha, decreased catalytic activity, and cross-resistance to topoisomerase II inhibitors. Characterization of isogenic cell lines expressing different levels of topoisomerase II proteins shows that cellular levels of topoisomerase IIalpha, but not the closely related topoisomerase IIbeta, directly influence the cell cycle kinetics in G2 and early mitosis as well as the resistance to nocodazole. These results underline the importance of topoisomerase IIalpha in late G2 and early M phases and provide evidence for an as-yet-unsuspected interaction between topoisomerase II and microtubule-directed agents.
Skladanowski
Andrzej
A
Group of Molecular and Clinical Cancer Therapeutics, INSERM U673, Hôpital Saint-Antoine, 75571 Paris 12, France.
Côme
Marie-George
MG
Sabisz
Michal
M
Escargueil
Alexandre E
AE
Larsen
Annette K
AK
D000951:Antigens, Neoplasm / Q000378:metabolism*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D002384:Catalysis; D002455:Cell Division; D002470:Cell Survival / Q000187:drug effects*; D002877:Chromosomes, Human; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm; D016195:G2 Phase; D018922:HL-60 Cells; D006801:Humans; D008870:Microtubules / Q000187:drug effects*; D008938:Mitosis; D015739:Nocodazole / Q000494:pharmacology; D014750:Vincristine / Q000494:pharmacology
D000951:Antigens, Neoplasm; D000972:Antineoplastic Agents, Phytogenic; D004268:DNA-Binding Proteins; D014750:Vincristine; D004250:DNA Topoisomerases, Type II; D015739:Nocodazole