DNA topoisomerase II is required for formation of mitotic chromosomes in Chinese hamster ovary cells: studies using the inhibitor 4'-demethylepipodophyllotoxin 9-(4,6-O-thenylidene-beta-D-glucopyranoside).
To study the biochemical processes which DNA topoisomerase II carries out in mammalian cells, which have not been identified, we have examined the effects on chromosome replication in Chinese hamster ovary cells of an agent which traps molecules of topoisomerase II when they are covalently integrated into DNA during their reaction. This agent, 4'-demethylepipodophyllotoxin 9-(4,6-O-thenylidene-beta-D-glucopyranoside) (VM-26), targets this enzyme specifically according to a compelling body of evidence. Using synchronously growing cells, we found that VM-26 at a cytotoxic concentration (0.08 microM) did not affect DNA replication during the S phase. The formation of mitotic chromosomes was delayed by 4 h, and its rate was reduced thereafter, causing a delay in mitosis of greater than 14 h in 65% of the cells; in some cells, the chromatin was aberrantly condensed, forming diffuse chromosomes or particles. Chromosome formation was completely inhibited at 0.32 microM VM-26. DNA fragments derived from topoisomerase II molecules covalently integrated in DNA and trapped by VM-26 were detected by FIGE analysis in the G2 period, but not during the S phase. The delay of chromosome formation appeared to be caused by two factors: first, a delay in the completion of DNA replication, because progress of some cells to mitosis after removal of VM-26 was prevented by aphidicolin, an inhibitor of DNA polymerases alpha and delta; and second, a delay of chromosome formation in cells which had apparently completed DNA replication. The observations reported here show that topoisomerase II carries out reactions which are essential for formation of mitotic chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)
Charron
M
M
Centre de Recherche en Cancérologie, l'Université Laval, Hôtel-Dieu Hospital, Québec, Canada.
Hancock
R
R
D000818:Animals; D016590:Aphidicolin; D002460:Cell Line; D002843:Chromatin / Q000187:drug effects / Q000648:ultrastructure; D002875:Chromosomes / Q000187:drug effects* / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D004261:DNA Replication / Q000187:drug effects*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004224:Diterpenes / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D007700:Kinetics; D008938:Mitosis / Q000187:drug effects*; D010053:Ovary; D013713:Teniposide / Q000494:pharmacology*; D013936:Thymidine / Q000378:metabolism; D059005:Topoisomerase II Inhibitors; D014316:Tritium
D002843:Chromatin; D004224:Diterpenes; D059005:Topoisomerase II Inhibitors; D014316:Tritium; D016590:Aphidicolin; D013713:Teniposide; D004250:DNA Topoisomerases, Type II; D013936:Thymidine