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)

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Abstract
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 .mu.M) did not affect DNA replication during the S phase. The formation of mitotoic chromosomes was delayed by 4 h, and its rate was reduced thereafter, causing a delay in mitosis of > 14 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 .mu.M VM-26. DNA fragments derived from topoisomerase II molecules covalently integrated in DNA an 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 carriers out which are essential for formation of mitotic chromosomes. They are compatible with a model in which topoisomerase II functions both during the completion of DNA replication and in a subsequent process which, by analogy with VM-26-sensitive steps in simian virus 40 DNA replication, may be the topological conversion of a series of replicated DNA loops or domains into two linear chromatid-length DNA molecules.