ARREST OF REPLICATION FORK PROGRESSION AT SITES OF TOPOISOMERASE II-MEDIATED DNA CLEAVAGE IN HUMAN LEUKEMIA CEM CELLS INCUBATED WITH VM-26

Recent studies have shown that the anticancer drugs VM-26 and mitoxant rone stabilize preferentially the binding of topoisomerase II alpha to replicating compared to nonreplicating DNA. To further understand the mechanisms by which cleavable complex-forming topoisomerase II inhibi tors interfere with DNA replication, we examined the effects of VM-26 on this process in human leukemia CEM cells. Both the inhibition of DN A synthesis and cell survival were directly related to the total amoun t of drug-stabilized cleavable complexes formed in VM-26-treated cells . DNA chain elongation was also inhibited in a concentration-dependent fashion in these cells, which suggested that VM-26-stabilized cleavab le complexes interfered with the movement of DNA replication forks. To test this hypothesis directly, we monitored replication fork progress ion at a specific site of VM-26-induced DNA cleavage. A topoisomerase II-mediated cleavage site was detected in the first exon of the c-myc gene in VM-26-treated cells. This cleavage site was downstream of a pu tative replication origin located in the 5' flanking region of the gen e. Replication forks, which moved through this region of the c-myc gen e in the 5' to 3' direction, were specifically arrested at this site i n VM-26-treated cells, but not in untreated or aphidicolin-treated cel ls. These studies provide the first direct evidence that a VM-26-stabi lized topoisomerase II-DNA cleavable complex acts as a replication for k barrier at a specific genomic site in mammalian cells. Furthermore, the data support the hypothesis that the replication fork arrest induc ed by cleavable complex-forming topoisomerase II inhibitors leads to t he generation of irreversible DNA damage and cytotoxicity in prolifera ting cells.