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 mitoxantrone stabilize preferentially the binding of topoisomerase IIalpha to replicating compared to nonreplicating DNA. To further understand the mechanisms by which cleavable complex-forming topoisomerase II inhibitors interfere with DNA replication, we examined the effects of VM-26 on this process in human leukemia CEM cells. Both the inhibition of DNA synthesis and cell survival were directly related to the total amount 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 cleavable complexes interfered with the movement of DNA replication forks. To test this hypothesis directly, we monitored replication fork progression 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 putative replication origin located in the 5' flanking region of the gene. Replication forks, which moved through this region of the c-myc gene in the 5' to 3' direction, were specifically arrested at this site in VM-26-treated cells, but not in untreated or aphidicolin-treated cells. These studies provide the first direct evidence that a VM-26-stabilized topoisomerase II-DNA cleavable complex acts as a replication fork barrier at a specific genomic site in mammalian cells. Furthermore, the data support the hypothesis that the replication fork arrest induced by cleavable complex-forming topoisomerase II inhibitors leads to the generation of irreversible DNA damage and cytotoxicity in proliferating cells.
Catapano
C V
CV
Department of Experimental Oncology, Hollings Cancer Center, Medical University of South Carolina, Charleston 29425, USA.
Carbone
G M
GM
Pisani
F
F
Qiu
J
J
Fernandes
D J
DJ
D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism / Q000494:pharmacology*; D004795:Enzyme Stability / Q000187:drug effects; D016259:Genes, myc / Q000187:drug effects; D006801:Humans; D015458:Leukemia, T-Cell / Q000188:drug therapy* / Q000235:genetics; D046911:Macromolecular Substances; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D013713:Teniposide / Q000494:pharmacology*; D059005:Topoisomerase II Inhibitors; D014407:Tumor Cells, Cultured
D046911:Macromolecular Substances; D019384:Nucleic Acid Synthesis Inhibitors; D059005:Topoisomerase II Inhibitors; D013713:Teniposide; D004250:DNA Topoisomerases, Type II