Induction of topoisomerase I cleavage complexes by 1-beta-D-arabinofuranosylcytosine (ara-C) in vitro and in ara-C-treated cells

1-beta-D-Arabinofuranosylcytosine (Ara-C) is a nucleoside analog commonly u sed in the treatment of leukemias. Ara-C inhibits DNA polymerases and can b e incorporated into DNA, Its mechanism of cytotoxicity is not fully underst ood. Using oligonucleotides and purified human topoisomerase I (top1), we f ound a 4- to 6-fold enhancement of top1 cleavage complexes when ara-e was i ncorporated at the +1 position (immediately 3') relative to a unique top1 c leavage site. This enhancement was primarily due to a reversible inhibition of top1-mediated DNA religation. Because ara-C incorporation is known to a lter base stacking and sugar puckering at the misincorporation site and at the neighboring base pairs, the observed inhibition of religation at the ar a-C site suggests the importance of the alignment of the 5'-hydroxyl end fo r religation with the phosphate group of the top1 phosphotyrosine bond. Thi s study also demonstrates that ara-C treatment and DNA incorporation trap t op1 cleavage complexes in human leukemia cells. Finally, we report that cam ptothecin-resistant mouse P388/ CPT45 cells with no detectable top1 are cro ssresistant to ara-C, which suggests that top1 poisoning is a potential mec hanism for ara-C cytotoxicity.