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Received January 17, 2001; accepted March 7, 2001.
Purpose. Topoisomerase II (Topo II) preferentially cuts DNA at alternating purine-pyrimidine repeats. Different Topo II poisons may affect Topo II to produce distinct drug-specific DNA cleavage patterns. GL331 is a new podophyllotoxin derivative exhibiting potent Topo II-poisoning activity. Therefore, the sequence selectivity of GL331-induced DNA cleavage was determined.
Methods. Human gastric adenocarcinoma SC-M1 cells were treated with GL331, and the resultant DNA fragments were isolated by SDSK+ precipitation. These DNA fragments were further cloned and sequenced to exhibit GL331-induced DNA cleavage sites. In addition, the telomere damage was detected by Southern blot analyses using a (TTAGGG)4 probe. GL331's effect on telomerase was examined using the TRAP assay.
Results. The selective sequences of GL331-induced DNA cleavage were analyzed. The first nucleotide 38-terminal to the cleavage sites was preferentially C or G and followed by the second nucleotide T. More than 50% of GL331-induced DNA cleavage fragments exhibited AT-rich sequences in the first 20 nucleotides. In addition, the telomeric damage was observed both from GL331-treated SC-M1 cells and in vitro incubation of genomic DNA with GL331 and purified human Topo II. Although GL331 treatment reduced cellular telomerase activity, in vitro reaction data suggested that GL331 was not a telomerase inhibitor.
Conclusions. GL331 preferentially induced Topo II-mediated DNA cleavage at (C/G)T sites. Because the telomeric repeat sequence contains GL331's GT preference site, the telomere was identified as one of the targets of GL331-induced DNA damage.
KEY WORDS: GL331; VP-16; topoisomerase II; telomere; DNA damage.
ABBREVIATIONS: Topo II, topoisomerase II; DIG, digoxigenin; TRAP, telomeric repeat amplification protocol.
INTRODUCTION
Podophyllotoxin is a naturally occurring toxin from the plant Podophyllum peltatum (1). Derivatives of podophyllotoxin, represented by etoposide (VP-16), have been used as antineoplastic agents because they act as topoisomerase II (Topo II) poisons (2,3). Topo II is a nuclear enzyme responsible for modulation of the topological change of DNA via double-stranded DNA breaks coupled with strand passage and re-ligation. VP-16 does not inhibit Topo II to cut DNA but inhibits the rejoining step, sustaining the covalent binding of Topo II to DNA breaks (4,5). Topo II cleaves DNA at nonrandom sites, as observed by a variety of experimental systems (6,7). Topo II preferentially cuts DNA at alternating purine-pyrimidine repeats, but...