Combined treatment with temozolomide and methoxyamine:: Blocking apurininc/pyrimidinic site repair coupled with targeting topoisomerase IIα

Purpose: Methoxyamine has been shown to potentiate the cytotoxic effect of temozolomide both in vitro and in human tumor xenograft models. We postulate that the enhanced cytotoxicity is mediated by methoxyamne-bound apurininc/pyrimidinic (MX-AP) site, a key lesion formed by the combination of temozolomide and methoxyamine. When located within topoisomerase II alpha (topo II) cleavage sites in DNA, MX-AP sites act as dual lethal targets, not only functionally disrupting the base excision repair (BER) pathway but also potentially poisoning topo II. Experimental Design: Using oligonucleotide substrates, in which a position-specific MX-AP site is located within topo II cleavage sites, we examined the effect of MX-AP site on both AP endonuclease - and topo II - mediated DNA cleavage in vitro. Results: MX-AP sites were refractory to the catalytic activity of AP endonuclease, indicating their ability to block BER. However, they were cleaved by either purified topo II or nuclear extracts from tumor cells expressing high levels of topo II, suggesting that MX-AP sites stimulate topo II - mediated DNA cleavages. In cells, treatment with temozolomide and methoxyamine increased the expression of topo II and enriched the formation of gamma H2AX foci, which were colocalized with up-regulated topo II, confirming that DNA double-strand breaks marked by gamma H2AX foci are associated with topo II in cells. Conclusions: Our findings identify a molecular mechanism of cell death whereby MX-AP sites that cumulated in cells due to resistance to BER potentially convert topo 11 into biotoxins, resulting in enzyme-mediated DNA scission and cell death.