title: Combined treatment with temozolomide and methoxyamine: blocking apurininc/pyrimidinic site repair coupled with targeting topoisomerase IIalpha.
abstract: OBJECTIVE
  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 methoxyamine-bound apurininc/pyrimidinic (MX-AP) site, a key lesion formed by the combination of temozolomide and methoxyamine. When located within topoisomerase IIalpha (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.
  
  
  METHODS
  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 gammaH2AX foci, which were colocalized with up-regulated topo II, confirming that DNA double-strand breaks marked by gammaH2AX 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 II into biotoxins, resulting in enzyme-mediated DNA scission and cell death.
authors.affiliation: Department of Medicine, Division of Hematology/Oncology, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Yan
authors.forename: Alina
authors.initials: A
authors.lastname: Bulgar
authors.forename: Yanling
authors.initials: Y
authors.lastname: Miao
authors.forename: Varun
authors.initials: V
authors.lastname: Mahajan
authors.forename: Jon R
authors.initials: JR
authors.lastname: Donze
authors.forename: Stanton L
authors.initials: SL
authors.lastname: Gerson
authors.forename: Lili
authors.initials: L
authors.lastname: Liu
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000187:drug effects* / Q000378:metabolism; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000187:drug effects / Q000473:pathology; D004247:DNA / Q000187:drug effects*; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects*; D004250:DNA Topoisomerases, Type II / Q000187:drug effects* / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000187:drug effects* / Q000378:metabolism; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004357:Drug Synergism; D005455:Fluorescent Antibody Technique; D006801:Humans; D006898:Hydroxylamines / Q000494:pharmacology*; D051379:Mice; D000077204:Temozolomide; D014407:Tumor Cells, Cultured
chemical_list: D000951:Antigens, Neoplasm; D018906:Antineoplastic Agents, Alkylating; D004268:DNA-Binding Proteins; D006898:Hydroxylamines; D003606:Dacarbazine; D004247:DNA; C005214:methoxyamine; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004250:DNA Topoisomerases, Type II; D000077204:Temozolomide