Combined treatment with temozolomide and methoxyamine: blocking apurininc/pyrimidinic site repair coupled with targeting topoisomerase IIalpha.
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.
Yan
Ling
L
Department of Medicine, Division of Hematology/Oncology, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Bulgar
Alina
A
Miao
Yanling
Y
Mahajan
Varun
V
Donze
Jon R
JR
Gerson
Stanton L
SL
Liu
Lili
L
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
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