Methoxyamine potentiates DNA single strand breaks and double strand breaksinduced by temozolomide in colon cancer cells

We have previously shown that human cancer cells deficient in DNA mismatch repair (MMR) are resistant to the chemotherapeutic methylating agent temozo lomide (TMZ) and can be sensitized by the base excision repair (BER) blocki ng agent methoxyamine (MX) [21]. To further characterize BER-mediated repai r responses to methylating agent-induced DNA damage, we have now evaluated the effect of MX on TMZ-induced DNA single strand breaks (SSB) by alkaline elution and DNA double strand breaks (DSB) by pulsed field gel electrophore sis in SW480 (O-6-alkylguanine-DNA-alkyltransferase [AGT]+, MMR wild type) and HCT116 (AGT+, MMR deficient) colon cancer cells. SSB were evident in bo th cell lines after a 2-h exposure to equitoxic doses of temozolomide. MX s ignificantly increased the number of TMZ-induced DNA-SSB in both cell lines . In contrast to SSB, TMZ-induced DNA-DSB were dependent on MMR status and were time-dependent. Levels of 50 kb double stranded DNA fragments in MMR p roficient cells were increased after TMZ alone or in combination with O-6-b enzylguanine or MX, whereas, in MMR deficient HCT116 cells, only TMZ plus M X produced significant levels of DNA-DSB. Levels of AP endonuclease, XBCC1 and polymerase beta were present in both cell lines and were not significan tly altered after MX and TMZ. However, cleavage of a 30-mer double strand s ubstrate by SW480 and HCT116 crude cell extracts was inhibited by MX plus T MZ. Thus, MX potentiation of TMZ cytotoxicity may be explained by the persi stence of apurinic/apyrimidinic (AP) sites not further processed due to the presence of MX. Furthermore, in MMR-deficient, TMZ-resistant HCT116 colon cancer cells, MX potentiates TMZ cytotoxicity through formation of large DS -DNA fragmentation and subsequent apoptotic signalling. (C) 2001 Elsevier S cience B.V. All rights reserved.