Pharmacologic disruption of base excision repair sensitizes mismatch repair-deficient and -proficient colon cancer cells to methylating agents

Abstract

Previously we showed that a mismatch repair (MMR)-deficient cell line, HCT116 (hMLH1 mut), unlike a MMR wild-type cell line, SW480, was more resistant to the therapeutic methylating agent, temozolomide (TMZ), because the MMR complex fails to recognize TMZ-induced O6-methylguanine DNA adduct mispairings with thymine that arise after replication. TMZ also produces N7-methylguanine and N3-methyladenine adducts that are processed efficiently by the base excision repair (BER) system. After removal of the methylated base by methylpurine glycosylase, which creates the abasic or apurinic-apyrimidinic (AP) site, the phosphodiester bond is hydrolyzed immediately by AP endonuclease, initiating the repair of the AP site. Methoxyamine (MX) reacts with the abasic site and prevents AP endonuclease cleavage, disrupting DNA repair. MX potentiated the cytotoxic effect of TMZ with a dose modification factor (DMF) of 2.3+/-0.12 in SW480 and 3.1+/-0.16 in HCT116. When combined with O6-benzylguanine (BG), MX and TMZ dramatically increased TMZ cytotoxicity (65.8-fold) in SW480, whereas no additive effect was seen in HCT116. This suggests that N7-methylguanine and N3-methyladenine adducts are cytotoxic lesions in MMR-deficient and wild-type cells when BER is interrupted. Because poly(ADP-ribose) polymerase (PARP) aids in processing of DNA strand breaks induced during MMR and BER, we asked whether PARP inhibitors would also affect BER-mediated cell killing. We found that PARP inhibitors PD128763, 3-aminobenzimide, and 6-aminonicotinamide increased the sensitivity to TMZ in both HCT116 MMR-deficient cells and SW480 MMR wild-type cells. In HCT116 cells, PD128763 remarkably decreased resistance to TMZ, with a DMF of 4.7+/-0.2. However, the combination of PD128763, BG, and TMZ had no greater effect, indicating that persistent O6-methylguanine had no effect on cytotoxicity. In SW480, the DMF for TMZ cytotoxicity was 3.1+/-0.12 with addition of PD128763 and 36 with addition of PD128763 and BG. Synergy analysis by median effect plots indicated a high degree of synergy between TMZ and MX or PD128763. In contrast, 1,3-bis(2-chloroethyl)-1-nitrosourea combined with either MX or PD128763 showed little if any potentiation observed in the absence of BG in either cell line, suggesting that BER pathway has little impact on cytotoxic processing of 1,3-bis(2-chloroethyl)-1-nitrosourea-induced adducts. These studies indicate that targeting BER with MX or PARP inhibitors enhances the cytotoxicity of methylating agents, even in MMR-deficient cells.