Liu L, Taverna P, Whitacre C M, Chatterjee S, Gerson S L
Department of Medicine and Ireland Cancer Center, Case Western Reserve University, Cleveland, Ohio 44106-4937, USA.
Clin Cancer Res. 1999 Oct;5(10):2908-17.
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.
此前我们发现，错配修复（MMR）缺陷的细胞系HCT116（hMLH1突变型）与MMR野生型细胞系SW480不同，对治疗性甲基化剂替莫唑胺（TMZ）更具抗性，因为MMR复合物无法识别复制后出现的TMZ诱导的O6 - 甲基鸟嘌呤DNA加合物与胸腺嘧啶的错配。TMZ还会产生N7 - 甲基鸟嘌呤和N3 - 甲基腺嘌呤加合物，这些加合物可由碱基切除修复（BER）系统有效处理。在甲基嘌呤糖基化酶去除甲基化碱基后，会产生无碱基或无嘌呤 - 无嘧啶（AP）位点，磷酸二酯键会立即被AP内切酶水解，从而启动AP位点的修复。甲氧基胺（MX）与无碱基位点反应并阻止AP内切酶切割，从而破坏DNA修复。MX增强了TMZ的细胞毒性作用，在SW480中的剂量修正因子（DMF）为2.3±0.12，在HCT116中为3.1±0.16。当与O6 - 苄基鸟嘌呤（BG）联合使用时，MX和TMZ显著增加了SW480中TMZ的细胞毒性（65.8倍），而在HCT116中未观察到相加效应。这表明当BER被中断时，N7 - 甲基鸟嘌呤和N3 - 甲基腺嘌呤加合物在MMR缺陷型和野生型细胞中都是细胞毒性损伤。由于聚（ADP - 核糖）聚合酶（PARP）有助于处理MMR和BER过程中诱导的DNA链断裂，我们询问PARP抑制剂是否也会影响BER介导的细胞杀伤。我们发现PARP抑制剂PD128763、3 - 氨基苯甲酰胺和6 - 氨基烟酰胺增加了HCT116 MMR缺陷细胞和SW480 MMR野生型细胞对TMZ的敏感性。在HCT116细胞中，PD128763显著降低了对TMZ的抗性，DMF为4.7±0.2。然而，PD128763、BG和TMZ的联合使用没有更大的效果，表明持续存在的O6 - 甲基鸟嘌呤对细胞毒性没有影响。在SW480中，添加PD128763时TMZ细胞毒性的DMF为3.1±0.12，添加PD128763和BG时为36。通过中位效应图进行的协同分析表明TMZ与MX或PD128763之间具有高度协同作用。相比之下，1,3 - 双（2 - 氯乙基） - 1 - 亚硝基脲与MX或PD128763联合使用时，在任一细胞系中，在不存在BG的情况下几乎未观察到增强作用，这表明BER途径对1,3 - 双（2 - 氯乙基） - 1 - 亚硝基脲诱导的加合物的细胞毒性处理影响很小。这些研究表明，用MX或PARP抑制剂靶向BER可增强甲基化剂的细胞毒性，即使在MMR缺陷细胞中也是如此。
