Department of Therapeutic Radiology, Yale School of Medicine, New Haven, Connecticut 06520-8040, USA.
Cancer Res. 2010 Jan 1;70(1):409-17. doi: 10.1158/0008-5472.CAN-09-1353. Epub 2009 Dec 22.
Base excision repair (BER) plays a critical role in the repair of bases damaged by oxidative metabolism or alkylating agents, such as those commonly used in cancer therapy. Incomplete BER generates intermediates that require activation of homology-dependent DNA repair to resolve. We investigated the effects of lithocholic acid (LCA), an inhibitor of the key BER enzyme DNA polymerase beta (pol beta), in cells deficient in expression of the homology-dependent repair factor BRCA2. In vitro studies show that LCA suppresses the DNA polymerase and 5'-deoxyribose phosphate lyase activities of DNA pol beta by preventing the formation of a stable pol beta-DNA complex, reducing BER effectiveness. Cytotoxicity assays based on colony formation revealed that LCA exhibits synergism with the alkylating agent temozolomide, which engages BER through DNA methylation, and that the degree of synergism is increased in cells lacking functional BRCA2. BRCA2-deficient cells also showed heightened susceptibility to both LCA and temozolomide individually. The potentiation of temozolomide cytotoxicity by LCA owes to the conversion of single-stranded DNA breaks generated through incomplete BER of methylated nucleotides into double-stranded breaks during DNA replication, as indicated by gammaH2AX immunofluorescence. Death seems to be induced in cotreated cells through an accumulation of persistent double-stranded DNA breaks. Mutations of the BRCA2 gene have been extensively characterized and are present in various cancers, implying that inhibition of BER may offer a means to augment tumor selectivity in the use of conventional cancer therapies.
碱基切除修复 (BER) 在修复氧化代谢或烷化剂（如癌症治疗中常用的那些）损伤的碱基方面起着至关重要的作用。不完全的 BER 会产生需要激活同源依赖性 DNA 修复来解决的中间产物。我们研究了胆酸（LCA）对同源依赖性修复因子 BRCA2 表达缺陷细胞中关键 BER 酶 DNA 聚合酶β（polβ）的影响。体外研究表明，LCA 通过防止稳定的 polβ-DNA 复合物的形成来抑制 polβ的 DNA 聚合酶和 5'-脱氧核糖磷酸解酶活性，从而降低 BER 效率。基于集落形成的细胞毒性测定表明，LCA 与烷化剂替莫唑胺表现出协同作用，后者通过 DNA 甲基化参与 BER，并且在缺乏功能性 BRCA2 的细胞中协同作用程度增加。BRCA2 缺陷细胞对 LCA 和替莫唑胺的敏感性也明显增加。LCA 增强替莫唑胺的细胞毒性归因于在 DNA 复制过程中，通过不完全 BER 将甲基化核苷酸产生的单链 DNA 断裂转化为双链断裂，如 γH2AX 免疫荧光所示。在共处理的细胞中，死亡似乎是通过持续双链 DNA 断裂的积累而诱导的。BRCA2 基因突变已被广泛研究，并存在于各种癌症中，这意味着抑制 BER 可能为提高传统癌症治疗的肿瘤选择性提供一种手段。
