CR-UK Drug-DNA Interactions Research Group, UCL Cancer Institute, London, WC1E 6BT, UK.
BMC Cancer. 2012 Sep 28;12:436. doi: 10.1186/1471-2407-12-436.
DNA interstrand cross-links (ICLs) are critical lesions produced by several cancer chemotherapy agents including platinum drugs and nitrogen mustards. We have previously shown in haematological (multiple myeloma) and solid tumours (ovarian cancer) that clinical sensitivity to such agents can result from a defect in DNA ICL processing leading to their persistence. Conversely, enhanced repair can result in clinical acquired resistance following chemotherapy. The repair of ICLs is complex but it is assumed that the 'unhooking' step is common to all ICLs.
Using a modification of the single cell gel electrophoresis (Comet) assay we measured the formation and unhooking of melphalan and cisplatin-induced ICLs in cell lines and clinical samples. DNA damage response in the form of γ-H2AX foci formation and the formation of RAD51 foci as a marker of homologous recombination were also determined. Real-time PCR of 84 genes involved in DNA damage signalling pathways was also examined pre- and post-treatment.
Plasma cells from multiple myeloma patients known to be clinically resistant to melphalan showed significant unhooking of melphalan-induced ICLs at 48 hours, but did not unhook cisplatin-induced ICLs. In ovarian cancer cells obtained from patients following platinum-based chemotherapy, unhooking of cisplatin-induced ICLs was observed at 48 hours, but no unhooking of melphalan-induced ICLs. In vitro, A549 cells were proficient at unhooking both melphalan and cisplatin-induced ICLs. γ-H2AX foci formation closely followed the formation of ICLs for both drugs, and rapidly declined following the peak of formation. RPMI8226 cells unhooked melphalan, but not cisplatin-induced ICLs. In these cells, although cross-links form with cisplatin, the γ-H2AX response is weak. In A549 cells, addition of 3nM gemcitabine resulted in complete inhibition of cisplatin-induced ICL unhooking but no effect on repair of melphalan ICLs. The RAD51 foci response was both drug and cell line specific. Real time PCR studies highlighted differences in the damage response to melphalan and cisplatin following equi-ICL forming doses.
These data suggest that the mechanisms by which melphalan and cisplatin-induced ICLs are 'unhooked' in vitro are distinct, and the mechanisms of clinical acquired resistance involving repair of ICLs, are drug specific.
DNA 链间交联（ICLs）是几种癌症化疗药物（包括铂类药物和氮芥类药物）产生的关键损伤。我们之前在血液学（多发性骨髓瘤）和实体瘤（卵巢癌）中表明，对这些药物的临床敏感性可能源于导致其持续存在的 DNA ICL 处理缺陷。相反，增强修复会导致化疗后获得临床耐药性。ICL 的修复很复杂，但假定“脱钩”步骤对所有 ICL 都是通用的。
我们使用单细胞凝胶电泳（彗星）试验的改良方法测量了细胞系和临床样本中氨甲喋呤和顺铂诱导的 ICL 的形成和脱钩。还测定了 DNA 损伤反应的形式，即 γ-H2AX 焦点的形成和 RAD51 焦点的形成，作为同源重组的标志物。还在治疗前和治疗后检查了涉及 DNA 损伤信号通路的 84 个基因的实时 PCR。
已知对氨甲喋呤临床耐药的多发性骨髓瘤患者的浆细胞在 48 小时时显示出氨甲喋呤诱导的 ICL 的明显脱钩，但不能脱钩顺铂诱导的 ICL。在接受铂类化疗的卵巢癌细胞中，在 48 小时时观察到顺铂诱导的 ICL 的脱钩，但未观察到氨甲喋呤诱导的 ICL 的脱钩。在体外，A549 细胞能够有效地脱钩氨甲喋呤和顺铂诱导的 ICL。γ-H2AX 焦点的形成与两种药物的 ICL 形成密切相关，并在形成高峰后迅速下降。RPMI8226 细胞可脱钩氨甲喋呤，但不能脱钩顺铂诱导的 ICL。在这些细胞中，尽管顺铂形成交联，但 γ-H2AX 反应较弱。在 A549 细胞中，添加 3nM 吉西他滨可完全抑制顺铂诱导的 ICL 脱钩，但对氨甲喋呤 ICL 的修复没有影响。RAD51 焦点反应具有药物和细胞系特异性。实时 PCR 研究强调了在等 ICL 形成剂量下对氨甲喋呤和顺铂的损伤反应的差异。
这些数据表明，体外氨甲喋呤和顺铂诱导的 ICL 脱钩的机制是不同的，涉及 ICL 修复的临床获得性耐药机制是药物特异性的。
