Institute of Toxicology, University Medical Center of the Johannes Gutenberg University, Mainz, Germany.
Mutat Res. 2009 Nov 2;670(1-2):32-41. doi: 10.1016/j.mrfmmm.2009.07.002. Epub 2009 Jul 16.
The present study aimed at elucidating mechanisms dictating cell death triggered by cisplatin-induced DNA damage. We show that CL-V5B hamster mutant cells, a derivative of V79B, are hypersensitive to cisplatin-induced apoptotic death. CL-V5B cells are characterized by attenuated cisplatin-induced early (2-6 h) stress response, such as phosphorylation of stress-activated protein kinases (SAPK/JNK), ATM and Rad3-related (ATR) protein kinase, histone H2AX and checkpoint kinase-1 (Chk-1). Human FANCC cells also showed a reduced phosphorylation of H2AX and SAPK/JNK at early time point after cisplatin treatment. This was not the case for BRCA2-defective VC-8 hamster cells, indicating that the FA core complex, rather than its downstream elements, is involved in early damage response. The alleviated early response of CL-V5B cells is not due to a general dysfunction in ATM/ATR-regulated signaling. It is rather due to a reduced formation of primary cisplatin-DNA adducts in the hypersensitive mutant as shown by analysis of DNA platination, DNA intra- and interstrand crosslink formation and DNA replication blockage. Despite of lower initial DNA damage and attenuated early DNA damage response (DDR), CL-V5B cells are characterized by an excessive G2/M arrest as well as an elevated frequency of DNA double-strand breaks (DSB) and chromosomal aberrations (CA) at late times (16-24h) after cisplatin exposure. This indicates that error-prone processing of cisplatin-induced lesions, notably interstrand crosslinks (ICL), and the formation of secondary DNA lesions (i.e. DSB), results in a powerful delayed DNA damage response and massive pro-apoptotic signaling in CL-V5B cells. The data provide an example that the initial level of cisplatin-DNA adducts and the corresponding early DNA damage response do not necessarily predict the outcome of cisplatin treatment. Rather, the accuracy of DNA damage processing and late checkpoint control mechanisms determine the extent of cell death triggered by cisplatin-induced DNA lesions.
本研究旨在阐明顺铂诱导的 DNA 损伤引发细胞死亡的机制。我们发现，CL-V5B 仓鼠突变细胞（V79B 的衍生物）对顺铂诱导的凋亡性死亡非常敏感。CL-V5B 细胞的特征是减弱了顺铂诱导的早期（2-6 小时）应激反应，如应激激活蛋白激酶（SAPK/JNK）、ATM 和 Rad3 相关（ATR）蛋白激酶、组蛋白 H2AX 和检查点激酶-1（Chk-1）的磷酸化。人 FANCC 细胞在用顺铂处理后早期也表现出 H2AX 和 SAPK/JNK 磷酸化减少。BRCA2 缺陷的 VC-8 仓鼠细胞则并非如此，这表明 FA 核心复合物而非其下游元件参与了早期损伤反应。CL-V5B 细胞早期反应的缓解并不是由于 ATM/ATR 调节信号的一般功能障碍。相反，如通过 DNA 铂化分析、DNA 链内和链间交联形成以及 DNA 复制阻滞所表明的，在超敏突变体中形成的初级顺铂-DNA 加合物减少。尽管初始 DNA 损伤较低且早期 DNA 损伤反应（DDR）减弱，但 CL-V5B 细胞的特征是 G2/M 期阻滞过度，以及在顺铂暴露后晚期（16-24 小时）出现 DNA 双链断裂（DSB）和染色体畸变（CA）的频率升高。这表明，顺铂诱导损伤的易错处理，特别是链间交联（ICL）的形成，以及次级 DNA 损伤（即 DSB）的形成，导致 CL-V5B 细胞中强大的延迟 DNA 损伤反应和大量促凋亡信号。这些数据提供了一个例子，即顺铂-DNA 加合物的初始水平和相应的早期 DNA 损伤反应不一定能预测顺铂治疗的结果。相反，DNA 损伤处理的准确性和晚期检查点控制机制决定了顺铂诱导的 DNA 损伤引发细胞死亡的程度。
