Kopnin Pavel B, Agapova Larissa S, Kopnin Boris P, Chumakov Peter M
Engelhardt Institute of Molecular Biology, Moscow, Russia.
Cancer Res. 2007 May 15;67(10):4671-8. doi: 10.1158/0008-5472.CAN-06-2466.
Oncogenic mutations within RAS genes and inactivation of p53 are the most common events in cancer. Earlier, we reported that activated Ras contributes to chromosome instability, especially in p53-deficient cells. Here we show that an increase in intracellular reactive oxygen species (ROS) and oxidative DNA damage represents a major mechanism of Ras-induced mutagenesis. Introduction of oncogenic H- or N-Ras caused elevated intracellular ROS, accumulation of 8-oxo-2'-deoxyguanosine, and increased number of chromosome breaks in mitotic cells, which were prevented by antioxidant N-acetyl-L-cysteine. By using Ras mutants that selectively activate either of the three major targets of Ras (Raf, RalGDS, and phosphatidylinositol-3-kinase) as well as dominant-negative Rac1 and RalA mutants and inhibitors of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases kinase-1 and p38 MAPKs, we have shown that several Ras effectors independently mediate ROS up-regulation. Introduction of oncogenic RAS resulted in repression of transcription from sestrin family genes SESN1 and SESN3, which encode antioxidant modulators of peroxiredoxins. Inhibition of mRNAs from these genes in control cells by RNA interference substantially increased ROS levels and mutagenesis. Ectopic expression of SESN1 and SESN3 from lentiviral constructs interfered with Ras-induced ROS increase, suggesting their important contribution to the effect. The stability of Ras-induced increase in ROS was dependent on a p53 function: in the p53-positive cells displaying activation of p53 in response to Ras, only transient (4-7 days) elevation of ROS was observed, whereas in the p53-deficient cells the up-regulation was permanent. The reversion to normal ROS levels in the Ras-expressing p53-positive cells correlated with up-regulation of p53-responsive genes, including reactivation of SESN1 gene. Thus, changes in expression of sestrins can represent an important determinant of genetic instability in neoplastic cells showing simultaneous dysfunctions of Ras and p53.
RAS基因中的致癌突变和p53的失活是癌症中最常见的事件。此前，我们报道激活的Ras会导致染色体不稳定，尤其是在p53缺陷细胞中。在此我们表明，细胞内活性氧（ROS）增加和氧化性DNA损伤是Ras诱导诱变的主要机制。致癌性H-Ras或N-Ras的导入导致细胞内ROS升高、8-氧代-2'-脱氧鸟苷积累以及有丝分裂细胞中染色体断裂数量增加，抗氧化剂N-乙酰-L-半胱氨酸可阻止这些现象。通过使用选择性激活Ras三个主要靶点（Raf、RalGDS和磷脂酰肌醇-3-激酶）之一的Ras突变体，以及显性负性Rac1和RalA突变体和丝裂原活化蛋白激酶（MAPK）/细胞外信号调节激酶激酶-1和p38 MAPK的抑制剂，我们表明几种Ras效应器独立介导ROS上调。致癌性RAS的导入导致sestrin家族基因SESN1和SESN3的转录受到抑制，这两个基因编码过氧化物酶的抗氧化调节剂。通过RNA干扰抑制对照细胞中这些基因的mRNA会显著增加ROS水平和诱变。慢病毒构建体异位表达SESN1和SESN3可干扰Ras诱导的ROS增加，表明它们对该效应有重要贡献。Ras诱导的ROS增加的稳定性取决于p53功能：在对Ras有反应而显示p53激活的p53阳性细胞中，仅观察到ROS短暂（4-7天）升高，而在p53缺陷细胞中上调是永久性的。在表达Ras的p53阳性细胞中ROS水平恢复正常与p53反应性基因的上调相关，包括SESN1基因的重新激活。因此，sestrin表达的变化可能是肿瘤细胞中遗传不稳定的重要决定因素，这些肿瘤细胞同时存在Ras和p53功能障碍。
