Repression of sestrin family genes contributes to oncogenic Ras-induced reactive oxygen species up-regulation and genetic instability
Repression of sestrin family genes contributes to oncogenic Ras-induced reactive oxygen species up-regulation and genetic instability
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DOI：
10.1158/0008-5472.can-06-2466
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发表时间：
2007-05-15
期刊：
影响因子：
11.2
通讯作者：
Chumakov, Peter M.
中科院分区：
文献类型：
--
作者：
Kopnin, Pavel B.;Agapova, Larissa S.;Chumakov, Peter M.
RAS基因的致癌突变和P53的失活是癌症中最常见的事件。早些时候，我们报道了激活的RAS导致染色体不稳定，特别是在P53缺失的细胞中。在这里，我们表明，细胞内活性氧簇(ROS)的增加和DNA氧化损伤是RAS诱导突变的主要机制。在有丝分裂细胞中引入致癌的H-或N-RAS可引起细胞内ROS升高、8-氧-2‘-脱氧鸟苷积聚和染色体断裂数目增加，而抗氧化剂N-乙酰-L-半胱氨酸可阻止这一过程。通过使用选择性地激活RAS的三个主要靶标(Raf、RalGDS和磷脂酰肌醇-3-激酶)之一的RAS突变体以及显性阴性的rac1和Rala突变体以及丝裂原活化蛋白激酶(MAPK)/细胞外信号调节激酶-1和p38MAPKs的抑制剂，我们已经证明了几种RAS效应独立地介导ROS的上调。癌基因RAS的引入导致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还蛋白的抗氧化调节剂。通过RNA干扰抑制对照细胞中这些基因的mRNAs，大大增加了ROS水平和突变。慢病毒载体中SESN1和SESN3的异位表达干扰了RAS诱导的ROS增加，表明它们在这一效应中起着重要作用。Ras诱导的ROS增加的稳定性依赖于P53的功能：在对Ras反应显示P53激活的P53阳性细胞中，仅观察到一过性(4-7天)的ROS升高，而在P53缺乏的细胞中，ROS的上调是永久性的。在表达Ras的P53阳性细胞中，ROS水平的恢复与P53反应基因的上调有关，包括SESN1基因的重新激活。因此，Sestrins表达的变化可以代表肿瘤细胞遗传不稳定性的重要决定因素，这些肿瘤细胞同时表现出RAS和P53功能障碍。
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.