AKT activity determines sensitivity to mammalian target of rapamycin (mTOR) inhibitors by regulating cyclin D1 and c-myc expression
AKT activity determines sensitivity to mammalian target of rapamycin (mTOR) inhibitors by regulating cyclin D1 and c-myc expression
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DOI：
10.1074/jbc.m309999200
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发表时间：
2004-01-23
影响因子：
4.8
通讯作者：
Lichtenstein, AK
中科院分区：
文献类型：
--
作者：
Gera, JF;Mellinghoff, IK;Lichtenstein, AK
先前的研究表明，AKT活性调节细胞对雷帕霉素（rapamycin）和CCI - 779等哺乳动物雷帕霉素靶蛋白（mTOR）抑制剂诱导的G₁期阻滞的敏感性。为了研究这一点，进行了一种新型的高通量微阵列多聚核糖体分析，以鉴定在mTOR抑制后其mRNA翻译效率受到差异影响的基因。该分析还能够评估稳态转录水平。我们鉴定出两种转录本，细胞周期蛋白D1和c - myc，它们以依赖AKT的方式表现出差异表达：高水平的活化AKT导致雷帕霉素诱导的表达下调，而低水平则导致表达上调。为了异位表达这些蛋白质，我们利用了这样一个发现：无论AKT活性如何，在mTOR抑制的情况下，p27(kip1) mRNA都能有效翻译。因此，将p27(kip1)的5' - 非翻译区与细胞周期蛋白D1和c - myc编码区融合，并在细胞中表达这些构建体。在转染的细胞中，雷帕霉素不会降低细胞周期蛋白D1或c - myc的表达。最重要的是，这完全将敏感细胞转变为对G₁期阻滞具有抗性的表型。此外，在体内用CCI - 779治疗后的小鼠异种移植模型中也观察到了这两个基因依赖AKT的差异表达模式。这些结果确定了两个关键的下游分子靶点，它们的表达受AKT活性调节，并且它们的下调是对雷帕霉素/CCI - 779敏感所必需的。
Prior work demonstrates that AKT activity regulates sensitivity of cells to G(1) arrest induced by mammalian target of rapamycin (mTOR) inhibitors such as rapamycin and CCI-779. To investigate this, a novel high-throughput microarray polysome analysis was performed to identify genes whose mRNA translational efficiency was differentially affected following mTOR inhibition. The analysis also allowed the assessment of steady-state transcript levels. We identified two transcripts, cyclin D1 and c-myc, which exhibited differential expression in an AKT-dependent manner: High levels of activated AKT resulted in rapamycin-induced down-regulation of expression, whereas low levels resulted in up-regulation of expression. To ectopically express these proteins we exploited the finding that the p27(kip1) mRNA was efficiently translated in the face of mTOR inhibition irrespective of AKT activity. Thus, the p27(kip1) 5'-untranslated region was fused to the cyclin D1 and c-myc coding regions and these constructs were expressed in cells. In transfected cells, expression of cyclin D1 or c-myc was not decreased by rapamycin. Most importantly, this completely converted sensitive cells to a phenotype resistant to G(1) arrest. Furthermore, the AKT-dependent differential expression patterns of these two genes was also observed in a mouse xenograft model following in vivo treatment with CCI-779. These results identify two critical downstream molecular targets whose expression is regulated by AKT activity and whose down-regulation is required for rapamycin/CCI-779 sensitivity.