Telomerase downregulation induced by the G-quadruplex ligand 12459 in A549 cells is mediated by hTERT RNA alternative splicing
Telomerase downregulation induced by the G-quadruplex ligand 12459 in A549 cells is mediated by hTERT RNA alternative splicing
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DOI：
10.1093/nar/gkh181
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Riou, JF
中科院分区：
文献类型：
--
作者：
Gomez, D;Lemarteleur, T;Riou, JF
配体12459是一种有效的G-四链体相互作用剂，属于三嗪系列，以前被证明可以下调人A549肺癌细胞系中的端粒酶活性。我们在这里表明，端粒酶活性的下调是由12459诱导的hTERT剪接模式的改变引起的，即活性（+ α，+ β）转录物几乎完全消失和非活性β转录物的过度表达。剪接的内含子6形成β hTERT转录本含有几个轨道的G-丰富的序列能够形成G-四链体。通过使用一个特定的PCR-停止测定，我们表明，12459是能够稳定这些G-四链体结构的形成。已经分析了针对12459抗性选择的A549细胞系克隆的hTERT剪接模式。抗性克隆能够在12459处理下维持活性hTERT转录物，表明能够绕过12459诱导的剪接改变的机制的出现。与12459相反，telomestatin和BRACO 19，另外两种G-四链体相互作用剂，对A549细胞中的hTERT剪接模式没有影响，对A549抗性克隆具有细胞毒性，并且显示出较低的稳定hTERT G-四链体的效率。这些结果使我们提出12459通过稳定位于hTERT内含子6中的四链体来损害hTERT的剪接机制。12459、BRACO 19和端粒抑制素对这些hTERT四链体的选择性的差异对于解释它们各自对hTERT剪接的活性和不活性可能是重要的。
Ligand 12459, a potent G-quadruplex-interacting agent that belongs to the triazine series, was previously shown to downregulate telomerase activity in the human A549 lung carcinoma cell line. We show here that the downregulation of telomerase activity is caused by an alteration of the hTERT splicing pattern induced by 12459, i.e. an almost complete disappearance of the active (+alpha,+beta) transcript and an over-expression of the inactive -beta transcript. Spliced intron 6 forming the -beta hTERT transcript contained several tracks of G-rich sequences able to form G-quadruplexes. By using a specific PCR-stop assay, we show that 12459 is able to stabilize the formation of these G-quadruplex structures. A549 cell line clones selected for resistance to 12459 have been analyzed for their hTERT splicing pattern. Resistant clones are able to maintain the active hTERT transcript under 12459 treatment, suggesting the appearance of mechanisms able to bypass the 12459-induced splicing alterations. In contrast to 12459, telomestatin and BRACO19, two other G-quadruplex-interacting agents, have no effect on the hTERT splicing pattern in A549 cells, are cytotoxic against the A549-resistant clones and display a lower efficiency to stabilize hTERT G-quadruplexes. These results lead us to propose that 12459 impairs the splicing machinery of hTERT through stabilization of quadruplexes located in the hTERT intron 6. Differences of selectivity between 12459, BRACO19 and telomestatin for these hTERT quadruplexes may be important to explain their respective activity and inactivity against hTERT splicing.