Suppression of survivin promoter activity by YM155 involves disruption of Sp1-DNA interaction in the survivin core promoter.
Suppression of survivin promoter activity by YM155 involves disruption of Sp1-DNA interaction in the survivin core promoter.
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发表时间：
2012
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通讯作者：
Q. Cheng;X. Ling;A. Haller;T. Nakahara;K. Yamanaka;Aya Kita;H. Koutoku;M. Takeuchi;M. Brattain-M.-Bratt
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作者：
Q. Cheng;X. Ling;A. Haller;T. Nakahara;K. Yamanaka;Aya Kita;H. Koutoku;M. Takeuchi;M. Brattain-M.-Bratt
YM155是一种新型的survivin抑制剂，对多种人类癌症具有有效的抗肿瘤活性，目前正在进行II期临床试验。在这项研究中，我们研究了YM155是否选择性地抑制了survivin的转录。我们假设抑制survivin转录在ym155介导的survivin抑制中起作用。我们在转染和荧光素酶活性实验中发现，YM155抑制survivin启动子活性，而对4个对照基因启动子的抑制作用最小，表明其具有选择性。转染各种survivin启动子-荧光素酶构建体，然后进行荧光素酶检测，结果表明survivin核心启动子（269 bp）在ym155介导的抑制作用中起主要作用。然而，流式细胞术分析表明，YM155对survivin启动子活性的抑制是细胞周期无关的，没有G1细胞阻滞。电泳迁移位移分析（EMSA）发现，YM155消除了与-149至-71区域结合的核蛋白，其中Sp1是主要的候选区域，并且YM155处理诱导Sp1重新亚细胞定位而不抑制其表达。Sp1的强制表达中和了ym155介导的survivin启动子活性下调。同样，在EMSA实验中，寡核苷酸探针中Sp1位点的突变降低了dna -蛋白质的相互作用，而survivin启动子-荧光素酶结构Sp1位点的突变降低了survivin启动子的活性。这些发现表明，YM155对survivin表达的抑制至少部分是通过破坏Sp1与survivin核心启动子-149至-71区域的相互作用来抑制survivin转录。
YM155, a novel survivin suppressant, shows potent antitumor activity against various human cancers and is currently in phase II clinical trials. In this study, we investigated whether YM155 selectively inhibits survivin transcription. We hypothesize that inhibition of survivin transcription plays a role in YM155-mediated survivin inhibition. We found that YM155 inhibited survivin promoter activity, while it showed minimal inhibitory effect on four control gene promoters in transfection and luciferase activity assay experiments, indicating its selectivity. Transfection of various survivin promoter-luciferase constructs followed by luciferase assays revealed that the survivin core promoter (269 bp) plays a major role in YM155-mediated inhibitory effects. However, flow cytometry analysis indicated that inhibition of survivin promoter activity by YM155 is cell cycle-independent without G1 cell arrests. Electrophoretic mobility shift assays (EMSA) identified that YM155 abrogates nuclear proteins binding to the region of -149 to -71, in which Sp1 is a major candidate, and that YM155 treatment induces Sp1 re-subcellular localization without inhibiting its expression. Forced expression of Sp1 neutralized YM155-mediated downregulation of survivin promoter activity. Consistently, mutation of the identified Sp1 sites in the oligonucleotide probe diminished DNA-protein interactions in EMSA experiments, and mutation of the Sp1 sites in the survivin promoter-luciferase construct diminished survivin promoter activity. These findings indicate that YM155 inhibition of survivin expression is at least in part through its inhibition of survivin transcription by disruption of Sp1 interaction with the region of -149 to -71 in the survivin core promoter.