Checkpoint kinase 1 down-regulation by an inducible small interfering RNA expression system sensitized in vivo tumors to treatment with 5-fluorouracil
Checkpoint kinase 1 down-regulation by an inducible small interfering RNA expression system sensitized in vivo tumors to treatment with 5-fluorouracil
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DOI：
10.1158/1078-0432.ccr-08-0304
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发表时间：
2008-08-15
影响因子：
11.5
通讯作者：
Damia, Giovanna
中科院分区：
文献类型：
--
作者：
Ganzinelli, Monica;Carrassa, Laura;Damia, Giovanna
目的：DNA损伤后，细胞中的检查点通路被激活以停止细胞周期，从而确保修复或诱导细胞死亡。为了更好地研究检查点激酶1（Chk 1）在对不同抗癌剂的细胞应答中的作用，通过使用小干扰RNA（siRNA）在HCT-116细胞系及其p53缺陷亚系中敲低Chk 1。Chk 1通过瞬时转染针对它的特异性siRNA而被废除，并用表达两种针对Chk 1的siRNA的质粒转染细胞，获得稳定的四环素诱导型Chk 1 siRNA克隆。经验证的诱导系统，然后在体内移植的诱导克隆在nukemic.Results翻译设置：瞬时Chk 1下调致敏HCT-116细胞，p53（-/-）比p53野生型对应，DNA损伤剂5-氟尿嘧啶（5-FU），阿霉素，依托泊苷治疗，没有修改紫杉醇和PS341细胞毒活性。诱导克隆中Chk 1蛋白水平的抑制与多西环素诱导相关，顺铂和5-FU活性增加。这种效应在p53缺陷的背景中更明显。将这些克隆移植到裸鼠体内，通过免疫组织化学检测Chk 1蛋白，在饮用水中给予四环素的小鼠的肿瘤样品中显示明显的Chk 1下调。更重要的是，在Chk 1和p53双沉默的肿瘤中发现了增加的5-FU抗肿瘤活性。结论：这些发现证实了以下事实，即Chk 1蛋白是在具有缺陷的G（1）检查点的肿瘤中被抑制以增加抗癌治疗的选择性的分子靶点。
Purpose: After DNA damage, checkpoints pathways are activated in the cells to halt the cell cycle, thus ensuring repair or inducing cell death. To better investigate the role of checkpoint kinase 1 (Chk1) in cellular response to different anticancer agents, Chk1 was knocked down in HCT-116 cell line and in its p53-deficient subline by using small interfering RNAs (siRNA).Experimental Design: Chk1 was abrogated by transient transfection of specific siRNA against it, and stable tetracycline-inducible Chk1 siRNA clones were obtained transfecting cells with a plasmid expressing two siRNA against Chk1. The validated inducible system was then translated in an in vivo setting by transplanting the inducible clones in nude mice.Results: Transient Chk1 down-regulation sensitized HCT-116 cells, p53(-/-) more than the p53 wild-type counterpart, to DNA-damaging agents 5-fluorouracil (5-FU), doxorubicin, and etoposide treatments, with no modification of Taxol and PS341 cytotoxic activities. Inhibition of Chk1 protein levels in inducible clones on induction with doxycycline correlated with an increased cisplatin and 5-FU activity. Such effect was more evident in a p53-deficient background. These clones were transplanted in nude mice and a clear Chk1 down-regulation was shown in tumor samples of mice given tetracycline in the drinking water by immunohistochemical detection of Chk1 protein. More importantly, an increased 5-FU antitumor activity was found in tumors with the double Chk1 and p53 silencing.Conclusions: These findings corroborate the fact that Chk1 protein is a molecular target to be inhibited in tumors with a defective G(1) checkpoint to increase the selectivity of anticancer treatments.