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Adenoviral-mediated Delivery of anti RET Hammerhead Ribozymes Inhibits Thyroid Medullary Carcinoma Cell Proliferation.

腺病毒介导的抗 RET 锤头核酶递送抑制甲状腺髓样癌细胞增殖。

基本信息

批准号：
14571133
负责人：
IMAI Tsuneo
金额：
$ 1.73万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571133/
关键词：
Ret oncogene ribozyme gene therapy adenovirus MEN type 1 MEN type 2 tyrosine kinase carcinogenesis ret遺伝子

项目摘要

Activating mutations of the RET proto-oncogene cause multiple endocrine neoplasia (MEN) 2A, MEN2B, familial medullary thyroid carcinoma (MTC). Down-regulation of RET expression may be an important strategy for cancer therapy. Indeed, we previously reported ribozyme targeting mutant RET suppress transformation of NIH 3T3 cells transfected with mutant RET. However, the most of mutant sequences are not compatible with requirement for cleavage by hammerhead ribozyme. In this study, we first designed hammerhead ribozymes to cleave the normal RET transcript. We select two target sites for cleavage of ribozymes targeting intracellular and extracellular domain, respectively. In vitro cleavage assay demonstrates that these ribozymes can cleave the artificial target sequence specifically and efficiently. In order to achieve efficient gene delivery, we developed recombinant adenoviruses encoding these ribozymes driven by the cytomegalovirus promoter. Human MTC cell line TT was infected with the adenoviruses. Reduction of RET expression in RNA and protein level was observed in active ribozyme-expressing adenovirus-infected cells. Concordantly, cell proliferation was also suppressed in the active ribozyme expressing adenovirus-infected TT cells. In order to demonstrate specificity of the ribozymes, infection into TPC-1 cells which harbor ret/PTC-1 rearrangement (where intracellular domain of RET is fused with irrelevant protein) exhibit that only ribozyme targeting intracellular domain has significant but much less prominent effect on the cell proliferation. Finally, caspase-3 activity was elevated in active ribozyme-expressing adenovirus infected TT cells, indicating that apoptotic pathways are involved in the effects of the ribozymes. These results provide a new avenue for inhibition of RET by selective mRNA degradation with its potential therapeutic application.

RET原癌基因的激活突变可导致多发性内分泌肿瘤(MEN)2A、MEN2B、家族性甲状腺髓样癌(MTC)。下调RET的表达可能是肿瘤治疗的重要策略。事实上，我们之前报道了针对突变RET的核酶抑制了转导突变RET的NIH 3T3细胞的转化。然而，大多数突变序列与锤头状核酶的切割要求不相容。在本研究中，我们首先设计了锤头状核酶来切割正常的RET转录本。我们选择了两个靶点来切割核酶，分别针对胞内和胞外结构域。体外切割实验表明，这些核酶能有效、特异地切割人工合成的靶序列。为了实现高效的基因传递，我们开发了编码巨细胞病毒启动子驱动的核酶的重组腺病毒。用重组腺病毒感染人MTC细胞系TT。在表达活性核酶的腺病毒感染细胞中，观察到RET在RNA和蛋白质水平的表达减少。相应地，在表达活性核酶的腺病毒感染TT细胞中，细胞增殖也受到抑制。为了证明核酶的特异性，感染存在ret/PTC-1重排的TPC-1细胞(RET的胞内区与无关蛋白融合)显示，只有靶向胞内区的核酶对细胞增殖有显著但不显著的影响。最后，在表达活性核酶的腺病毒感染的TT细胞中，caspase-3的活性升高，表明核酶的作用与细胞的凋亡途径有关。这些结果为选择性降解mRNA抑制RET提供了新的途径，具有潜在的治疗应用前景。