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Dominant negative action of SXR AF-2 mutant for multidrug-resistant cancer gene therapy.

SXR AF-2 突变体对多重耐药癌症基因治疗的显着负面作用。

基本信息

批准号：
14571079
负责人：
TAKESHITA Akira
金额：
$ 1.66万
依托单位：
Okinaka Memorial Institute for Medical Research
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

项目摘要

The multidrug resistance (MDR) is a major cause of failure of cancer chemotherapy. The overexpression of the MDR1 gene that encodes P-glycoprotein is responsible for the development of drug-resistant tumor cells. Recent study revealed that the orphan nuclear receptor SXR regulates MDR1 gene expression. A variety of compounds such as rifampicin (RFP) have been shown to bind to SXR as ligands and stimulate transcription. We speculate that the inhibition of SXR-mediated transcription may be an effective approach to suppress MDR1 gene expression, whereby tumor cells may turn to response to the chemotherapy.We created artificial deletion mutant of human SXR (SXRΔAF2), lacking 15 residues in the helix 12 of the C-terminus. We examined transcriptional activity and dominant negative activity of SXRΔAF2, and found that SXRΔAF2 had strong dominant negative activity. A mammalian two-hybrid assay revealed that the dominant negative activity by SXRΔAF2 is likely due to lack of coactivator binding and constitutive recruitment of the corepressors.In real time PCR analysis, mRNA expression of MDR1 in colon cancer cell line, LS 174T cells, was in creased in RFP-dependent manner. Then, we created stable cell line, which express SXRΔAF2 mutant to examine whether SXRΔAF2 can inhibit RFP-stimulated MDR1 mRNA expression in this cell line. Currently, we are under investigation.The use of SXRΔAF2 may be a novel therapeutic approach using mutant nuclear receptor for the treatment of multidrug-resistant cancer.

多药耐药（MDR）是癌症化疗失败的主要原因。编码p糖蛋白的MDR1基因的过度表达与耐药肿瘤细胞的发展有关。最近的研究发现孤儿核受体SXR调控MDR1基因的表达。各种化合物如利福平（RFP）已被证明可以作为配体与SXR结合并刺激转录。我们推测，抑制sfr介导的转录可能是抑制MDR1基因表达的有效途径，从而使肿瘤细胞对化疗产生反应。我们构建了人类SXR的人工缺失突变体（SXRΔAF2），该突变体在c端螺旋12中缺少15个残基。我们检测了SXRΔAF2的转录活性和显性负活性，发现SXRΔAF2具有很强的显性负活性。一项哺乳动物双杂交实验显示，SXRΔAF2的主要负活性可能是由于缺乏辅激活因子结合和辅抑制因子的组成性募集。实时PCR分析显示，结肠癌细胞系LS 174T细胞中MDR1 mRNA表达呈rfp依赖性升高。然后，我们建立了稳定的细胞系，表达SXRΔAF2突变体，以检测SXRΔAF2是否可以抑制rfp刺激的MDR1 mRNA的表达。目前，我们正在接受调查。SXRΔAF2的使用可能是利用突变核受体治疗多药耐药癌症的一种新的治疗方法。