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The Significance of NFATp Expression in Human Chondrosarcomas and Its Application for Gene Therapy

NFATp在人软骨肉瘤中表达的意义及其在基因治疗中的应用

基本信息

批准号：
13671506
负责人：
UEDA Takafumi
金额：
$ 2.24万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671506/
关键词：
chondrosarcoma gene therapy NFATp tissue-specific promoter type 11 collagen thymidine kinase II型コラーゲン

项目摘要

NFATs (Nuclear factor of activated T cells) are very important transcriptional regulatory factors of signal transduction through T lymphocyte receptors in lymphoid system. A recent experimental study using knock-out mice has demonstrated that one of the NFAT family, NFATp is an important suppressor of chondrogenesis in adult mice, and indicated that it is working as an inhibitory factor for the differentiation and proliferation of human mesenchymal stem cells (MSC) to chondrocytes. Thus, it is suggested that NFATp gene rearrangement might be involved in the oncogenesis and/or development of human chondrosarcomas, and we hypothesized that NFATp could be used as a molecular target for gene therapy in chondrosarcoma, which is a problematic malignant bone tumor because of its resistance against adjuvant chemotherapy and radiation therapy. To investigate the expression of NFATp gene in human chondrosarcomas, we have performed the screening of the expression of NFATp in frozen tumor samples obtained from 10 patients with chondrosarcoma using RT-PCR method However, all the 10 cases showed positive expression of NFATp gene. Moreover, Aoyama et al. from Kyoto University also reported that seven out of 26 cases of human chondrosarcoma showed NFATp gene mutations in 4 exons, but they were only silent mutation or polymorphism, and concluded that NFATp is not implicated in the pathogenesis of chondrosarcoma. From these results, we have changed the strategy of experimental gene therapy for chondrosarcoma from NFATp to the use of herpes simplex virus thymidine kinase (HS V-TK) and chondrocyte-specific type 11 collagen a2 gene (Col11a2) promoter, which is recently cloned by our colleague, Dr Tsumaki, targeting to chondrosarcomas. We have, therefore, designed and made a plasmid vector inserted HSV-TK and Col11a2 promoter, and transfected it into a human chondrosarcoma cell line to perform in vitro and in vivo experimental gene therapy for chondrosarcoma as a further project.

NFAT（激活T细胞核因子）是淋巴系统中通过T淋巴细胞受体进行信号转导的非常重要的转录调节因子。最近一项使用基因敲除小鼠的实验研究表明，NFAT家族之一的NFATp是成年小鼠软骨形成的重要抑制因子，并表明它是人间充质干细胞（MSC）向软骨细胞分化和增殖的抑制因子。因此，表明NFATp基因重排可能参与人类软骨肉瘤的肿瘤发生和/或发展，并且我们假设NFATp可以用作软骨肉瘤基因治疗的分子靶点，软骨肉瘤是一种有问题的恶性骨肿瘤，因为它对辅助化疗和放射治疗具有抵抗力。为了探讨NFATp基因在人软骨肉瘤中的表达情况，我们采用RT-PCR方法对10例软骨肉瘤患者的冷冻肿瘤样本中NFATp的表达进行了筛查，结果10例均呈阳性表达。此外，青山等人。京都大学的研究人员也报道，26例人类软骨肉瘤中有7例显示NFATp基因4个外显子突变，但只是沉默突变或多态性，并得出NFATp与软骨肉瘤发病无关的结论。根据这些结果，我们将软骨肉瘤的实验性基因治疗策略从 NFATp 改为使用单纯疱疹病毒胸苷激酶 (HS V-TK) 和软骨细胞特异性 11 型胶原蛋白 a2 基因 (Col11a2) 启动子，该启动子最近由我们的同事 Tsumaki 博士克隆，针对软骨肉瘤。因此，我们设计并制作了插入HSV-TK和Col11a2启动子的质粒载体，并将其转染到人软骨肉瘤细胞系中，作为进一步的项目进行软骨肉瘤的体外和体内实验基因治疗。