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Basic study for Analysis of Intractable Resistant Tumor Cells and their Overcoming in Cancer Gene Therapy

难治性耐药肿瘤细胞分析及其在癌症基因治疗中克服的基础研究

基本信息

批准号：
14571125
负责人：
TAKEDA Yasutaka
金额：
$ 2.24万
依托单位：
The University of Tokyo
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 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571125/
关键词：
Cancer gene therapy Resistant cells Fas Mutation frequency Apoptosis cDNA depletion cDNA methylation anti-Fas mAb DNA欠落 DNAメチル化抗体療法

项目摘要

It is important for more effective gene therapies to clarify the mechanisms by which cDNA integrated intc cells can maintain or lose its function in vivo. However, the fate of the cDNA introduced has not been well studied.Solid tumors (MH134) formed by CD95 (Fas/Apo-1) cDNA-transfected hepatoma cells (F6b) were clinically completely cured by single treatment with anti-CD95 monoclonal antibody (mAb) but relapsed after some latency in mice. Relapsed tumors were resistant to be repeated the mAb treatment. The content of resistant cells in tumors was estimated to 2-8 per 10^8 cells. Resistant cells also appeared from ascites F6b tumors treated with the mAb. Of 5 typical single cell clones isolated from them, 3 did not express surface CD95 at all and lost integrated cDNA and 2 retained cDNA but expressed CD95 at lower levels than F6b cells. In these 2 clones, integrated cDNA was heavily methylated, and treatment in vitro with a demethylation reagent, azadeoxycytidine, restored CD95 expression and sensitivity to the mAb, indicating that DNA methylation was responsible for reduced CD95 expression and resistance to the mAb. Re-treatment of ascites tumors from these 2 clones with the mAb further reduced CD95 expression and caused still heavier methylation but not deletion of cDNA. The results clearly indicate that CD95^+ tumor cells transfected with CD95 cDNA can resist against the attack with the mAb via CD95-mediated apoptosis pathway by eliminating and methylating the integrated cDNA. Elimination and methylation of integrated cDNA appear to occur through different mechanisms.Our study of resistant tumor provides us important and fundamental information for improving the efficiency of gene-therapy.

阐明整合cDNA的intc细胞在体内维持或丧失其功能的机制，对于更有效的基因治疗具有重要意义。由CD 95（Fas/Apo-1）cDNA转染的肝癌细胞（F6 b）形成的实体瘤（MH 134）通过抗CD 95单克隆抗体（mAb）的单次治疗在临床上完全治愈，但在小鼠中经过一些潜伏期后复发。复发的肿瘤对重复mAb治疗具有抗性。肿瘤中耐药细胞的含量估计为2-8/10^8个细胞。用mAb处理的腹水F6 b肿瘤也出现抗性细胞。在5个典型的单细胞克隆中，3个克隆完全不表达表面CD 95，缺失整合的cDNA，2个克隆保留cDNA，但表达水平低于F6 b细胞。在这2个克隆中，整合的cDNA高度甲基化，体外用去甲基化试剂氮脱氧胞苷处理，恢复了CD 95表达和对mAb的敏感性，表明DNA甲基化是导致CD 95表达降低和对mAb耐药的原因。用mAb再处理来自这2个克隆的腹水肿瘤进一步降低了CD 95表达，并导致更重的甲基化，但不导致cDNA缺失。结果清楚地表明，转染CD 95 cDNA的CD 95 ^+肿瘤细胞可以通过CD 95介导的细胞凋亡途径消除和甲基化整合的cDNA来抵抗mAb的攻击。整合cDNA的甲基化和消除可能通过不同的机制发生，我们对耐药肿瘤的研究为提高基因治疗的效率提供了重要的基础信息。