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

阐明整合到细胞中的cDNA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。然而，引入的cDNA的命运尚未得到很好的研究。经CD95 (Fas/Apo-1) cdna转染的肝癌细胞（F6b）形成的实体瘤（MH134）经抗CD95单克隆抗体（mAb）单次治疗在临床上完全治愈，但在小鼠体内潜伏期后复发。复发肿瘤对重复单抗治疗有耐药性。肿瘤中耐药细胞的含量估计为每10^8个细胞2-8个。用单抗治疗的腹水F6b肿瘤也出现耐药细胞。从它们分离的5个典型单细胞克隆中，3个完全不表达表面CD95，丢失了完整的cDNA， 2个保留了cDNA，但表达CD95的水平低于F6b细胞。在这两个克隆中，整合的cDNA被严重甲基化，用去甲基化试剂azadeoxycytidine体外处理后，CD95的表达和对单抗的敏感性得以恢复，这表明DNA甲基化是CD95表达降低和对单抗耐药的原因。用mAb再次处理这2个克隆的腹水肿瘤进一步降低了CD95的表达，导致更重的甲基化，但没有缺失cDNA。结果清楚地表明，转染CD95 cDNA的CD95^+肿瘤细胞可以通过CD95介导的凋亡途径，通过消除和甲基化整合的cDNA来抵抗mAb的攻击。整合cDNA的消除和甲基化似乎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。

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.