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

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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.