INHIBITION OF TRANSCRIPTION OF THE HUMAN EGFR GENE IN GLIOMA BY SITE-SPECIFIC OLIGONUCLEOTIDES DESIGNED TO FORM DNA TRIPLE HELICES

设计了嘌呤-嘧啶混合型抗基因寡脱氧核苷酸，与人表皮生长因子受体（EGFR）基因启动子中富含嘧啶的元件形成共线DNA三链体，作为基因密码子阻断剂。使用人鳞状细胞癌（A431）和人胶质瘤（U251 MG和U87 MG）细胞系评价它们作为EGFR基因转录抑制剂的作用。凝胶位移分析表明，寡核苷酸在Sp-1结合位点内形成共线三链体。体外试验揭示了三链体形成和EGFR转录抑制之间的相关性。我们假设鸟嘌呤残基并不总是最佳的同位G-C对形成三重螺旋的目标。我们发现，设计成与EGFR基因启动子的增强子元件形成三螺旋的寡核苷酸可以抑制mRNA的形成，也可以抑制人脑胶质瘤细胞系的增殖。与DNA双链体结合的抗基因可以作为体外基因控制的替代方案的基础。我们正试图开发允许表达抗基因的逆转录病毒载体。我们还考虑了抗基因策略作为胶质瘤辅助治疗的可行性。

Mixed purine-pyrimidine anti-gene oligodeoxynucleotides were designed to form collinear DNA triplexes with pyrimidine-rich elements in the human epidermal growth factor receptor (EGFR) gene promoter as gene code blocker. Their effects as suppressors of the EGFR gene transcription were evaluated using human squamous cell carcinoma (A431) and human glioma (U251MG and U87MG) cell lines. Gel shift analyzes indicated that the oligonucleotide forms a collinear triplex within the Sp-1 binding site. An in vitro assay revealed a correlation between the triplex formation and the suppression of EGFR transcription. We postulate that guanine residues are not always optimum in apposition to G-C pairs to form triple helices in the target. We found that oligonucleotides designed to form a triple helix with enhancer elements of the EGFR gene promoter can suppress mRNA formation and also the proliferation of a human glioma cell line. Anti-gene binding to a DNA duplex may serve as a basis for an alternative program of gene control in vitro. We are trying to develope retrovirus vectors which allow expression of anti-genes. We also consider the feasibility of an anti-gene strategy as adjuvant therapy of glioma.