OLIGONUCLEOTIDE INHIBITION OF ONCOGENE EXPRESSION

这项工作的目标是阐明的机制， 反义寡核苷酸抑制细胞增殖 由人类c-myc和cHaras原癌基因转化。 人cmyc编码一种保守的核蛋白p65， 可能参与基因表达调控，c-Ha-ras 编码保守的内质膜蛋白p21， 可能参与转导生长因子信号。 细胞 转变可能是由于不恰当的表达或 细胞增殖过程中癌基因产物的反应，因此， 降低c-myc和cHa-ras基因表达水平可能是 原则上足以逆转转型。 计算 cmyc和cHaras mRNA的二级结构预测 在帽和起始密码子之间有许多环和凸起， 对反义寡脱氧核苷酸杂交阻滞敏感。 向HL 60细胞中加入抗cmyc十五核苷酸， 培养抑制增殖并刺激分化， 剂量依赖性和序列特异性的方式和拮抗剂， Haras十五核苷酸抑制肿瘤细胞增殖 T24转化NIH 3 T3细胞。 在这个项目中，剂量 反义寡核苷酸对c-myc的依赖性作用 和cHa-ras mRNA转录、稳定性、翻译、DNA 复制和细胞形态将在正常和 转化细胞 寡脱氧核苷酸摄取， 区室化和降解将在相同的环境中进行研究。 细胞系 然而，寡脱氧核苷酸不够稳定， 用于静脉内或口服给药。 寡脱氧核苷 甲基膦酸酯是不带电的和核酸酶抗性的， 动物细胞，并保护它们免受病毒的挑战，但 在体外的效果出乎意料地低于正常 寡脱氧核苷酸 R和S立体异构体存在于每个 磷原子，但只有S型具有相同的构象 正常的寡脱氧核苷酸。 立体特异性allS 将合成寡脱氧核苷甲基膦酸酯， 测试它们在杂交阻滞中的功效。

The goal of this work is to elucidate the mechanism by which antisense oligodeoxynucleotides inhibit proliferation of cells transformed by human c-myc and cHaras proto-oncogneses. Human cmyc codes for a conserved nuclear protein, p65, which may be involved in regulation of gene expression, and c-Ha-ras codes for a conserved inner plasma membrane protein, p21, which may be involved in transducing growth factor signals. Cell transformation may be caused by inappropriate expression or response of oncogene products during cell proliferation, so that reducing the level of cmyc and cHa-ras expression may in principle be sufficient to reverse transformation. Calculated secondary structures of cmyc and cHaras mRNAs predict many loops and bulges between the cap and the initiation codon, susceptible to antisense oligodeoynucleotide hybridization arrest. Addition of an anti-cmyc pentadecanucleotide to HL60 cells in culture inhibits proliferation and stimulates differentiation in a dose dependent and sequence specific manner and an antic- Haras pentadecanucleotide inhibits proliferation of T24transformed NIH 3T3 cells. In this project, the dose dependent effects of antisense oligodeoxynucleotides on cmyc and cHa-ras mRNA transcription, stability, translation, DNA replication, and cell morphology will be determined in normal and transformed cells. Oligodeoxynucleotide uptake, compartmentalization and degradation will be studied in the same cell lines. Oligodeoxynucleotides, however, are not stable enough for intravenous or oral administration. Oligodeoxynucleoside methylphosphonates are uncharged and nuclease resistant, enter animal cells and protect them from viral challenge, but are unexpectedly less effective in vitro than normal oligodeoxynucleotides. R and S stereoisomers occur at each phosphorus atom, but only the S form has the same conformation as a normal oligodeoxyonucleotide. Stereospecific allS oligodeoxynucleoside methylphosphonates will be synthesized and tested for their efficacy in hybridization arrest.