ANTISENSE RNA AND DNA INHIBITION OF KI-RAS TRANSLATION

已观察到21 Kda膜蛋白（p21）的过度产生， 几种哺乳动物肿瘤，并与ras基因扩增相关。 在 研究中的实验系统，Y1小鼠肾上腺癌， 大约有30个明显正常的Ki-ras基因拷贝， 高水平的p21。 本研究的主要目的是 开发特异性抑制p21合成的系统， 这种抑制可以产生转化表型的逆转。 具体目标如下：（1）优化全文转录 长度的反义Ki-ras序列（来自于 初步实验）在Y1细胞中，并证实这些杂交 以特异性方式与Y1 Ki-ras mRNA结合并抑制p21翻译。 （二） 构建额外的转录部分反义的抗ras质粒 Ki-ras序列对mRNA的某些区域具有特异性，并测试其 在体外和培养细胞中对p21抑制的有效性。 （三） 构建针对不同区域的序列特异性抗ras寡核苷酸， Ki-ras mRNA的表达，并测试它们对p21抑制的有效性， 和培养的细胞中。 (4)研究p21减少的后果 通过评估Y1细胞的特性， 转型 其中包括增长率、增长因素 要求，核型（例如，DM或HSR丢失）恶性程度， 在裸鼠中诱导肿瘤的能力。 (5)确定的有效性 7反义杂交（Ki-ras质粒构建体或寡核苷酸） p21在其他细胞系中的翻译，包括那些过表达的 Ki-ras mRNA，但Ki-ras基因未扩增和ras突变的人 基因

Overproduction of a 21 Kda membrane protein (p21) has been observe in several mammalian tumors and is correlated with ras gene amplification. In the experimental system under study, the Y1 mouse adrenal carcinoma, there are approximately 30 copies of an apparently normal Ki-ras gene and a correspondingly high level of p21. The main goals of this study are to develop systems to specifically inhibit p21 synthesis and to test whether such inhibition can produce a reversal of the transformed phenotype. Specific goals are as follows: (1) Optimize the transcription of full- length antisense Ki-ras sequences (from the plasmid pIFN-ras described in the preliminary experiments) in Y1 cells and confirm that these hybridize in a specific manner with Y1 Ki-ras mRNA and inhibit p21 translation. (2) Construct additional anti-ras plasmids which transcribe partial antisense Ki-ras sequences specific for certain areas of the mRNA and test their effectiveness on p21 inhibition both in vitro and in cultured cells. (3) Construct sequence specific anti-ras oligonucleotides to various regions of Ki-ras mRNA and test their effectiveness on p21 inhibition both in vitro and in cultured cells. (4) Examine the consequences of p21 reduction through assessment of properties of Y1 cells associated with their transformation. Among these would be changes in growth rate, growth factor requirements, karyotype (e.g., loss of DMs or HSR) degree of malignancy and ability to induce tumors in nude mice. (5) Determine the effectiveness of 7antisense hybridons (Ki-ras plasmid constructs or oligonucleotides) on the translation of p21 in other cell lines including those with overexpressed Ki-ras mRNA but an unamplified Ki-ras gene and those with a mutated ras gene.