ANTISENSE RNA AND DNA INHIBITION OF KI-RAS TRANSLATION

反义 RNA 和 DNA 对 KI-RAS 翻译的抑制

• 批准号: 3458593
• 负责人: MARGARET B PENNO
• 金额: $ 9.75万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-03-01 至 1993-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3458593
• 关键词: adrenal neoplasm; athymic mouse; enzyme induction /repression; genetic translation; growth factor; laboratory mouse; membrane proteins; messenger RNA; natural gene amplification; neoplastic growth; nucleic acid sequence; oncogenes; tissue /cell culture

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.

已观察到 21 Kda 膜蛋白 (p21) 的过量产生 多种哺乳动物肿瘤，并且与 ras 基因扩增相关。 在 正在研究的实验系统，Y1小鼠肾上腺癌，有 大约有 30 个明显正常的 Ki-ras 基因拷贝和一个 p21 的水平相应较高。 这项研究的主要目标是 开发专门抑制 p21 合成的系统并测试是否 这种抑制可以产生转化表型的逆转。 具体目标如下：（1）优化全转录 长度反义 Ki-ras 序列（来自质粒 pIFN-ras，描述于 Y1细胞中的初步实验）并确认这些杂交 以特定方式与 Y1 Ki-ras mRNA 结合并抑制 p21 翻译。 (2) 构建转录部分反义的额外抗 ras 质粒 Ki-ras 序列特异于 mRNA 的某些区域并测试其 在体外和培养细胞中对 p21 抑制的有效性。 (3) 构建针对不同区域的序列特异性抗 ras 寡核苷酸 Ki-ras mRNA 并在体外测试其对 p21 抑制的有效性 以及在培养的细胞中。 (4)考察p21减少的后果 通过评估 Y1 细胞与其相关的特性 转变。 其中包括增长率、生长因子的变化 要求、核型（例如，DM 或 HSR 缺失）、恶性程度和 具有在裸鼠中诱导肿瘤的能力。 (5) 确定有效性 7 反义杂交子（Ki-ras 质粒构建体或寡核苷酸） p21 在其他细胞系（包括过表达细胞系）中的翻译 Ki-ras mRNA，但未扩增的 Ki-ras 基因和 ras 突变的基因 基因。