SMALL MOLECULE REGULATION OF BREAST CANCER EXPRESSION

乳腺癌表达的小分子调控

• 批准号: 6329102
• 负责人: JOEL M. GOTTESFELD
• 金额: $ 34.88万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-22 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6329102
• 关键词: DNA footprinting; RNase protection assay; amides; antineoplastics; binding proteins; binding sites; breast neoplasms; drug design /synthesis /production; gel mobility shift assay; gene expression; genetic enhancer element; genetic promoter element; genetic regulation; laboratory mouse; messenger RNA; monoclonal antibody; neoplasm /cancer genetics; neoplastic cell; neoplastic growth; oncogenes; oncoproteins; polymerase chain reaction; tissue /cell culture; transcription factor; western blottings

The proposed research plan involves the development of new drugs for the treatment of human breast cancer. We plan to design small molecule ligands to target the promoter and enhancer elements of-the Her-2/neu oncogene and other genes that encode proteins involved in breast cancer proliferation. Since overexpression and/or mutation of Her-2/neu is correlated with a poor prognosis in breast and ovarian carcinomas, downregulation of the Her-2/neu gene (and protein) may prove effective in combination therapy for these cancers. Synthetic pyrrole-imidizole (Py-lm) polyamides have been shown to bind a wide range of DNA sequences with subnanomolar affinities; moreover, these molecules can inhibit transcription factor-DNA interactions and gene expression both in vitro and in cultured cells. A series of Py-Im polyamides will be designed and synthesized to target the region immediately flanking the TATA and CCAAT box elements and the binding sites for Sp1, AP-2, ESX, and RBPJkappa within the promoter and proximal enhancer of the human Her-2/neu oncogene. Using DNase footprinting methods and gel mobility shift assays, we will determine whether these polyamides inhibit binding of the TATA-box binding protein, TBP, to the Her-2/neu TATA element and other transcription factors to their cognate sites in this promoter. The effects of the polyamides on basal and activated transcription will be monitored using cell-free extracts from breast carcinoma cells and transcription assays with purified factors and RNA polymerase II. We will monitor the effects of the polyamides, individually and in combination, on Her-2/neu mRNA expression in human breast carcinoma cell lines. The levels of Her-2/neu protein in polyamide-treated and control cells will be monitored by Western blotting with a monoclonal antibody to Her-2/neu. The effects of down-regulation of Her-2/neu mRNA and protein levels on cell viability, cellular motility and tumorogenicity will be monitored in both cell-based assays and in small animal models. Toxicity of the polyamides will be assessed in cell-based assays and non-specific effects of the polyamides on genome-wide gene expression will be monitored using high density DNA array/hybridization methodology.

提出的研究计划涉及开发用于治疗人类乳腺癌的新药。 我们计划设计小分子配体，以针对HER-2/NEU癌基因的启动子和增强子元素，以及编码参与乳腺癌增殖的蛋白质的其他基因。 由于HER-2/NEU的过表达和/或突变与乳腺癌和卵巢癌的预后不良相关，因此HER-2/NEU基因（和蛋白质）的下调可能在这些癌症的结合治疗中有效。 已证明合成吡咯 - 硅质（PY-LM）聚酰胺与亚洋摩尔亲和力结合了多种DNA序列。此外，这些分子可以在体外和培养细胞中抑制转录因子-DNA相互作用和基因表达。 将设计和合成一系列的PY-IM聚酰胺，以瞄准立即侧翼TATA和CCAAT盒元素以及SP1，AP-2，ESX，ESX和RBPJKAPPA的结合位点，并在人类HER-2/NEU ONCONECON的启动子和近代增强子中内部的增强子内的sp1，ap-2，esx和rbpjkappa。 使用DNase足迹方法和凝胶迁移率转移测定，我们将确定这些聚酰胺是否抑制TATA-box结合蛋白TBP与HER-2/NEU TATA元素的结合以及对该启动子中同源位点的其他转录因子的结合。 将使用乳腺癌细胞中的无细胞提取物以及具有纯化因子和RNA聚合酶II的转录测定法对聚酰胺对基础和激活转录的影响进行监测。 我们将单独和组合监测聚酰胺对人乳腺癌细胞系中HER-2/NEU mRNA表达的影响。 聚酰胺处理和对照细胞中HER-2/NEU蛋白的水平将通过蛋白质印迹使用与HER-2/NEU的单克隆抗体进行监测。 HER-2/NEU mRNA和蛋白质水平下调对细胞活力，细胞运动性和肿瘤性的影响将在基于细胞的测定中和小动物模型中受到监测。 聚酰胺的毒性将在基于细胞的测定中进行评估，并且将使用高密度DNA阵列/杂交方法来监测聚酰胺对全基因组表达的非特异性作用。