Regulation of the Human MDR1 Gene

人类 MDR1 基因的调控

• 批准号: 6483913
• 负责人: BRANIMIR I SIKIC
• 金额: $ 27.95万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-08 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6483913
• 关键词: DNA binding protein; MCF7 cell; acute myelogenous leukemia; antisense nucleic acid; clinical research; complementary DNA; flow cytometry; gel mobility shift assay; gene expression; genetic promoter element; genetic regulation; human tissue; immunoprecipitation; interleukin 6; microarray technology; multidrug resistance; neoplasm /cancer genetics; neoplastic cell; phosphorylation; polymerase chain reaction; protein protein interaction; protein structure function; protooncogene; transcription factor; transfection; western blottings; yeast two hybrid system

DESCRIPTION (PROVIDED BY APPLICANT): MDR1 gene expression is an important prognostic marker in many human cancers. This application focuses on the underlying mechanisms responsible for regulating the expression of MDR1, particularly involving the NF-IL6 family of transcriptional regulators. Aim 1. To Study the Role of NF-IL6 in Activating or Suppressing MDR1 Expression. The hypothesis is that altered expression of NF-IL6 family members in human cancer cells may be responsible for MDR1 activation in these cells. Experimental approaches will include co-transfection of MDR1 promoter constructs and different forms of NF-IL6, quantitative analysis of NF-IL6 family members in nuclear and cytoplasmic extracts, and studies of the phosphorylation status of NF-IL6 species in cellular models. Aim 2. To Study Protein-Protein Interactions Mediated by NF-IL6 in MDR1. An NF-IL6-2 interacting site was mapped in MCF-7 cells within -128 to -75 of the MDR1 P1 promoter, a region which lacks NF-IL6 binding motifs. NF-IL6 may activate the MDR1 promoter through multiple interaction sites. Physical interactions among NF-IL6 family members, the Y-box-associated factors (NF-Y and YB-1), and AP1 (c-fos and c-jun) will be verified in vitro by GST pull down experiments utilizing a GST-NF-IL6 fusion protein to precipitate factors in nuclear extracts of MDR cell lines. Once protein interactions are established, their functional role in regulating the chromosomal MDR1 gene will be examined in stable transfectants containing MDR1 constructs. Aim 3. To Investigate a Novel Activator Involved in MDR1 Regulation. The hypothesis is that that there is a novel binding protein, other than NF-IL6, responsible for maintaining basal promoter activity in MCF-7 cells. The plan is to identify the protein and its gene binding to the -148 to -140 element by mobility shift assays as well as the yeast one-hybrid system. Sense and antisense cDNAs for this binding protein will be transfected into both MCF-7 and MCF-7/ADR cells to test their capacity to activate or modulate MDR1 expression. Aim 4. To Study MDR1 Regulation in Clinical Specimens. The focus will be on acute myeloid leukemia (AML) as a clinical model for MDR1 expression. MDR1 will be analyzed by rtPCR and flow cytometry. NF-IL6 members will be quantitatively analyzed in both nudear and cytoplasmic extracts of AML.

描述（由申请人提供）：MDR1 基因表达是一个重要的 许多人类癌症的预后标志物。该应用程序重点关注 负责调节 MDR1 表达的潜在机制， 特别是涉及转录调节因子 NF-IL6 家族。目标1。 研究 NF-IL6 在激活或抑制 MDR1 表达中的作用。这 假设是人类癌症中 NF-IL6 家族成员的表达发生改变 细胞可能负责这些细胞中 MDR1 的激活。实验性的 方法将包括 MDR1 启动子构建体的共转染和 不同形式的NF-IL6，NF-IL6家族成员的定量分析 核和细胞质提取物，以及磷酸化状态的研究 细胞模型中的 NF-IL6 种类。目标 2. 研究蛋白质-蛋白质相互作用 由 MDR1 中的 NF-IL6 介导。 MCF-7 中映射了 NF-IL6-2 相互作用位点 MDR1 P1 启动子 -128 至 -75 范围内的细胞，该区域缺乏 NF-IL6 结合图案。 NF-IL6可能通过多种途径激活MDR1启动子 互动网站。 NF-IL6 家族成员之间的身体相互作用 Y-box 相关因子（NF-Y 和 YB-1）和 AP1（c-fos 和 c-jun）将 利用 GS​​T-NF-IL6 融合体通过 GST Pull down 实验进行体外验证 蛋白质沉淀 MDR 细胞系核提取物中的因子。一次 蛋白质相互作用已建立，它们在调节中的功能作用 将在含有 MDR1 的稳定转染子中检查染色体 MDR1 基因 构造。目标 3. 研究涉及 MDR1 的新型激活剂 规定。假设存在一种新的结合蛋白，其他 比 NF-IL6 强，负责维持 MCF-7 中的基础启动子活性 细胞。该计划是鉴定与-148结合的蛋白质及其基因 -140 元素通过迁移率变化测定以及酵母单杂交系统。 该结合蛋白的有义和反义 cDNA 将被转染到 MCF-7 和 MCF-7/ADR 细胞测试其激活或调节能力 MDR1 表达。目标 4. 研究临床样本中的 MDR1 调控。这 重点将放在急性髓系白血病 (AML) 作为 MDR1 的临床模型上 表达。 MDR1 将通过 rtPCR 和流式细胞术进行分析。 NF-IL6成员 将在 AML 的核提取物和细胞质提取物中进行定量分析。