Regulation of the Human MDR1 Gene

描述(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记物。此应用程序侧重于 负责调控MDR1表达的潜在机制， 尤其涉及转录调控因子NF-IL6家族。目标1。 研究核因子-IL6在激活或抑制多药耐药基因表达中的作用。这个 假设核因子-IL-6家族成员在人类肿瘤中的表达改变 这些细胞中的多药耐药基因的激活可能由细胞负责。实验 方法将包括共转染MDR1启动子构建体和 不同形态的核因子-白介素6及其家族成员的定量分析 细胞核和细胞质提取液，以及对其磷酸化状态的研究 细胞模型中的核因子-白介素6。目的2.研究蛋白质与蛋白质的相互作用 由多药耐药基因mdr1中的NF-IL6介导。在MCF-7中定位了一个核因子-白介素6-2相互作用位点 Mdr1 P1启动子-128至-75内的细胞，该区域缺少核因子-IL6 具有约束力的主题。核因子-IL-6可能通过多种途径激活MDR1启动子 互动网站。核因子-IL-6家族成员之间的物理相互作用 Y-box相关因子(NF-Y和YB-1)和AP1(c-fos和c-jun)将是 利用GST-NF-IL6融合的GST下拉实验在体外验证 多药耐药细胞系核提取液中沉淀因子的蛋白质。一次 蛋白质之间的相互作用已经确立，它们在调节 染色体mdr1基因将在含有mdr1的稳定转染体中进行检测 构造。目的3.研究一种新的多药耐药相关激活剂 监管。这个假设是存在一种新的结合蛋白，其他 而不是NF-IL6，负责维持MCF-7的基本启动子活性 细胞。该计划是确定与-148结合的蛋白质及其基因 -140元件通过迁移率变化分析以及酵母单杂交系统。 该结合蛋白的正义和反义cDNA将被导入 MCF-7和MCF-7/ADR细胞，以测试其激活或调节能力 Mdr1表达。目的4.研究临床标本中多药耐药基因的调控。这个 焦点将集中在急性髓系白血病(AML)作为MDR1的临床模型上 表情。采用逆转录聚合酶链式反应和流式细胞术分析mdr1基因。核因子-IL6成员 将在AML的核提取液和细胞质提取液中进行定量分析。

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.