Regulation of the Human MDR1 Gene

描述（申请人提供）：MDR 1基因表达是一个重要的 许多人类癌症的预后标志物。本申请集中于 负责调节MDR 1表达的潜在机制， 特别是涉及转录调节因子的NF-IL 6家族。目标1。 研究NF-IL-6激活或抑制MDR 1表达的作用。的 假设在人类癌症中NF-IL 6家族成员表达改变 这些细胞中的MDR 1活化可能是由这些细胞引起的。实验 方法将包括MDR 1启动子构建体的共转染， 不同形式的NF-IL 6，NF-IL 6家族成员的定量分析， 细胞核和细胞质提取物，以及磷酸化状态的研究， 细胞模型中的NF-IL 6种类。目标2.研究蛋白质-蛋白质相互作用 在MDR 1中由NF-IL 6介导。在MCF-7中定位了NF-IL 6 -2相互作用位点 在MDR 1 P1启动子的-128至-75范围内的细胞，该区域缺乏NF-IL 6 结合基序NF-IL 6可以通过多种途径激活MDR 1启动子。 互动网站NF-IL 6家族成员之间的物理相互作用， Y-盒相关因子（NF-Y和YB-1）和AP 1（c-fos和c-jun）将被 通过利用GST-NF-IL 6融合的GST下拉实验在体外验证 蛋白质沉淀MDR细胞系的核提取物中的因子。一旦 蛋白质相互作用的建立，它们在调节蛋白质相互作用中的功能作用， 将在含有MDR 1的稳定转染子中检查染色体MDR 1基因 结构。目标3：一种新的MDR 1激活剂的研究 调控假设存在一种新的结合蛋白， 比NF-IL 6，负责维持MCF-7中的基础启动子活性 细胞该计划是确定蛋白质及其与-148结合的基因， -140元件的迁移率变动分析以及酵母单杂交系统。 将该结合蛋白的正义和反义cDNA转染入 MCF-7和MCF-7/ADR细胞以测试它们激活或调节 MDR 1表达。目标4。研究临床标本中MDR 1的调控。的 重点将是急性髓性白血病（AML）作为MDR 1的临床模型 表情将通过rtPCR和流式细胞术分析MDR 1。NF-IL 6成员 将在AML的细胞核和细胞质提取物中进行定量分析。

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.