The Role of MEF1 in Multidrug Resistance

描述：（应用摘要）P-糖蛋白（P-gp）， MDR 1基因是导致肿瘤细胞产生多药耐药（MDR）的重要基因。急性 骨髓性白血病（AML），其中P-gp发生在20%的新发和约 75%的继发性病例，P-gp表达为阴性预后 功能.我们最近发现了一种新的130 kDa转录因子， 作为MDR 1启动子增强因子1（MEF 1），与GTCAATCC结合 MDR 1启动子元件并增加其在HL-60/VCR细胞中的活性， AML细胞系HL-60的MDR变体。此外，162 kDa的MEF 1相关的 MEF 1-AP蛋白与MEF 1相互作用，但其功能尚不清楚。 该应用程序的总体目标是解开的结构， 这些蛋白质增强MDR 1启动子活性的机制， 并利用这些知识来阻止MDR 1基因的表达。具体目标是 （1）纯化、克隆和进一步表征MEF 1和MEF 1-AP;（2）继续 探讨MEF 1和MEF 1-AP对MDR 1启动子的调控作用 活性;以及（3）确定MEF 1和MEF 1-AP是否在MEF 1和MEF 1-AP之间起作用。 MDR相关药物对MDR 1基因的转录激活， 调节剂，并分析其亚细胞定位和正常组织 表情具体目标1被设计为（a）纯化这些蛋白质的氨基 酸测序;（B）克隆MEF 1和MEF 1-AP的全长cDNA， 从HL-60/VCR细胞中筛选cDNA文库;和（c）产生抗体， 这些蛋白质。在具体目标2中，我们将（a）确定MEF 1是否与 使用酵母双杂交筛选系统与MEF 1-AP体内杂交;和（B） 用MEF 1和MEF 1-AP cDNA克隆到双顺反子载体中， 表达载体，并确定MEF 1单独或与MEF 1-AP一起是否增强了MEF 1-AP的表达。 MDR 1启动子/荧光素酶活性、MDR 1 mRNA和P-gp表达。具体目标 3的目的是（a）用MEF 1和MEF-AP转染HL-60和HL-60/VCR细胞 以有义或反义方向克隆到表达载体中的cDNA， 确定这些因素是否参与MDR 1基因的激活 （B）检测它们的转录水平; 用诱导型MEF 1和MEF 1-AP转染的细胞中的亚细胞定位 通过共聚焦显微镜观察表达载体;以及（c）确定这些表达载体是否 蛋白质在各种MDR细胞和某些正常组织中表达。 解开这些蛋白质的分子结构和机制 增强MDR 1启动子活性有助于设计策略， 抑制MDR 1表达。

DESCRIPTION: (Application Abstract) P-glycoprotein (P-gp), the product of the MDR1 gene, causes multidrug resistance (MDR) in cancer cells. In acute myelogenous leukemia (AML) where P-gp occurs in 20 percent of de novo and about 75 percent of secondary cases, expression of P-gp is a negative prognostic feature. We recently discovered a novel 130 kDa transcription factor, referred to as the MDR1 promoter-enhancing factor 1 (MEF1), which binds to the GTCAATCC element of the MDR 1 promoter and increases its activity in HL-60/VCR cells, a MDR variant of the AML cell line HL-60. Moreover, a 162 kDa MEF1-associated protein (MEF1-AP) interacts with MEF1 but its function remains to be found. The overall goal of the application is to unravel the structures of and mechanism(s) by which these proteins enhance the activity of the MDR1 promoter, and use this knowledge to prevent MDR1 gene expression. The Specific Aims are to (1) purify, clone, and further characterize MEF1 and MEF1-AP; (2) continue to investigate the roles of MEF1 and MEF1-AP in regulating MDR1 promoter activity; and (3) determine whether MEF1 and MEF1-AP play roles in the transcriptional activation of the MDR1 gene by MDR-related drugs and modulators, and analyze their subcellular localization and normal tissue expression. Specific Aim 1 is designed to (a) purify these proteins for amino acid sequencing; (b) clone the full-length cDNAs for MEF1 and MEF1-AP by screening the cDNA library from HL-60/VCR cells; and (c) produce antibodies for these proteins. In Specific Aim 2 we will (a) determine whether MEF1 interacts with MEF1-AP in vivo using the yeast-two hybrid screening system; and (b) transfect cancer cells with MEF1 and MEF1-AP cDNAs cloned into bicistronic expression vectors and determine whether MEF1 alone or with MEF1-AP enhances MDR1 promoter/luciferase activity, MDR1 mRNA and P-gp expression. Specific Aim 3 is designed to (a) transfect HL-60 and HL-60/VCR cells with MEF1 and MEF-AP cDNAs cloned in the sense or antisense orientation into expression vectors and determine whether these factors are involved in the activation of MDR1 gene transcription by MDR-related drugs and modulators; (b) examine their subcellular localization in cells transfected with inducible MEF1 and MEF1-AP expression vectors by confocal microscopy; and (c) determine whether these proteins are expressed in various MDR cells and certain normal tissues. Unraveling the molecular structures of and mechanism(s) by which these proteins enhance the MDR1 promoter activity should aid in the design of strategies to prevent MDR1 expression.