The Role of MEF1 in Multidrug Resistance

描述：(应用摘要)P-糖蛋白(P-gp)，是 MDR1基因，导致肿瘤细胞产生多药耐药(MDR)。在急性期 粒细胞白血病(AML)，其中P-gp出现在20%的初治患者中，约 在75%的继发性病例中，P-gp的表达是一种阴性预后 特写。我们最近发现了一种新的130 kDa转录因子，参考 TO为mdr1启动子增强因子1(MEF1)，与GTCAATCC结合 并增加其在HL-60/VCR细胞中的活性， AML细胞系HL-60的多药耐药变异体。此外，162 kDa的MEF1相关基因 蛋白质(MEF1-AP)与MEF1相互作用，但其功能尚不清楚。这个 应用程序的总体目标是解开和的结构 这些蛋白质增强mdr1启动子活性的机制(S)， 并利用这一知识来阻止MDR1基因的表达。具体目标是 (1)纯化、克隆并进一步鉴定MEF1和MEF1-AP；(2)继续 MEF1和MEF1-AP在mdr1启动子调控中的作用 以及(3)确定MEF1和MEF1-AP是否在 多药耐药相关药物对多药耐药基因转录激活的影响 并分析它们的亚细胞定位和正常组织 表情。特异性目标1是为了(A)提纯这些蛋白质中的氨基 (B)克隆MEF1和MEF1-AP的全长cDNA 从HL-60/VCR细胞中筛选cDNA文库；及(C)产生抗体 这些蛋白质。在具体目标2中，我们将(A)确定MEF1是否相互作用 在体内使用酵母双杂交筛选系统进行MEF1-AP；和(B) 克隆入双顺反子的MEF1和MEF1-AP基因转染癌细胞 表达载体，并确定MEF1单独或与MEF1-AP一起增强 Mdr1启动子/荧光素酶活性、mdr1mRNA和P-gp表达。特定目标 3的目的是(A)用MEF1和MEF-AP转染HL-60和HL-60/VCR细胞 正、反义定向克隆入表达载体和 确定这些因素是否参与多药耐药基因的激活 MDR相关药物和调节剂的转录；(B)检查其 诱导型MEF1和MEF1-AP转基因细胞的亚细胞定位 通过共聚焦显微镜检测表达载体；以及(C)确定这些 蛋白质在各种多药耐药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作用机制(S) 增强mdr1启动子的活性应有助于制定 防止MDR1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.