MULTIDRUG RESISTANCE--P GLYCOPROTEIN GENES

在癌症的不同类型的耐药性中，这一现象 多药耐药构成了一个特别重要的 临床问题，因为它涉及对许多常用药物的耐药性 抗癌药物，包括长春花碱、蒽环类和 表鬼臼毒素。人类细胞产生多药耐药性 由于一种名为mdrl的基因表达增加。Mdrl基因 是多基因家族的成员，该家族至少包括一个 另一个表达基因是mdr2。Mdrl基因的产物是一种 大膜蛋白(P-糖蛋白)，很可能 充当外排泵，提供更少的药物 在多药耐药细胞中积累。一种改变的模式 在一个病例中发现了对不同药物的交叉耐药性 从mdrl基因点突变导致单一氨基 P-糖蛋白中的酸取代。P的许多方面- 对糖蛋白的功能知之甚少。尤其是，它是 未知是什么决定了P-糖蛋白的特异性 与不同结构药物的相互作用，以及mdrl基因是如何 表达是受调控的。在拟议的研究中，内含子/外显子 Mdrl基因的结构将通过比较cdna来确定。 和基因组克隆。多药耐药基因中存在点突变 不同多药耐药细胞系中的基因将 用核糖核酸酶保护试验进行研究。美国政府的角色 初步鉴定的药物结合、核苷酸结合和 P-糖蛋白的糖硅化位点将通过 寡核苷酸定向诱变。局部随机诱变 将被用来识别参与该过程的其他氨基酸残基 单抗、药物与P-糖蛋白的结合 抑制剂。Mdrl基因表达的调节将通过以下方式进行研究 鉴定诱导该基因表达的化合物 在组织培养中通过鉴定和分析顺式调控 Mdrl基因的序列。Mdr2基因的作用将是 通过对mdr2全长cDNA的克隆和测序， MDR2在不同类型细胞中的表达及基因分析 Mdr2的转移和表达。DNA的特异性和作用 在mdr2基因中观察到的重排也将被分析。 临床上检测mdrl表达的方法 基于原位RNA杂交的肿瘤样本将被 为临床研究开发和优化。

Among different types of drug resistance in cancer, the phenomenon of multidrug resistance constitutes a particularly important clinical problem since it involves resistance to many commonly used anticancer agents, including Vinca alkaloids, anthracyclines and epypodophyllotoxins. Multidrug resistance in human cells results from increased expression of a gene designated mdrl. The mdrl gene is a member of a multigene family, which includes at least one other expressed gene, mdr2. The product of the mdrl gene is a large membrane protein (P-glycoprotein), which most probably functions as an efflux pump, providing for decreased drug accumulation in multidrug-resistant cells. An altered pattern of cross-resistance to different drugs was found in one case to result from point mutations in the mdrl gene leading to a single amino acid substitution in P-glycoprotein. Many aspects of P- glycoprotein function are poorly understood. In particular, it is unknown what determines the specificity of P-glycoprotein interaction with structurally different drugs, and how mdrl gene expression is regulated. In the proposed studies the intron/exon structure of the mdrl gene will be determined by comparison of cDNA and genomic clones. The presence of point mutations in the mdrl gene in different multidrug-resistant cell lines will be investigated by RNase protection assays. The role of the tentatively identified drug-binding, nucleotide-binding and glycosilation sites of P-glycoprotein will be analyzed by oligonucleotide-directed mutagenesis. Localized random mutagenesis will be used to identify other amino acid residues involved in the binding of monoclonal antibodies, drugs and P-glycoprotein inhibitors. Regulation of mdrl gene expression will be studied by identifying the compounds which induce the expression of this gene in tissue culture and by identifying and analyzing cis-regulatory sequences in the mdrl gene. The role of the mdr2 gene will be studied by cloning and sequencing of full-length mdr2 cDNA, analyzing mdr2 expression in different types of cells and by gene transfer and expression of mdr2. The specificity and role of DNA rearrangements observed in the mdr2 gene will also be analyzed. Finally, protocols for detection of mdrl expression in clinical tumor samples, based on in situ RNA hybridization, will be developed and optimized for clinical studies.