MULTIDRUG RESISTANCE--P GLYCOPROTEIN GENES

多药耐药性--P糖蛋白基因

• 批准号: 3180147
• 负责人: IGOR B RONINSON
• 金额: $ 22.12万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-01 至 1993-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3180147
• 关键词: aminoacid; anthracyclines; antineoplastics; chemical binding; chemical structure function; clone cells; combination chemotherapy; complementary DNA; drug resistance; gene expression; genetic regulation; genome; glycoproteins; glycosylation; human tissue; in situ hybridization; membrane proteins; molecular cloning; molecular oncology; natural gene amplification; neoplasm /cancer genetics; neoplasm /cancer pharmacology; neoplastic cell; nucleic acid hybridization; oligonucleotides; pharmacogenetics; podophyllin; point mutation; vinca alkaloids

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.

在不同类型的癌症耐药性中， 多药耐药性的研究构成了一个特别重要的 临床问题，因为它涉及到许多常用的耐药性 抗癌剂，包括阿托伐他汀生物碱、蒽环类和 表鬼臼毒素 人类细胞的多药耐药性结果 来自于一种称为mdr 1的基因表达的增加。 mdrl基因 是多基因家族的成员，其包括至少一个 其他表达基因，mdr 2. mdrl基因的产物是 大膜蛋白（P-糖蛋白），最有可能 作为外排泵，提供减少的药物 在多药耐药细胞中积累。 一种改变了的 对不同药物的交叉耐药性在一个病例中被发现， 从mdrl基因的点突变导致单个氨基酸 P-糖蛋白中的酸取代。 P的许多方面- 糖蛋白的功能知之甚少。 特别是 不知道是什么决定了P-糖蛋白的特异性 与结构不同药物的相互作用，以及mdrl基因 表达受到调控。 在拟议的研究中，内含子/外显子 mdr 1基因的结构将通过比较cDNA 和基因克隆。 mdrl中存在点突变 基因在不同的多药耐药细胞系将是 通过RNA酶保护测定进行研究。 的作用 初步鉴定的药物结合、核苷酸结合和 P-糖蛋白的糖基化位点将通过 抗坏血酸定向诱变。 局部随机诱变 将被用于鉴定参与蛋白质合成的其他氨基酸残基。 单克隆抗体、药物和P-糖蛋白的结合 抑制剂的 mdrl基因表达的调节将通过以下方法进行研究： 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节 MDR 1基因中的序列。 mdr 2基因的作用将是 通过全长mdr 2cDNA的克隆和测序研究， 通过基因分析不同类型细胞中的mdr 2表达 mdr 2的转移和表达。 DNA的特性和作用 还将分析在mdr 2基因中观察到的重排。 最后，为临床应用中mdrl表达的检测提供了方案。 肿瘤样本，基于原位RNA杂交，将 为临床研究开发和优化。