MECHANISMS OF NON-P-GLYCOPROTEIN MULTIDRUG RESISTANCE

非P-糖蛋白多药耐药机制

• 批准号: 3460525
• 负责人: WILLIAM T BELLAMY
• 金额: $ 9.81万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3460525
• 关键词: antineoplastics; biomarker; gene expression; genetic strain; glycoproteins; immunocytochemistry; laboratory mouse; membrane proteins; molecular cloning; monoclonal antibody; multidrug resistance; neoplasm /cancer pharmacology; northern blottings; nucleic acid sequence

Resistance to chemotherapy continues to be a major impediment to the successful treatment of many types of cancer. Failure of chemotherapy may be in part due to the emergence of drug resistance. Attention has been directed towards the multiple drug resistance phenotype (MDR) in which cells selected for resistance to a given antineoplastic agent simultaneously develop resistance to other drugs which are structurally and functionally unrelated. This phenotype appears to be associated with the expression of a 170 kD membrane glycoprotein designated as the P-glycoprotein (PGP) which is the product of the mdr-1 gene. Recent studies suggest that tumor cells may also exhibit an MDR phenotype without expression of the mdr-1 gene. The focus of this proposal is to identify and characterize those mechanisms of multidrug resistance alternative to mdr-1. The principle model system to be studied is the human breast carcinoma cell line MCF-7 and a subline selected for resistance to mitoxantrone, MCF-7/MITOX. Selection for resistance to this agent in vitro results in cells which are cross resistant to anthracyclines, vinca alkaloids, and epipodophyllotoxins. There is no overexpression of PGP nor of the mdr-1 gene in these cells although there is a marked decrease in drug accumulation which is energy dependent. No evidence for an alteration in the intracellular target of this drug, DNA topoisomerase II, has been found to date. Considering the similar patterns of drug resistance and transport in non-PGP MDR as compared to PGP-mediated MDR, we postulate that there may be an alternative drug transport system operating in the non-PGP cell lines. To identify and characterize this system, we have constructed cDNA expression libraries from the drug-resistant MCF-7/MITOX and the drug-sensitive MCF-7/S cell lines and are screening them for differentially expressed genes through the use of subtractive hybridization techniques. In addition, we have produced monoclonal antibodies directed against proteins uniquely expressed in the drug-resistant cell line and will use them to screen the cDNA libraries as well. Differentially expressed cDNA clones identified by our screening procedures will be characterized by northern blot analysis and DNA sequencing. Their relationship to known genes and proteins will be ascertained through screening currently existing data bases such as GenBank and the National Biomedical Research Foundation protein data base and their relationship to previously cloned genes ascertained. To demonstrate a causative role in the drug resistant phenotype, full length cDNA clones will be isolated and transfected into drug-sensitive host cells. By studying this form of drug resistance means may be developed which will lead to increased diagnostic capabilities and a potential target for alternative therapy with chemosensitizers.

化疗耐药仍然是化疗的主要障碍 成功治疗多种癌症。 化疗失败 可能部分是由于耐药性的出现。 注意有 已针对多重耐药表型（MDR） 哪些细胞选择对给定的抗肿瘤药物产生耐药性 同时对其他药物产生耐药性，这些药物在结构上 并且功能上不相关。 该表型似乎与 170 kD 膜糖蛋白的表达 P-糖蛋白（PGP）是mdr-1基因的产物。 最近的 研究表明肿瘤细胞也可能表现出 MDR 表型 不表达 mdr-1 基因。 该提案的重点是 识别和表征多药耐药机制 MDR-1 的替代品。 要研究的原理模型系统是 人乳腺癌细胞系MCF-7及其亚系 对米托蒽醌、MCF-7/MITOX 的耐药性。 选择抗性 这种药物在体外会导致细胞交叉耐药 蒽环类、长春花生物碱和表鬼臼毒素。 没有 尽管存在 PGP 或 mdr-1 基因在这些细胞中的过度表达 是能量依赖性药物蓄积的显着减少。 不 该药物的细胞内靶标 DNA 发生改变的证据 迄今为止已发现拓扑异构酶II。 考虑到类似 与非 PGP MDR 相比的耐药性和转运模式 PGP介导的MDR，我们假设可能有替代药物 运输系统在非 PGP 细胞系中运行。 识别和 为了表征该系统，我们构建了 cDNA 表达库 来自耐药 MCF-7/MITOX 和药物敏感 MCF-7/S 细胞 品系，并通过以下方式筛选它们的差异表达基因 使用消减杂交技术。 此外，我们还有 产生独特的针对蛋白质的单克隆抗体 在耐药细胞系中表达，并将用它们来筛选 cDNA文库也是如此。鉴定出差异表达的 cDNA 克隆 我们的筛选程序将通过 Northern blot 进行表征 分析和 DNA 测序。 它们与已知基因的关系 将通过筛选现有数据来确定蛋白质 GenBank、国家生物医学研究基金会等基地 蛋白质数据库及其与先前克隆的基因的关系 确定。 证明耐药性中的致病作用 表型，全长 cDNA 克隆将被分离并转染到 药物敏感的宿主细胞。 通过研究这种形式的耐药性 可能会开发出更多的诊断手段 的能力和替代疗法的潜在目标 化学增敏剂。