MECHANISMS OF NON-P-GLYCOPROTEIN MULTIDRUG RESISTANCE

非P-糖蛋白多药耐药机制

• 批准号: 3460524
• 负责人: WILLIAM T BELLAMY
• 金额: $ 9.61万
• 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/3460524
• 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

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.

对化疗的耐药性仍然是化疗的主要障碍。 成功治疗多种癌症。 化疗失败 部分原因可能是抗药性的出现。 关注 针对多药耐药表型（MDR）， 这些细胞被选择用于抵抗给定的抗肿瘤剂， 同时对其他药物产生耐药性， 和功能无关。 这种表型似乎与 一种170 kD的膜糖蛋白的表达，称为 P-糖蛋白（PGP）是mdr-1基因的产物。 最近 研究表明，肿瘤细胞也可能表现出MDR表型， 而不表达MDR-1基因。 本提案的重点是 确定和描述多药耐药机制 MDR-1的替代物。 要研究的原理模型系统是 人乳腺癌细胞系MCF-7和选择用于 对米托蒽醌耐药，MCF-7/MITOX。 抗性选择 这种药剂在体外会导致细胞对以下物质产生交叉耐药性 蒽环类、长春花生物碱和表鬼臼毒素。 没有 在这些细胞中，PGP和mdr-1基因的过表达，尽管存在 是能量依赖性的药物积累的显著减少。 没有 这种药物的细胞内靶点DNA发生改变的证据 拓扑异构酶II，迄今已发现。 考虑到类似的 非PGP MDR的耐药和转运模式与 PGP介导的MDR，我们推测可能存在替代药物， 在非PGP细胞系中运行的转运系统。 识别和 我们构建了cDNA表达文库， 从耐药MCF-7/MITOX和药物敏感MCF-7/S细胞 并通过以下方法筛选它们的差异表达基因： 使用消减杂交技术。 另外我们有 产生了针对蛋白质的单克隆抗体， 在耐药细胞系中表达，并将使用它们来筛选 cDNA文库。鉴定的差异表达cDNA克隆 通过我们的筛选程序将其特征在于北方印迹 分析和DNA测序。 它们与已知基因的关系， 蛋白质将通过筛选现有数据来确定 国家生物医学研究基金会（National Biomedical Research Foundation） 蛋白质数据库及其与先前克隆基因的关系 确定。 为了证明在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抗药性 可能会开发出一种方法， 能力和替代疗法的潜在目标， 化学增敏剂。