MECHANISMS OF NON-P-GLYCOPROTEIN MULTIDRUG RESISTANCE

对化疗的抗药性仍然是影响人类健康的主要障碍。 成功地治疗了多种癌症。化疗失败 可能部分原因是出现了抗药性。注意力已经 多药耐药表型(MDR) 选择哪些细胞对给定的抗肿瘤药物产生抗药性 同时对其他结构上的药物产生耐药性 而且在功能上不相关。这种表型似乎与 170kD膜糖蛋白的表达 P-糖蛋白(P-gp)是MDR-1基因的产物。近期 研究表明，肿瘤细胞也可能表现出多药耐药表型。 不表达MDR-1基因。这项提议的重点是 识别和描述这些多药耐药的机制 MDR-1的替代品。待研究的原则性模型体系为 人乳腺癌细胞系MCF-7及其亚系 对米托蒽醌、MCF-7/Mitox耐药。选择抗病品种 这种药物在体外会产生交叉抗性的细胞 蒽环类、长春花碱和表鬼臼毒素。没有 Pgp或MDR-1基因在这些细胞中的过度表达 是药物积累的显着减少，这是能量依赖的。不是 这种药物的细胞内靶点DNA发生变化的证据 拓扑异构酶II，迄今已被发现。考虑到相似的 非PGP多药耐药患者耐药和转运模式的比较 Pgp介导的MDR，我们推测可能存在一种替代药物 在非PGP细胞系中运行的运输系统。以识别和 对该系统进行了鉴定，构建了表达文库 耐药细胞株MCF7/Mitox和耐药细胞株MCF7/S 并正在通过以下途径筛选差异表达基因 差减杂交技术的使用。此外，我们还有 产生了针对蛋白质的独一无二的单抗 在耐药细胞系中表达，并将其用于筛选 也构建了相应的cDNA文库。鉴定差异表达的cDNA克隆 我们的筛选程序将以Northern印迹为特征 分析和DNA测序。它们与已知基因和 蛋白质将通过筛选目前存在的数据来确定 基因库和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基因的关系 已经确定了。证明在抗药性中起到致病作用 表型、全长cdna克隆将被分离并导入 药物敏感的宿主细胞。通过研究这种形式的耐药性 可以开发出能够提高诊断率的方法 替代治疗的能力和潜在目标 化学增敏剂。

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.