GENETIC ANALYSIS OF THE MULTIDRUG RESISTANCE PHENOTYPE IN TUMOR CELLS

我们继续分析多药的作用机制。 运输商，并致力于制定新的战略 规避癌症的多药耐药并开发分子 多药转运体的知识，以设计新的癌症治疗方法。 多药转运蛋白(P-糖蛋白)已纯化至近 同质性和高活性的药物依赖的ATPase 重组为蛋白脂质体后的比活性。水泡 含有能够转运的P-糖蛋白具有非常活性的P-糖蛋白 糖蛋白激酶，这种活性是由GTP刺激的。至少一个 新的质膜相关P-糖蛋白-激酶已部分 纯化，但它在调节多药转运体活性中的作用 尚未确定。动力学研究表明， 转运蛋白与药物在脂质双层内相互作用，并且是间接的 有证据表明，药物可以从内部和外部移除。 双层的传单。P-糖蛋白的分子调控 与ATP其他成员的点突变和嵌合体的分析- 结合盒(ABC)超家族转运蛋白已经揭示了多个 跨膜结构域附近或内部的分子区域 影响底物专一性，并表明了 Mdr1和mdr2之间的ABC，一种未知特异性的相关转运体。 插入失活研究P-糖蛋白的功能 Mdr1b基因在小鼠肾上腺Y-1细胞中的缺失，并失去 这些细胞分泌高于基础水平的类固醇。我们一直在继续 开发MDR-1基因作为基因治疗的显性可选择标记。 表达人多药耐药基因的逆转录病毒载体能够提供 转导和移植小鼠骨髓对紫杉醇的耐药性 细胞，这一策略正在考虑用于人类的基因治疗 在癌症大剂量化疗期间保护骨髓。另外两个 多药耐药基因系统正在开发中，以帮助 多药耐药的其他机制分析：(1)人黑色素瘤 对表鬼臼毒素(Vp-16和Vm-26)和 在拓扑异构酶II中缺失ALA 428的蒽环类药物；以及 (2)高水平顺铂耐药人肝癌和KB腺癌 有多种蛋白质变化的细胞。

We have continued to analyze the mechanism of action of the multidrug transporter and have worked on the development of new strategies to circumvent multidrug resistance in cancer and to exploit molecular knowledge of the multidrug transporter to design new cancer treatments. The multidrug transporter (P-glycoprotein) has been purified to near homogeneity and shown to be an active drug-dependent ATPase of high specific activity after reconstitution into proteoliposomes. Vesicles containing P-glycoprotein capable of transport have very active P- glycoprotein kinases, and this activity is stimulated by GTP. At least one novel plasma membrane associated P-glycoprotein-kinase has been partially purified, but its role in regulating activity of the multidrug transporter has not yet been determined. Kinetic studies demonstrate that the transporter interacts with drugs within the lipid bilayer, and indirect evidence suggests that drug may be removed from both the inner and outer leaflets of the bilayer. Molecular manipulation of P-glycoprotein by analysis of point mutations and chimeras with other members of the ATP- binding cassette (ABC) superfamily of transporters has revealed multiple regions of the molecule near or within the transmembrane domains which affect substrate specificity, and has indicated the interchangeability of ABCs between MDR1 and MDR2, a related transporter of unknown specificity. Function of P-glycoprotein has been explored by insertional inactivation of the mdr1b gene in mouse adrenal Y- 1 cells, with loss of ability of these cells to secrete steroids above basal levels. We have continued to develop the MDR 1 gene as a dominant selectable marker for gene therapy. Retroviral vectors expressing the human MDR1 cDNA are able to confer resistance to taxol on transduced and transplanted mouse bone marrow cells, and this strategy is under consideration for gene therapy in humans to protect bone marrow during high dose chemotherapy for cancer. Two other multidrug resistant genetic systems are under development to aid in the analysis of other mechanisms of multidrug resistance: (1) A human melanoma line cross-resistant to epipodophyllotoxins (VP-16 and VM-26) and anthracyclines which has a deletion of Ala 428 in topoisomerase II; and (2) High level cis-platinum resistant human hepatoma and KB adenocarcinoma cells with multiple protein alterations.