GENETIC ANALYSIS OF THE MULTIDRUG RESISTANCE PHENOTYPE IN TUMOR CELLS

肿瘤细胞多药耐药表型的遗传分析

• 批准号: 3774310
• 负责人: M GOTTESMAN
• 金额: --
• 依托单位: DIVISION OF CANCER BIOLOGY AND DIAGNOSIS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3774310
• 关键词: P glycoprotein; adenocarcinoma; adenosinetriphosphatase; antineoplastics; chemical kinetics; chimeric proteins; enzyme mechanism; gene therapy; genetic markers; genetic transduction; hepatocellular carcinoma; human genetic material tag; laboratory mouse; lipid bilayer membrane; liposomes; melanoma; multidrug resistance; mutant; neoplasm /cancer chemotherapy; neoplastic cell; paclitaxel; point mutation; protein purification; tissue /cell culture; transfection /expression vector

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.

我们继续分析了多药治疗的作用机制， 运输商，并致力于制定新的战略， 克服癌症的多药耐药性， 多药转运蛋白的知识来设计新的癌症治疗方法。 多药转运蛋白（P-糖蛋白）已被纯化至接近 并显示为高活性药物依赖性ATP酶 重组为脂蛋白体后的比活性。囊泡 含有能够转运的P-糖蛋白，具有非常活性的P- 糖蛋白激酶，并且这种活性由GTP刺激。至少一 新的质膜相关P-糖蛋白激酶已部分 纯化，但其在调节多药转运蛋白活性中的作用 尚未确定动力学研究表明， 转运蛋白与脂双层内的药物相互作用，并间接 有证据表明，药物可以从内部和外部移除。 双层的小叶。P-糖蛋白的分子操作 分析点突变和与ATP其他成员的嵌合体- 结合盒（ABC）转运蛋白超家族已经揭示了多种 在跨膜结构域附近或内部的分子区域， 影响底物特异性，并已表明 MDR 1和MDR 2之间的ABC，一种特异性未知的相关转运蛋白。 通过插入失活探索P-糖蛋白的功能 mdr 1b基因在小鼠肾上腺Y- 1细胞中的表达， 这些细胞分泌高于基础水平的类固醇。我们继续 MDR 1基因可作为基因治疗的显性选择标记。 表达人MDR 1 cDNA的逆转录病毒载体能够赋予 转导和移植的小鼠骨髓对紫杉醇的抗性 细胞，这种策略正在考虑用于人类的基因治疗 在癌症的大剂量化疗期间保护骨髓。另外两 多药耐药遗传系统正在开发中，以帮助 多药耐药的其他机制分析：（1）人黑素瘤 对表鬼臼毒素（VP-16和VM-26）交叉耐药的品系， 在拓扑异构酶II中缺失Ala 428的蒽环类;和 (2)高水平顺铂耐药的人肝癌和KB腺癌 有多种蛋白质改变的细胞。