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: 6433042
• 负责人: MICHAEL M GOTTESMAN
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/6433042
• 关键词: P glycoprotein; adenocarcinoma; adenosinetriphosphatase; antineoplastics; biological transport; chemical binding; cis platinum compound; drug delivery systems; drug receptors; enzyme activity; enzyme structure; fungal genetics; gene expression; gene therapy; genetic markers; human genetic material tag; multidrug resistance; neoplastic cell; transfection /expression vector; vaccinia virus

Resistance to chemotherapy occurs in cancer cells because of intrinsic or acquired changes in expression of specific proteins. We have studied resistance to natural product chemotherapeutic agents such as doxorubicin, Vinca alkaloids, and taxol, and to the synthetic drug cisplatin. In both cases, cells become simultaneously resistant to multiple drugs because of reductions in intracellular drug concentrations. For the natural product drugs this cross-resistance is due to expression of an energy-dependent drug efflux system known as P-glycoprotein (P-gp), the product of the MDR1 gene. For cisplatin, cross-resistance to methotrexate, some nucleoside analogs, heavy metals, and toxins is due to a reduction in drug influx resulting from a pleiotropic defect in uptake systems. Recent evidence suggests a global defect in endocytosis in these cisplatin resistant cells, including both receptor-mediated and fluid phase endocytosis. Studies on mechanism of action of P-gp have focused on the manner in which many different substrates and inhibitors are recognized by the transporter, how substrate interaction results in activation of ATPase, and how ATPase results in drug translocation and efflux. We have made molecular alterations in various domains of the 12 transmembrane (TM), 2 ATP-site MDR1 transporter, including chimeras with MDR2, and characterized the effects of these mutations on transport function after high-level transient expression of P-gp in a vaccinia virus-based system in cultured cells. Mutational and biochemical analysis of the two ATP sites demonstrates that both are essential, but their ATP binding and catalytic activities differ. These studies and others have led to the following major conclusions: (1) there are multiple, probably overlapping sites for interaction of substrates and inhibitors primarily formed by TM segments from both the amino-terminal (TM5,6) and carboxy-terminal (TM11,12) halves of P-gp; (2) substrate interaction sites include a high affinity "on" site, a lower affinity "off" site, and an allosteric site which affects ability of substrates to bind to the "on" and"off" sites; (3) both amino- and carboxy-terminal ATP sites are essential for function of P-gp and the sites are partially interchangeable, but not identical; (4) both ATP sites are not utilized simultaneously, supporting a model of alternating activation of ATPase during substrate transport; and (5) activation of ATPase results in a reduction of substrate binding to P-gp, consistent with translocation of substrate from the "on" site to the "off" site. A second molecule of ATP may need to be hydrolyzed to return the transporter to its native high affinity state. Studies on the normal function of P-gp suggest that it is involved in normal uptake and distribution of many drugs, and that its expression reduces infectivity of CD4 positive cells by HIV. Use of the MDR1 gene as a dominant selectable marker in gene therapy has focused on the development of SV40 as a vector for delivery of MDR1. MDR1 can be efficiently packaged into SV40 vectors both in vivo and in vitro and delivered into hematopoietic cells and many other cell types.

癌细胞对化疗产生耐药性是由于特定蛋白表达的内在或获得性变化。我们研究了天然产物化疗药物如阿霉素、长春花碱和紫杉醇以及合成药物顺铂的耐药性。在这两种情况下，由于细胞内药物浓度的降低，细胞同时对多种药物产生抗药性。对于天然产物药物，这种交叉耐药性是由于mdr1基因的产物P-糖蛋白(P-gp)的能量依赖的药物外排系统的表达。对于顺铂，对甲氨蝶呤、一些核苷类似物、重金属和毒素的交叉耐药性是由于摄取系统的多效性缺陷导致药物流入减少。最近的证据表明，这些顺铂耐药细胞的内吞作用存在全局性缺陷，包括受体介导的内吞作用和液相内吞作用。对P-gp作用机制的研究主要集中在转运蛋白识别多种不同底物和抑制剂的方式，底物相互作用如何导致ATPase的激活，以及ATPase如何导致药物的转运和外排。我们在12跨膜(TM)、2个ATP位点的mdr1转运蛋白的不同区域进行了分子改变，包括与mdr2的嵌合体，并研究了这些突变对P-gp在基于痘苗病毒的培养细胞系统中高水平瞬时表达后对转运蛋白功能的影响。突变和生化分析表明，这两个ATP位点都是必需的，但它们的ATP结合和催化活性不同。这些研究和其他研究得出了以下主要结论：(1)底物与抑制剂的相互作用有多个可能重叠的部位，主要由P-gp的氨基末端(TM5，6)和羧基末端(TM11，12)的TM片段形成；(2)底物相互作用部位包括高亲和力的“开”部位，低亲和力的“关”部位，以及影响底物与“开”和“关”部位结合能力的变构部位；(3)氨基和羧基末端的ATP位点都是P-gp功能所必需的，并且这两个位点是部分可互换的，但不是完全相同的；(4)两个ATP位点不是同时利用的，支持底物转运过程中ATPase交替激活的模型；(5)ATPase的激活导致底物与P-gp的结合减少，这与底物从“开”位到“关”位的移位是一致的。第二个ATP分子可能需要被水解，以使转运蛋白返回到其天然的高亲和力状态。对P-gp正常功能的研究表明，P-gp参与了多种药物的正常摄取和分配，其表达可降低HIV对CD4阳性细胞的感染力。在基因治疗中，MDR1基因作为显性可选择标记的应用主要集中在发展SV40作为MDR1载体的研究。MDR1可以在体内和体外有效地包装成SV40载体，并被输送到造血细胞和许多其他类型的细胞中。