Genetic Analysis of the Multidrug Resistance Phenotype in Tumor Cells

化疗耐药发生在癌细胞中，是因为特定蛋白质表达的内在或获得性变化。我们研究了对天然产物化疗药物的耐药性，如阿霉素、长春花生物碱和紫杉醇，以及对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降低，细胞同时对多种药物产生耐药性。对于天然产物药物，这种交叉耐药是由于被称为p -糖蛋白（P-gp）的能量依赖性药物外排系统的表达，p -糖蛋白是MDR1基因的产物。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是由于摄取系统的多效性缺陷导致药物内流减少。最近的证据表明，这些顺铂耐药细胞的内吞作用存在全球性缺陷，包括受体介导和液相内吞作用。对P-gp作用机制的研究主要集中在转运体识别许多不同底物和抑制剂的方式，底物相互作用如何导致atp酶的激活，以及atp酶如何导致药物易位和外排。我们对12个跨膜（TM）， 2个atp位点的MDR1转运体的不同结构域进行了分子改变，包括与MDR2的嵌合体，并在培养细胞中以痘苗病毒为基础的系统中高水平瞬时表达P-gp后，表征了这些突变对转运功能的影响。对这两个ATP位点的突变和生化分析表明，它们都是必需的，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)存在多个可能重叠的底物和抑制剂相互作用位点，主要由P-gp的氨基端（TM5,6）和羧基端（TM11,12）一半的TM片段形成；(2)底物相互作用位点包括高亲和力的“on”位点、低亲和力的“off”位点和影响底物与“on”和“off”位点结合能力的变构位点；(3)氨基端和羧基端ATP位点对P-gp的功能都是必需的，它们部分可互换，但不完全相同；(4)两个ATP位点不是同时被利用的，这支持了在底物运输过程中ATP酶交替激活的模型；(5) atp酶的激活导致底物与P-gp的结合减少，这与底物从“on”位点转移到“off”位点一致。第二个ATP分子可能需要水解，以使转运体恢复其天然的高亲和力状态。对P-gp正常功能的研究表明，它参与许多药物的正常摄取和分布，其表达可降低HIV对CD4阳性细胞的感染性。在基因治疗中，将MDR1基因作为显性选择标记物的重点是将SV40作为MDR1的载体。在体内和体外，MDR1都可以有效地包装成SV40载体，并递送到造血细胞和许多其他细胞类型。

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.