Genetic Analysis of the Multidrug Resistance Phenotype in Tumor Cells

由于特定蛋白质表达的内在或获得性变化，癌细胞对化疗产生抗性。 我们已经研究了对天然产物化疗药物如阿霉素、紫杉醇和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耐药性。 对于天然产物药物，这种交叉耐药性是由于称为P-糖蛋白（P-gp）的能量依赖性药物外排系统（MDR 1基因的产物）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中的多效性缺陷导致的药物流入减少。最近的证据表明，在这些顺铂耐药细胞的内吞作用，包括受体介导的和流体相的内吞作用的整体缺陷。 对P-gp作用机制的研究主要集中在转运蛋白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酶激活以及ATP酶如何导致药物转运和外排。 我们已经在12个跨膜（TM），2个ATP位点的MDR 1转运蛋白，包括与MDR 2的嵌合体的各个结构域的分子改变，并表征了这些突变对在培养细胞中基于牛痘病毒的系统中高水平瞬时表达P-gp后的转运功能的影响。两个ATP位点的突变和生化分析表明，两者都是必不可少的，但它们的ATP结合和催化活性不同。 这些研究和其他研究导致了以下主要结论：（1）存在多个可能重叠的底物和抑制剂相互作用位点，主要由来自P-gp的氨基端（TM 5，6）和羧基端（TM 11，12）的TM片段形成;（2）底物相互作用位点包括高亲和力“开”位点、低亲和力“关”位点和影响底物结合“开”和“关”位点的能力的变构位点;（3）氨基端和羧基端ATP位点对P-gp的功能都是必需的，这两个位点是部分可互换的，但不是完全相同的：（4）两个ATP位点不是同时被利用的，支持ATP酶在底物转运过程中交替激活的模型;（5）ATP酶的激活导致底物与P-gp结合的减少，这与底物从“on”位点到“off”位点的移位一致。第二个ATP分子可能需要水解，以使转运蛋白恢复到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，并且它的表达降低了HIV对CD 4阳性细胞的感染性。在基因治疗中使用MDR 1基因作为显性选择性标记已经集中于开发SV 40作为递送MDR 1的载体。MDR 1可以在体内和体外有效地包装到SV 40载体中，并递送到造血细胞和许多其他细胞类型中。

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.