Genetic Analysis of Multidrug Resistance Phenotype

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，包括受体介导的和流体期的内吞作用，以及细胞内蛋白质运输和细胞骨架的缺陷。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。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) atp酶的激活导致底物与P-gp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。已经检测到P-gp的常见多态性变异，但编码多态性似乎不会改变P-gp的药物转运功能。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。利用重组SV40衣壳蛋白，可以在体外包装DNA，包括P-gp和含有绿色荧光蛋白（GFP）的载体。使用体外包装的DNA转导P-gp和GFP是非常高效的，并且允许转移多达15 kb的DNA，而不需要在包装的DNA中使用SV40序列。这种方法为将P-gp转移到造血细胞和其他细胞中进行基因治疗提供了希望。我们还在犬模型中表明，用编码P-gp和人类白细胞介素受体共同γ链的嵌合载体转导骨髓干细胞可产生紫杉醇抗性骨髓，其中大多数循环血细胞表达γ链和P-gp。

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, and defects in intracellular protein trafficking and the cytoskeleton. 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. 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; and (2) 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. Common polymorphic variants of P-gp have been detected, but coding polymorphisms do not appear to alter the drug transport functions of P-gp. 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. Using recombinant SV40 capsid proteins, it is possible to package DNA in vitro, including P-gp and green fluorescent protein (GFP) containing vectors. Transduction of P-gp and GFP using in vitro packaged DNA is highly efficient, and allows transfer of up to 15 kb of DNA without the need for SV40 sequences in the packaged DNA. This approach offers promise for transfer of P-gp into hematopoietic and other cells for gene therapy. We have also shown in a canine model that transduction of bone marrow stem cells with a chimeric vector encoding P-gp and the human common gamma chain of interleukin receptors results in taxol-resistant bone marrow in which most circulating blood cells express the gamma chain and P-gp.