GENETIC ANALYSIS OF THE MULTIDRUG RESISTANCE PHENOTYPE IN TUMOR CELLS

癌细胞逃避化疗是因为特定蛋白质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、一些核苷类似物和重金属的交叉耐药性是由于摄取系统的多效性缺陷导致的药物内流减少。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个跨膜(TM)，2个ATP位点的MDR1转运蛋白的不同区域进行了分子改变，并研究了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，这些突变对转运功能的影响。Mdr1和mdr2的功能嵌合体揭示了mdr2的TM6的微小变化允许mdr2的N-一半支持mdr1骨架中的多药转运，证明了TM6在决定底物特异性方面的重要性。此外，我们已经证明，TM12中的特定突变改变了MDR识别包括氟戊醇在内的特定类型底物的能力。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)底物和抑制剂的相互作用存在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部位、低亲和力部位和影响底物与部位结合能力的变构部位；(3)氨基和羧基末端的ATP部位都是P-gp发挥作用所必需的，这些部位部分可以互换，但不完全相同；(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结合的减少，与底物从现场到非现场的移位一致。将mdr1基因作为显性的可选择标记用于基因治疗，最近的重点是开发SV40作为mdr1的载体。MDR1可以被有效地包装成SV40载体并输送到造血细胞中。顺铂，基因治疗，多药耐药，磷酸化，痘苗病毒，载体，非人类受试者或人类组织

Cancer cells elude chemotherapy 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, and heavy metals is due to a reduction in drug influx resulting from a pleiotropic defect in uptake systems. 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 and characterize 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. Functional chimeras of MDR1 and MDR2 have revealed that minor alterations in TM6 of MDR2 allow the N-half of MDR2 to support multidrug transport in an MDR1 backbone, demonstrating the importance of TM6 in determining substrate specificity. In addition, we have shown that a specific mutation in TM12 alters ability of MDR to recognize a specific class of substrates including flupentixol. 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 site; (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. Use of the MDR1 gene as a dominant selectable marker in gene therapy has recently focused on the development of SV40 as a vector for delivery of MDR1. MDR1 can be efficiently packaged into SV40 vectors and delivered into hematopoietic cells. - cisplatin, gene therapy, multidrug resistance, phosphorylation, vaccinia virus, vectors, - Neither Human Subjects nor Human Tissues