Genetic Analysis of the Multidrug Resistance Phenotype i

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，这种交叉耐药性通常是由于称为P-糖蛋白（P gp）的能量依赖性药物外排系统（ABC转运蛋白）的表达，即MDR 1或ABCB 1基因的产物，或ABC转运蛋白家族的其他成员。为了探索ABC转运蛋白家族的其他成员可能参与癌症耐药性的可能性，我们开发了实时PCR检测48种已知ABC转运蛋白中的大多数;这些技术已用于关联已知耐药性的癌细胞系中新型ABC转运蛋白的表达。已显示约30种ABC转运蛋白的表达与对特定细胞毒性药物的抗性相关。这些转运蛋白中的几种的转染已经证实，它们赋予在相关性研究中检测到的药物抗性。此外，该分析揭示了一些药物对P-gp表达细胞的毒性比对非表达细胞的毒性更大，这提示了治疗MDR癌症的新方法。已经鉴定出具有这种性质的几种不同的化学类别，包括氨基硫脲。一种化合物，NSC 73306，已被详细研究，并显示通过阻断它们在S期以高特异性杀死P-gp表达细胞。存活的细胞不表达P-gp，并且对天然产物药物如蒽环类、紫杉醇和苦碟子生物碱的化疗敏感。对P-gp正常功能的研究表明，P-gp参与多种药物的正常摄取和分布。已检测到P-gp的常见多态性变体，但编码多态性似乎不会改变P-gp的药物转运功能。然而，在特定P-gp单倍型的背景下的同义多态性（C3435 T，无氨基酸变化）可以通过改变蛋白质折叠的节奏和改变底物和抑制剂与P-gp的相互作用来影响P-gp泵的效率。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。使用重组SV 40衣壳蛋白，可以在体外包装DNA和RNA。特别地，siRNA可以以高效率和比脂质转染所需的低得多的浓度递送。递送毒性DNA，如假单胞菌外毒素cDNA，可用于体外和小鼠异种植物模型中靶向癌症。我们还发现了黑色素瘤细胞中ABC转运蛋白的独特特征。这些转运蛋白之一ABCB 5与P-gp（MDR 1）密切相关，似乎有助于黑素瘤细胞中的MDR。对顺铂在黑素瘤细胞中的细胞处理的详细分析表明，它积聚在黑素体中（而不是细胞核中），然后可以被细胞挤出。黑色素瘤对化疗的一些顽固性可能归因于其独特的ABC转运蛋白集和黑色素体隔离和细胞毒性药物的挤出。顺铂耐药突变体显示，对甲氨蝶呤、某些核苷类似物、重金属和毒素的交叉耐药是由于摄取系统多效性缺陷导致的药物流入减少。最近的证据表明，这些顺铂耐药细胞的内吞作用存在全面缺陷，细胞内蛋白质运输、细胞骨架和线粒体能量产生也存在缺陷

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 frequently due to expression of an energy-dependent drug efflux system (ABC transporter) known as P-glycoprotein (P gp), the product of the MDR1 or ABCB1 gene, or to other members of the ABC transporter family. To explore the possibility that other members of the ABC family of transporters may be involved in drug resistance in cancer, we have developed real-time PCR for detection of most of the 48 known ABC transporters; these techniques have been used to correlate expression of novel ABC transporters in cancer cell lines of known drug resistance. Expression of approximately 30 ABC transporters has been shown to correlate with resistance to specific cytotoxic drugs. Transfection of several of these transporters has confirmed that they confer resistance to the drugs detected in the correlation studies. Furthermore, this analysis has revealed that some drugs are more toxic to P-gp expressing cells than to non-expressors, suggesting a novel approach to treatment of MDR cancers. Several different chemical classes with this property, including thiosemicarbazides, have been identified. One compound, NSC73306, has been studied in detail and shown to kill P-gp-expressing cells with high specificity by blocking them in S phase. Surviving cells do not express P-gp and are sensitive to chemotherapy with natural product drugs such as anthracyclines, paclitaxel and Vinca alkaloids. 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. However, a synonymous polymorphism (C3435T, no amino acid change) in the setting of a specific P-gp haplotype can affect efficiency of P-gp pumping by altering the rhythm of protein folding and changing substrate and inhibitor interactions with 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 and RNA in vitro. In particular, siRNA can be delivered with high efficiency and at much lower concentrations than are needed for lipofection. Delivery of toxic DNAs, such as Pseudomonas exotoxin cDNA, can be used to target cancers in vitro and in mouse xenoplant models. We have also found a unique signature of ABC transporters in melanoma cells. One of these transporters, ABCB5, is closely related to P-gp (MDR1) and appears to contribute to MDR in melanoma cells. Detailed analysis of the cellular handling of cisplatin in melanoma cells has indicated that it accumulates in melanosomes (and not in nuclei) that can then be extruded by the cells. Some of the intractability of melanomas to chemotherapy may be attributable to both its unique set of ABC transporters and melanosomal sequestration and extrusion of cytotoxic drugs. Cisplatin-resistant mutants have revealed that 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 and defects in intracellular protein trafficking, the cytoskeleton and mitochondrial energy production