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。在大多数情况下，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，这种交叉耐药通常是由于被称为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，P-糖蛋白是MDR1或ABCB1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的可能性，我们开发了实时PCR检测已知的48种ABC转运蛋白中的大多数；这些技术已被用于在已知耐药的癌细胞系中关联新的ABC转运蛋白的表达。在NCI-60细胞系中，大约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。此外，该分析还揭示了一些药物对表达P-gp的细胞的毒性比不表达P-gp的细胞更大，这提示了一种治疗耐多药癌症的新方法。已经确定了几种具有这种性质的不同化学类别，包括硫代氨基脲。一种化合物NSC73306已被详细研究，并显示出通过在S期阻断它们以高特异性杀死表达p- gp的细胞。用NSC73306和相关药物治疗也会导致ABCB1 mRNA的周转增加。在NSC73306治疗下存活的细胞不表达P-gp，并且对使用天然产物药物（如蒽环类药物、紫杉醇和长春花生物碱）的化疗敏感。通过对NSC73306类似物的定量结构活性分析和对NCI-60细胞系的进一步相关性分析，我们发现了许多具有相似杀伤p- gp表达细胞能力的化合物，但其溶解度得到了改善。在与NCI发展治疗项目（DTP）的合作下，NSC73306在小鼠模型的临床前测试中得到了合适的配方。高通量筛选底物、抑制剂或特异性杀死p- gp表达细胞的新药物的技术已经开发出来。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。我们的合作者Robert Innis在NIMH开发了c11 -去甲氧基-洛哌丁胺，在使用或不使用强效P-gp抑制剂（如tariquar）治疗的情况下，在癌症和大脑中对这种特异性P-gp底物的PET图像分布进行了研究。我们已经证明，在血脑屏障的三种最重要的转运蛋白（ABCB1, ABCC1, ABCG2）中，该化合物对ABCB1 （P-gp）具有特异性。P-gp的常见多态性变异也已被发现，但编码多态性似乎并未改变P-gp的药物转运功能。然而，在特定P-gp单倍型的环境中，同义多态性（C3435T，没有氨基酸变化）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的泵浦效率。这种单倍型似乎改变了mRNA折叠，并导致主要的翻译延迟，导致P-gp构象的改变。具有P-gp单倍型形式的稳定转染的猪LLC-PK1细胞与野生型P-gp转染相比，显示出不同的耐药性和抑制敏感性。我们使用TaqMan低密度阵列创建了一种高度敏感的人类癌症样本中ABC转运蛋白mrna定量分析方法。在对几种以NCI-60细胞系为代表的癌细胞的初步研究中，我们发现临床癌症中ABC转运体基因的表达模式与已建立的细胞系有很大不同。这一结果表明，目前的体外癌细胞模型可能不足以研究癌症的耐药性。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。利用重组SV40衣壳蛋白，可以在体外包装DNA和RNA。特别是，siRNA和化学修饰的siRNA可以以比脂肪转染所需的低得多的浓度高效递送。递送有毒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的靶向癌症。

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. In most 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 in the NCI-60 cell lines with resistance to specific cytotoxic drugs. 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. Treatment with NSC73306 and related drugs also results in increased turnover of ABCB1 mRNA. Cells that survive NSC73306 treatment do not express P-gp and are sensitive to chemotherapy with natural product drugs such as anthracyclines, paclitaxel and Vinca alkaloids. A quantitative structure activity analysis of NSC73306 analogs and a further correlation analysis in the NCI-60 cell lines has yielded many additional compounds with a similar ability to kill P-gp-expression cells, but improved solubility properties. In collaboration with the NCI Developmental Therapeutics Program (DTP), appropriate formulation of NSC73306 for pre-clinical testing in mouse models has been achieved. Technology enabling a high-throughput screen for new agents that are substrates, inhibitors or specifically kill P-gp-expressing cells has been developed. Studies on the normal function of P-gp suggest that it is involved in normal uptake and distribution of many drugs. C11-desmethoxy-loperamide has been developed by our collaborator Robert Innis in NIMH to PET image distribution of this specific P-gp substrate in cancers and in the brain, with and without treatment with potent inhibitors of P-gp such as tariquidar. We have shown that among three most prominent transporters at the blood-brain barrier (ABCB1, ABCC1, ABCG2), this compound is specific for ABCB1 (P-gp). Common polymorphic variants of P-gp have also 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. This haplotype appears to change mRNA folding, and cause a major translational delay which results in altered conformation of P-gp. Stable transfectants of porcine LLC-PK1 cells with the haplotype form of P-gp show altered drug resistance and inhibitory sensitivity compared to wild-type P-gp transfectants. We have created a highly sensitive, quantitative assay for ABC transporter mRNAs in human cancer samples using TaqMan Low Density Arrays. In initial studies of several cancers whose cell lines are represented in the NCI-60 cell lines we have found that patterns of expression of ABC transporter genes in clinical cancers differ substantially from those of the established cell lines. This result suggests that current in vitro cancer cell models may not be adequate to study drug resistance in cancer. 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 and chemically modified siRNAs 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.