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，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，这种交叉耐药通常是由于被称为P-糖蛋白（P -糖蛋白）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<i>MDR</i>1或<i>ABCB</i>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。大约30种ABC转运蛋白的表达与对特定细胞毒性药物的耐药性有关。转染这些转运蛋白中的一些已证实它们赋予了对相关研究中检测到的药物的耐药性。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。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，我们发现了几种具有类似杀伤p- gp表达细胞能力的化合物，但其溶解度得到了改善。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。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。使用<i>MDR</i>1基因作为基因治疗中的显性选择标记，重点是开发SV40作为传递<i>MDR</i>1的载体。利用重组SV40衣壳蛋白，可以在体外包装</i>的DNA和RNA。特别是，siRNA可以以比脂肪感染所需的低得多的浓度高效递送。递送毒性dna，如<i>假单胞菌外毒素</i> cDNA，可用于在体外</i>和小鼠异种植物模型中靶向肿瘤<i>。细胞内药物积累的最终水平既取决于药物摄取的速率，也取决于药物排出的速率。我们已经开始探索已知的溶质载体（SLC和SLCO）转运体在癌细胞药物敏感性和耐药性模式中的作用。我们最初的方法是将NCI-60细胞系中SLC和SLCO转运体的表达与药物敏感性程度联系起来。获得了几个命中，表明摄取转运蛋白的表达与药物敏感性有关。

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 <i>MDR</i>1 or <i>ABCB</i>1 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. A quantitative structure activity analysis of NSC73306 analogs has yielded several additional compounds with a similar ability to kill P-gp-expression cells, but improved solubility properties. 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 <i>MDR</i>1 gene as a dominant selectable marker in gene therapy has focused on the development of SV40 as a vector for delivery of <i>MDR</i>1. Using recombinant SV40 capsid proteins, it is possible to package DNA and RNA <i>in vitro</i>. 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 <i>Pseudomonas exotoxin</i> cDNA, can be used to target cancers <i>in vitro</i> and in mouse xenoplant models. The ultimate level of drug accumulation in a cell depends on both the rate of drug uptake as well as the rate of efflux. We have begun to explore the role of known solute carrier (SLC and SLCO) transporters as contributors to patterns of drug sensitivity and resistance in cancer cells. Our initial approach has been to correlate expression of SLC and SLCO transporters with degree of drug sensitivity in the NCI-60 cell lines. Several hits were obtained, indicating that expression of uptake transporters is associated with drug sensitivity.