Genetic Analysis of the Multidrug Resistance Phenotype in Tumor Cells

癌细胞对化疗的耐药性是由于内在的或获得性的 特定蛋白质表达的变化。我们研究了对天然产物的抗性 化学治疗剂，如阿霉素、阿米卡星生物碱和紫杉醇，以及合成的 药物顺铂。在这两种情况下，细胞同时对多种药物产生耐药性， 细胞内药物浓度的降低。对于天然产物药物， 交叉耐药通常是由于能量依赖性药物外排系统的表达 (ABC转运蛋白）称为P-糖蛋白（P-gp），其产物为 <i>MDR</i>1或<i>ABCB</i>1基因，或 ABC转运蛋白家族的其他成员。探索其他成员 ABC转运蛋白家族的一个成员可能与癌症的耐药性有关， 开发了实时PCR，用于检测48种已知ABC转运蛋白中的大多数;这些 技术已经被用于关联癌细胞中新的ABC转运蛋白的表达 已知的抗药性已显示约30种ABC转运蛋白的表达 与对特定细胞毒性药物的耐药性相关。其中几种的转染 转运体已经证实，它们赋予了对在 相关性研究此外，这项分析表明，有些药物的毒性更大， P-糖蛋白表达细胞的耐药率明显高于非表达细胞，提示了一种新的治疗MDR的方法 癌的具有这种性质的几种不同的化学类别，包括氨基硫脲， 已经被确认。一种化合物，NSC 73306，已被详细研究，并显示出杀死 通过将其阻断在S期，使表达P-gp的细胞具有高特异性。存活的细胞 不表达P-gp，对天然产物药物化疗敏感， 蒽环类、紫杉醇类和双氢类生物碱。的定量结构活性分析 NSC 73306类似物产生了几种具有类似杀伤能力的其他化合物 P-gp表达细胞，但改善了溶解性。正常功能的研究 P-gp参与多种药物的正常摄取和分布。共同 已检测到P-gp的多态性变体，但编码多态性似乎不 改变P-gp的药物转运功能。然而，一个同义多态性（C3435 T，no 氨基酸改变）在特定P-gp单倍型的设置中可影响P-gp的效率。 通过改变蛋白质折叠的节奏和改变底物和抑制剂来泵送 与P-gp的相互作用<i>MDR</i>1基因作为显性基因的应用 基因治疗中的选择性标记已经集中于开发SV 40作为用于 交付<i>MDR</i>1。使用重组SV 40衣壳蛋白， 可以<i>在体外</i>包装DNA和RNA。在 特别地，siRNA可以以高效率并且以比siRNA低得多的浓度递送。 是脂质转染所必需的递送有毒DNA，如<i>假单胞菌 外毒素</i>cDNA，可用于靶向癌症<i>， 体外</i>和小鼠异种植物模型中。药物累积的最终水平 在细胞中的作用取决于药物摄取的速率以及外排的速率。我们已经开始 探索已知溶质载体（SLC和SLCO）转运蛋白作为 癌细胞的药物敏感性和耐药性模式。我们最初的做法是 SLC和SLCO转运蛋白的表达与药物敏感程度相关， NCI-60细胞系。获得了几个匹配结果，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.