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 化疗剂，如阿霉素、苦马豆生物碱和紫杉醇。在大多数情况下，细胞 由于细胞内药物减少， 浓度的对于天然产物药物，这种交叉耐药性通常是由于 表达能量依赖性药物外排系统（ABC转运蛋白），称为P-糖蛋白 （P gp），MDR 1的产物，或 ABCB 1基因或ABC转运蛋白的其他成员 家人为了探索ABC转运蛋白家族的其他成员可能 与癌症耐药性有关，我们开发了实时聚合酶链反应， (PCR)用于检测48种已知ABC转运蛋白中的大多数;这些技术已被用于 将已知药物的癌细胞系中新型ABC转运蛋白的表达与 阻力大约30种ABC转运蛋白的表达已被证明与 对特定细胞毒性药物的耐药性。这些转运蛋白中的几种的转染具有 证实它们赋予了对相关性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P-gp表达的毒性更大， 细胞比非表达者，这表明了一种治疗多药耐药的新方法。 (MDR)癌的具有这种性质的几种不同的化学类别，包括 氨基硫脲，已被确定。对其中一种化合物NSC 73306进行了详细研究 并显示通过阻断这些细胞在S 相位存活细胞不表达P-gp，对化疗敏感， 产品药物，如蒽环类，紫杉醇和生物碱。数量结构 NSC 73306类似物的活性分析已经产生了几种具有类似活性的另外的化合物。 杀死表达P-gp的细胞的能力，但改善了溶解性。科技赋能 高通量筛选作为底物、抑制剂或特异性杀伤的新试剂 已经开发了表达P-gp的细胞。对P-gp正常功能的研究表明， 它参与许多药物的正常吸收和分布。常见的多态性变体 已检测到P-gp，但编码多态性似乎不改变药物转运 P-gp的功能然而，在C3435 T，无氨基酸变化的同义多态性， 特定P-gp单倍型的设定可以通过改变P-gp泵的效率， 蛋白质折叠和改变底物和抑制剂与P-gp相互作用的节律。这 单倍型似乎改变mRNA折叠，并导致主要的翻译延迟， P-gp构象改变MDR 1基因作为抗肿瘤药物的用途 基因治疗中的显性选择标记集中在SV 40作为载体的开发上 用于MDR 1的交付。使用重组SV 40衣壳 蛋白质，可以在体外包装DNA和RNA。 特别地，siRNA和化学修饰的siRNA可以以高效率递送， 其浓度远低于脂转染所需的浓度。有毒DNA的传递，例如 假单胞菌外毒素cDNA，可用于靶向癌症 在体外和小鼠异种植物模型中。

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 MDR 1 or ABCB 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 polymerase chain reaction (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 multi-drug resistant (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 these cells 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-expressing cells, but improved solubility properties. 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. 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. This haplotype appears to change mRNA folding, and cause a major translational delay which results in altered conformation of P-gp. Use of the MDR 1 gene as a dominant selectable marker in gene therapy has focused on the development of SV40 as a vector for delivery of MDR 1. 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.