Genetic Analysis of the Multidrug Resistance Phenotype i

多药耐药表型的遗传分析i

• 批准号: 7337913
• 负责人: MICHAEL M GOTTESMAN
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7337913
• 关键词: Genetic; Analysis; Multidrug; Resistance; Phenotype

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

癌细胞对化疗产生耐药性是由于特定蛋白表达的内在或获得性变化。我们研究了天然产物化疗药物如阿霉素、长春花碱和紫杉醇以及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抗药性。对于天然产物药物，这种交叉耐药性通常是由于被称为P-糖蛋白(P-gp)的能量依赖的药物外排系统(ABC转运体)的表达，P-糖蛋白(P-gp)是MDR1或ABCB1基因的产物，或者是ABC转运体家族的其他成员。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，我们开发了实时荧光聚合酶链式反应(Real-time PCR)来检测48个已知的ABC转运蛋白中的大多数；这些技术已经被用来关联新的ABC转运蛋白在已知耐药的癌细胞系中的表达。大约30个ABC转运蛋白的表达已被证明与特定细胞毒药物的耐药性有关。对其中几种转运蛋白的转染已经证实，它们对相关性研究中检测到的药物具有耐药性。此外，这一分析还表明，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，这为治疗MDR癌症提供了一种新的方法。具有这种性质的几种不同的化学类别，包括氨基硫脲，已经被确定。对一种化合物NSC73306进行了详细的研究，结果表明，通过将P-gp表达的细胞阻断在S期，NSC73306可以高度特异性地杀死P-gp表达的细胞。存活的细胞不表达P-gp，对天然产物药物化疗敏感，如蒽环类药物、紫杉醇和长春花碱。对P-gp正常功能的研究表明，它与许多药物的正常摄取和分布有关。已检测到P-gp的常见多态变异，但编码多态似乎不会改变P-gp的药物转运功能。然而，特定P-gp单倍型设置中的同义多态(C3435T，不改变氨基酸)可以通过改变蛋白质折叠的节奏以及改变底物和抑制物与P-gp的相互作用来影响P-gp的泵送效率。在基因治疗中，MDR1基因作为显性可选择标记的应用主要集中在发展SV40作为MDR1载体的研究。利用重组SV40衣壳蛋白，在体外包装DNA和RNA是可能的。特别是，siRNA可以以高效率和比脂质体所需的低得多的浓度输送。有毒DNA的传递，如假单胞菌外毒素基因，可用于在体外和小鼠异种移植模型中靶向癌症。我们还在黑色素瘤细胞中发现了ABC转运蛋白的独特特征。其中一种转运蛋白ABCB5与P-gp(Mdr1)密切相关，似乎与黑色素瘤细胞的多药耐药有关。对细胞处理顺铂在黑色素瘤细胞中的详细分析表明，顺铂在黑素小体(而不是细胞核)中积累，然后被细胞挤出。黑色素瘤对化疗的一些难治性可能是由于其独特的ABC转运蛋白以及黑素体的隔离和细胞毒药物的排出。顺铂耐药突变株揭示，对甲氨蝶呤、一些核苷类似物、重金属和毒素的交叉耐药性是由于摄取系统的多效性缺陷导致药物内流减少。最近的证据表明，这些顺铂耐药细胞的内吞作用存在全球性缺陷，细胞内蛋白运输、细胞骨架和线粒体能量产生也存在缺陷