Genetic Analysis of the Multidrug Resistance Phenotype in Tumor Cells

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、长春花碱和紫杉醇的抗药性。在大多数情况下，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NCI-60细胞系对特定细胞毒药物的耐药性有关。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。NSC73306及相关药物治疗后，ABCB1mRNA的转换率也增加。在NSC73306治疗中存活的细胞不表达P-gp，并且对天然产物药物如蒽环类药物、紫杉醇和长春花碱的化疗敏感。对NSC73306类似物的定量结构活性分析和在NCI-60细胞系中的进一步相关性分析已经产生了许多额外的化合物，它们具有更强的选择性杀伤P-gp表达细胞的能力，但也具有更好的溶解性。此外，硫普罗宁是一种临床上用于治疗胱氨酸病的巯基供体，其化合物及其一些衍生物已被证明是杀死多药耐药细胞的强大选择性药物。能够高通量筛选作为底物、抑制剂或专门杀死P-gp表达细胞的新试剂的技术已经开发出来。在Kaposi肉瘤患者的细胞系中，暴露于HIV蛋白酶抑制剂会导致ABCB1的过度表达，而蛋白酶抑制剂是其重要的底物。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。C11-去甲氧基-氯哌丁胺是与Robert Inennis在NIMH上合作开发的，目的是对这种特定的P-gp底物在癌症和脑中的分布进行PET成像，在使用和不使用有效的P-gp抑制剂(如Tariquidar)治疗的情况下。作为弱碱基的PET配体被困在溶酶体中，放大大脑、一些正常组织和癌症中的摄取信号。我们已经证明，在血脑屏障最突出的三种转运蛋白(ABCB1，ABCC1，ABCG2)中，这种化合物是ABCB1(P-gp)的特异性化合物。P-gp的常见多态变异也已被检测到，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。这种单倍型似乎改变了mRNA的折叠，并导致了主要的翻译延迟，从而导致P-gp的构象改变。与野生型P-gp转染体相比，单倍型P-gp稳定转染组的猪LLC-PK1细胞显示出不同的耐药性和抑制敏感性。我们已经使用TaqMan低密度阵列创建了一种对人类癌症样本中的ABC转运蛋白mRNAs进行高灵敏的定量分析。在对几种癌症的初步研究中，我们发现ABC转运蛋白基因在临床癌症中的表达模式与已建立的细胞系的表达模式显著不同，无论这些细胞的培养方式(单层、3D培养或异种移植)。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，正在努力开发新的体外癌细胞生长条件，以维持体内MDR基因的表达模式。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和化学修饰的siRNA，如肽核酸(PNA)，可以高效地传递，并且浓度比脂质体所需的浓度低得多。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。

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 improved ability to kill selectively P-gp-expressing cells, but also with improved solubility properties. In addition, the compound tiopronin, which is a sulfhydryl donor used clinically to treat cystinosis, and some of its derivatives have been shown to be powerful selective agents for killing MDR cells. Technology enabling a high-throughput screen for new agents that are substrates, inhibitors or specifically kill P-gp-expressing cells has been developed. In cell lines derived from patients with Kaposi sarcoma, exposure to HIV protease inhibitors results in overexpression of ABCB1, for which the protease inhibitors are important substrates. 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 in collaboration with 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. PET ligands that are weak bases are trapped in lysosomes, amplifying the uptake signal in the brain, in some normal tissues, and in cancers. 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, irrespective of the manner in which these cells are cultured (monolayer, 3D culture or xenografts). This result indicates that current in vitro cancer cell models may not be adequate to study drug resistance in cancer and efforts are underway to develop novel ex vivo cancer cell growth conditions that maintain in vivo patterns of expression of MDR genes. 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 such as peptide nucleic acids (PNAs) 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.