Genetic Analysis of the Multidrug Resistance Phenotype in Tumor Cells

由于特定蛋白质表达的内在或获得性变化，癌细胞对化疗产生抗性。我们已经研究了对天然产物化疗剂如阿霉素、紫杉醇和紫杉醇的耐药性。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耐药性。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）的能量依赖性药物外排系统（ABC转运蛋白）的表达，其是MDR 1或ABCB 1基因的产物，或ABC转运蛋白家族的其他成员，包括ABCB 5。为了探索ABC转运蛋白家族的其他成员可能参与癌症耐药性的可能性，我们开发了实时PCR检测48种已知ABC转运蛋白中的大多数;这些技术已用于关联已知耐药性的癌细胞系中新型ABC转运蛋白的表达。已显示约30种ABC转运蛋白的表达在NCI-60细胞系中与对特异性细胞毒性药物的抗性相关。此外，该分析揭示了一些药物对P-gp表达细胞的毒性比对非表达细胞的毒性更大，这提示了治疗MDR癌症的新方法。具有这种性质的几种不同的化学类别，包括氨基硫脲（例如，NSC 73306），已被鉴定。NSC 73306类似物的定量结构活性分析、NCI-60细胞系中的进一步相关性分析以及美国药典中对杀死表达P-gp的细胞的化合物的高通量筛选已经产生了许多另外的化合物，其具有改善的选择性杀死表达P-gp的细胞的能力，而且还具有改善的溶解性。此外，化合物硫普罗宁（其是临床上用于治疗胱氨酸病的巯基供体）及其一些衍生物已被证明是杀死MDR细胞的强选择性试剂。在这种情况下，多药耐药细胞对硫普罗宁敏感不需要P-gp的表达，硫普罗宁似乎通过靶向谷胱甘肽作为过氧化物酶来杀死。对P-gp正常功能的研究表明，P-gp参与多种药物的正常摄取和分布。C11-去甲基洛哌丁胺是与NIMH的Robert Innis合作开发的，用于PET成像这种特定P-gp底物在癌症和大脑中的分布，使用和不使用强效P-gp抑制剂（如tariquidar）治疗。作为弱碱的PET配体被捕获在溶酶体中，放大脑、一些正常组织和癌症中的摄取信号。我们已经表明，在血脑屏障的三种最突出的转运蛋白（ABCB 1，ABCC 1，ABCG 2）中，该化合物对ABCB 1（P-gp）具有特异性。我们已经开发了一种用于分析ABCG 2在血脑屏障上的表达的系统，该系统基于以下事实：白藜芦醇是血脑屏障上的ABCG 2底物，并且可以在其中荧光素酶在血脑屏障上表达的转基因小鼠中检测到其进入脑的通道。我们现在正在扩展这个模型，以研究转运蛋白在脑肿瘤中的作用，以及它们与血脑屏障的相互作用。我们还表明小鼠和人ABCG 2的特异性非常相似，这意味着小鼠模型充分反映了人ABCG 2的功能。在特定P-gp单倍型的背景下，P-gp的同义多态性（C3435 T，无氨基酸变化）可通过改变蛋白质折叠的节律以及改变底物和抑制剂与P-gp的相互作用来影响P-gp泵送的效率。C3435 T单倍型似乎改变了mRNA的折叠，并导致主要的翻译延迟，从而导致P-gp构象的改变。与野生型P-gp转染子相比，具有P-gp单倍型形式的猪LLC-PK 1细胞的稳定转染子显示出改变的耐药性和抑制剂敏感性。我们已经建立了一个高度敏感的，定量分析ABC转运蛋白mRNA和其他mRNA相关的耐药性培养的癌细胞。我们已经详细研究了来自人类卵巢癌、肝细胞癌（HCC）和急性髓性白血病（AML）的样本。在卵巢癌中，有一个11基因MDR标记与化疗反应不良相关。在HCC中，签名更复杂，但准确区分预后不良与预后较好的癌症。将基因表达模式从不良预后模式改变为更好预后模式的试剂也使培养的HCC细胞对抗癌药物敏感。对于AML，研究了化疗前后来自相同患者的样品。在这种情况下，每种情况下的耐药性显示出ABC基因和其他MDR基因表达的不同模式，这表明需要个体化的方法来治疗耐药性。与Kevin Chen（NINDS）合作的关于发育期间ABCG 2表达调控的研究表明，它在发育期间由靶向ABCG 2 mRNA的3个主要非编码区的microRNA（miRNA）转录后调控。ABCG 2 mRNA水平在多能干细胞中很高，但直到细胞开始分化才表达为蛋白质。在组织培养中经常与ABCB 1表达相关的有趣表型之一是对诱导细胞凋亡的试剂的反应改变。使用tet诱导的P-gp KB细胞系的研究表明，当P-gp在KB细胞中表达时，凋亡诱导配体TRAIL的表达降低。这提供了KB细胞对外源性TRAIL的敏感性改变的潜在机制。

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, including ABCB5. 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 (e.g., NSC73306), have been identified. A quantitative structure activity analysis of NSC73306 analogs, a further correlation analysis in the NCI-60 cell lines, and a high-throughput screen for compounds in the U.S. Pharmacopeia that kill P-gp-expressing cells have 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. In this case, expression of P-gp is not required for multidrug-resistant cells to be sensitive to tiopronin, which appears to kill by targeting glutathione as peroxidases. Studies on the normal function of P-gp suggest that it is involved in normal uptake and distribution of many drugs. C11-desmethyl-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). We have developed a system for analysis of ABCG2 expression at the blood-brain barrier based on the fact that luciferin is an ABCG2 substrate at the blood-brain barrier and its passage into the brain can be detected in transgenic mice in which luciferase is expressed at the blood-brain barrier. We are now extending this model to examine the role of transporters in brain tumors, and their interaction with the blood-brain barrier. We have also shown that the specificity of mouse and human ABCG2 are very similar, meaning that mouse models adequately reflect the function of human ABCG2. A synonymous polymorphism of P-gp (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. The C3435T 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 inhibitor sensitivity compared to wild-type P-gp transfectants. We have created a highly sensitive, quantitative assay for ABC transporter mRNAs and other mRNAs associated with drug resistance in cultured cancer cells. We have studied samples from human ovarian cancer, hepatocellular cancer (HCC), and acute myelogenous leukemia (AML) in some detail. In ovarian cancer, there is an 11-gene MDR signature associated with poor response to chemotherapy. In HCC, the signature is more complex, but accurately distinguishes poor prognosis vs. better prognosis cancer. Agents that change the pattern of gene expression from poor prognosis to better prognosis patterns also sensitize cultured HCC cells to anti-cancer drugs. For AML, samples from the same patients before and after chemotherapy were studied. In this case, resistance in each case shows a different pattern of expression of ABC genes and other MDR genes, suggesting that individualized approaches to resistance to therapy will be needed. Studies in collaboration with Kevin Chen (NINDS) on regulation of expression of ABCG2 during development indicate that it is regulated during development post-transcriptionally by a microRNA (miRNA) that targets the 3 prime non-coding region of ABCG2 mRNA. ABCG2 mRNA levels are high in pluripotent stem cells, but are not expressed as protein until the cells begin to differentiate. One of the interesting phenotypes frequently associated in tissue culture with expression of ABCB1 is altered response to agents that induce apoptosis. Studies using tet-inducible P-gp KB cell lines indicate that the apoptosis-inducing ligand TRAIL is decreased in expression when P-gp is expressed in KB cells. This provides a potential mechanism for altered sensitivity of KB cells to exogenous TRAIL.