Genetic Analysis of the Multidrug Resistance Phenotype in Tumor Cells

化疗耐药发生在癌细胞中，是因为特定蛋白质表达的内在或获得性变化。我们研究了对天然产物化疗药物的耐药性，如阿霉素、长春花生物碱和紫杉醇。在大多数情况下，由于细胞内药物浓度降低，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，p -糖蛋白是MDR1或ABCB1基因的产物，或ABC转运蛋白家族的其他成员，包括ABCB5。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PCR检测已知的48种ABC转运蛋白中的大多数；这些技术已被用于在已知耐药的癌细胞系中关联新的ABC转运蛋白的表达。在NCI-60细胞系中，大约30种ABC转运蛋白的表达与对特定细胞毒性药物的抗性相关。此外，该分析表明，一些药物对表达p- gp的细胞的毒性比不表达p- gp的细胞更大，这提示了一种治疗耐多药癌症的新方法。已经确定了几种具有这种性质的不同化学类别，包括硫代氨基脲（如NSC73306）。通过对NSC73306类似物的定量结构活性分析，对NCI-60细胞系的进一步相关性分析，以及对美国药书中杀死表达p- gp细胞的化合物的高通量筛选，我们发现了许多额外的化合物，它们具有更好的选择性杀死表达p- gp细胞的能力，而且还具有更好的溶解度。此外，复方硫普罗宁是一种巯基供体，用于临床治疗胱氨酸病，它的一些衍生物已被证明是杀死耐多药耐药细胞的强大选择性药物。在这种情况下，多重耐药细胞对硫普罗蛋白敏感并不需要P-gp的表达，硫普罗蛋白似乎是通过靶向谷胱甘肽作为过氧化物酶来杀死细胞的。对P-gp正常功能的研究表明，它参与许多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，用于在癌症和大脑中使用或不使用强效P-gp抑制剂（如tariquar）治疗的这种特异性P-gp底物的PET图像分布。弱碱基的PET配体被困在溶酶体中，放大了大脑、一些正常组织和癌症中的摄取信号。我们已经证明，在血脑屏障的三种最重要的转运蛋白（ABCB1, ABCC1, ABCG2）中，该化合物对ABCB1 （P-gp）具有特异性。基于荧光素是ABCG2在血脑屏障中的底物，并且在荧光素酶在血脑屏障中表达的转基因小鼠中可以检测到其进入大脑的通道，我们开发了一种分析血脑屏障中ABCG2表达的系统。我们现在正在扩展这个模型来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。我们还发现小鼠和人类ABCG2的特异性非常相似，这意味着小鼠模型充分反映了人类ABCG2的功能。P-gp的同义多态性（C3435T，无氨基酸变化）可以通过改变蛋白质折叠的节奏和改变底物和抑制剂与P-gp的相互作用来影响P-gp的泵浦效率。C3435T单倍型似乎改变mRNA折叠，并导致主要的翻译延迟，从而导致P-gp构象的改变。具有P-gp单倍型形式的稳定转染的猪LLC-PK1细胞与野生型P-gp转染相比，显示出改变的耐药性和抑制剂敏感性。我们已经创建了一种高度敏感的定量检测方法，用于检测ABC转运蛋白mrna和其他与培养癌细胞耐药相关的mrna。我们对人类卵巢癌、肝细胞癌（HCC）和急性髓性白血病（AML）的样本进行了一些详细的研究。在卵巢癌中，有一个11个基因的耐多药特征与化疗反应差有关。在HCC中，信号更为复杂，但能准确区分预后差和预后好的肿瘤。将基因表达模式从不良预后模式转变为良好预后模式的药物也使培养的HCC细胞对抗癌药物敏感。对于AML，研究了同一患者化疗前后的样本。在这种情况下，每个病例的耐药表现出ABC基因和其他耐多药基因的不同表达模式，这表明需要个性化的治疗耐药方法。与Kevin Chen （NINDS）合作的关于发育过程中ABCG2表达调控的研究表明，在发育过程中，ABCG2受靶向ABCG2 mRNA 3 '非编码区的microRNA （miRNA）的转录后调控。ABCG2 mRNA水平在多能干细胞中很高，但直到细胞开始分化时才以蛋白质形式表达。在组织培养中，与ABCB1表达相关的一个有趣表型是对诱导细胞凋亡的药物的改变反应。使用et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的表达减少。这为KB细胞对外源性TRAIL的敏感性改变提供了潜在的机制。

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.