Genetic Analysis of the Multidrug Resistance Phenotype in Tumor Cells

肿瘤细胞多药耐药表型的遗传分析

• 批准号: 9556203
• 负责人: Michael Gottesman
• 金额: $ 81.82万
• 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/9556203
• 关键词: ABCB1 gene; ABCC1 gene; ABCG2 gene; ATP Hydrolysis; ATP-Binding Cassette Transporters; Acute Myelocytic Leukemia; Adrenocortical carcinoma; Antineoplastic Agents; Antipsychotic Agents; Binding; Binding Sites; Biological Assay; Blood; Blood - brain barrier anatomy; Brain; Cancer Prognosis; Cell Line; Cells; Chemicals; Collaborations; Complex; Crystallography; Cytotoxic agent; Doxorubicin; Drug Efflux; Drug resistance; Eye; Failure; Family; Fetus; Gene Expression Profile; Genes; Goals; Haloperidol; Histone Deacetylase Inhibitor; Human; Knockout Mice; Laboratories; Luciferases; Malignant Neoplasms; Malignant neoplasm of ovary; Messenger RNA; Modeling; Molecular; Molecular Conformation; Multi-Drug Resistance; Multidrug Resistance Gene; Mus; Natural Product Drug; Natural Products; New Zealand; P-Glycoprotein; Paclitaxel; Patients; Pattern; Pharmaceutical Preparations; Pharmacopoeias; Phenotype; Physiological; Pigments; Primary carcinoma of the liver cells; Property; Proteins; Research Personnel; Resistance; Role; Sampling; Sampling Studies; Site; Solubility; Specificity; Structure; Structure-Activity Relationship; System; Systems Analysis; Thiosemicarbazones; Time; Transgenic Mice; Universities; Vinca Alkaloids; Work; Xenograft Model; Yeasts; Zebrafish; analog; base; cancer cell; chemotherapeutic agent; chemotherapy; genetic analysis; high throughput screening; human tissue; improved; killings; kinase inhibitor; luciferin; medical schools; melanoma; member; multi drug transporter; neoplastic cell; novel strategies; outcome forecast; personalized approach; resistance mechanism; response; targeted agent; therapy resistant; tool; uptake

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 more recently, histone deacetylase inhibitors and targeted kinase inhibitors. 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 ABCG2 and ABCB5. Work from our laboratory and others 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 thiosemicarbazones (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 order to study the effect of these agents that target cancer cells expressing P-gp, we have developed a mouse xenograft model of human adrenocortical carcinoma, a cancer that intrinsically expresses high level of P-gp. Not only are ABC transporters responsible for drug resistance in cancer, but they are a major component of the blood-brain barrier (BBB) and blood-placental barrier. The three most prominent transporters at the blood-brain barrier are ABCB1, ABCC1, and ABCG2. We have developed a system for analysis of ABCG2 expression at the blood-brain and the blood-placental barriers based on the fact that luciferin is an ABCG2 substrate at these barriers and its passage into the brain or into developing fetuses can be detected in transgenic mice in which luciferase is expressed at the blood-brain barrier or blood-placental barrier. Because studies of the BBB in mice are time-consuming and expensive, we are developing a parallel analysis in zebrafish, as components of the zebrafish BBB appear to be very similar to those of the mammalian BBB. To understand how the structure of P-gp determines its polyspecificity and how specificity is altered with changes in folding, we have collaborated with other senior investigators in the LCB, including Di Xia, Suresh Ambudkar, and Sriram Subramaniam. Cryo-EM studies have demonstrated that apo P-gp has a dynamic structure in which the two ATP-binding sites are either separated or close together. Binding of ATP fixes the conformation of P-gp in the latter state and ATP hydrolysis results in separation of the ATP sites. Crystallography studies using mouse P-gp as a model show that the separation between the ATP sites determines the pitch of the transmembrane (TM) helices where substrates bind, suggesting the hypothesis that as the ATP sites move together and apart, the TM helices expose different residues that enable binding to many different substrates. Studies on mouse-human chimeric P-gps have revealed similar structure-function relationships for these two evolutionarily related transporters. 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. ABCB5 is a close molecular relative of ABCB1. It is expressed in pigmented cells in the brain and eye, and in melanoma. In collaboration with Richard Cannon (University of Otega, New Zealand) we have shown that when expressed in yeast, ABCB5 is a multidrug transporter. ABCB5 knock-out mice are sensitive to the major tranquilizer haloperidol, consistent with a role of this transporter in the brain (with Gary Peltz, Stanford School of Medicine).

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。我们已经研究了天然产物化疗药物的耐药性，如阿霉素、长春花碱和紫杉醇，以及最近的组蛋白脱乙酰酶抑制剂和靶向激酶抑制剂。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(P-gp)是MDR1或ABCB1基因的产物，或者ABC转运体家族的其他成员，包括ABCG2和ABCB5。我们实验室和其他实验室的工作表明，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，这表明了一种治疗MDR癌症的新方法。具有这种性质的几种不同的化学类别，包括缩氨基硫脲(例如，NSC73306)已被鉴定。对NSC73306类似物的定量结构活性分析、在NCI-60细胞系中的进一步相关性分析以及对美国药典中杀死P-gp表达细胞的化合物的高通量筛选，已经产生了许多额外的化合物，这些化合物具有更强的选择性杀伤P-gp表达细胞的能力，而且具有更好的溶解性。为了研究这些针对表达P-gp的癌细胞的药物的作用，我们建立了人肾上腺皮质癌的小鼠异种移植模型，人肾上腺皮质癌是一种固有地高表达P-gp的癌症。ABC转运蛋白不仅对癌症的耐药性负责，而且是血脑屏障(BBB)和血胎盘屏障的主要组成部分。血脑屏障上最突出的三种转运蛋白是ABCB1、ABCC1和ABCG2。我们已经开发了一种分析ABCG2在血脑和血-胎盘屏障中表达的系统，这是基于这样一个事实，即荧光素是这些屏障中的ABCG2底物，在转基因小鼠中可以检测到它进入大脑或进入发育中的胎儿，其中荧光素酶在血脑屏障或血-胎盘屏障中表达。由于对小鼠血脑屏障的研究既耗时又昂贵，我们正在开发一种针对斑马鱼的平行分析，因为斑马鱼血脑屏障的成分似乎与哺乳动物的血脑屏障成分非常相似。为了了解P-gp的结构如何决定其多特异性，以及特异性如何随着折叠的变化而改变，我们与LCB的其他高级研究人员合作，包括Di Xia，Suresh Ambukar和Sriram Subramaniam。冷冻-EM研究表明，apo P-gp具有两个ATP结合位点分离或紧密结合的动态结构。ATP的结合使P-gp的构象固定在后一种状态，而ATP的水解会导致ATP位点的分离。以小鼠P-gp为模型进行的结晶学研究表明，ATP位点之间的分离决定了底物结合的跨膜(TM)螺旋的间距，这表明当ATP位点一起和分开移动时，TM螺旋暴露出不同的残基，从而能够与许多不同的底物结合。对鼠-人嵌合P-GPS的研究表明，这两个进化上相关的转运蛋白具有相似的结构-功能关系。我们已经建立了一种高灵敏度的定量检测ABC转运蛋白mRNAs和其他与培养的癌细胞耐药性相关的mRNAs的方法。我们对人卵巢癌、肝细胞癌和急性髓系白血病的样本进行了较为详细的研究。在卵巢癌中，有11个基因的多药耐药信号与化疗反应差有关。在肝细胞癌中，征象更复杂，但准确地区分预后不良和预后较好的癌症。将基因表达模式从较差的预后模式改变为较好预后模式的药物也会使培养的肝癌细胞对抗癌药物敏感。对于急性髓细胞白血病，研究对象是化疗前后相同患者的样本。在这种情况下，每个病例的耐药情况都显示出ABC基因和其他MDR基因的不同表达模式，这表明需要对治疗耐药采取个性化的方法。ABCB5是ABCB1的近亲。它在大脑和眼睛的色素细胞以及黑色素瘤中表达。在与新西兰奥特加大学的理查德·坎农的合作中，我们已经证明，当在酵母中表达时，ABCB5是一种多药物转运体。ABCB5基因敲除小鼠对主要的镇静剂氟哌啶醇敏感，这与这种转运蛋白在大脑中的作用一致(斯坦福医学院的加里·佩尔茨)。