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).

由于特定蛋白质表达的内在或获得的变化，癌细胞中对化疗的抗性发生在癌细胞中。我们研究了对天然产物化学治疗剂的耐药性，例如阿霉素，芬卡生物碱和紫杉醇，以及最近的组蛋白脱乙酰基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具有抗性。对于天然产物药物，这种交叉耐药通常是由于能量依赖性药物外排系统（ABC转运蛋白）称为P-糖蛋白（P-GP），MDR1或ABCB1基因的乘积，或其他ABC Transporter家族的成员，包括ABCG2和ABCB5。我们实验室和其他实验室的工作表明，某些药物对表达P-gp的细胞的毒性比对非表达者的毒性更具毒性，这表明一种新型的MDR癌症治疗方法。已经确定了与该特性的几种不同的化学类别，包括硫代性氨基酮（例如NSC73306）。 NSC73306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在美国Pharmacopeia的化合物的高通量屏幕，可杀死P-gp表达P-gp的细胞，从而产生了许多其他化合物，具有改进的能力，可杀死选择性地表达P-gp-gp-gp-gp-gp-Exparts-Exparts-Exparts-Express-Express-Express-Sexpress-Selubility Propties。为了研究这些靶向表达P-gp的癌细胞的药物的作用，我们开发了人类肾上腺皮质癌的小鼠异种移植模型，该模型本质上表达了高水平的P-gp。 ABC转运蛋白不仅负有癌症的耐药性，而且是血脑屏障（BBB）和血液斑势垒的主要组成部分。血脑屏障的三个最突出的转运蛋白是ABCB1，ABCC1和ABCG2。我们已经开发了一个系统，用于分析血脑屏上ABCG2表达和血液隔离屏障，基于以下事实：荧光素是这些障碍物的ABCG2底物，并且可以在转基因小鼠中检测到其进入大脑或发育中的胎儿，在该障碍物中，在该障碍物中以荧光素酶在血液中表达在血脑壁垒或血液中。由于在小鼠中对BBB的研究很耗时且昂贵，因此我们正在斑马鱼中进行平行分析，因为斑马鱼BBB的组成部分似乎与哺乳动物BBB的组件非常相似。为了了解P-gp的结构如何确定其多元特异性以及如何随着折叠的变化而改变特异性，我们已经与LCB的其他高级研究人员合作，包括Di Xia，Suresh Ambudkar和Sriram Subramaniam。冷冻EM研究表明，APO P-GP具有动态结构，其中两个ATP结合位点要么分开或靠近。 ATP的结合固定了后一种状态和ATP水解的P-gp构象会导致ATP位点分离。使用小鼠P-gp作为模型的晶体学研究表明，ATP位点之间的分离决定了底物结合的跨膜（TM）螺旋的间隔，这表明当ATP位点一起移动并与TM螺旋分开时，TM螺旋揭露了不同的残基，使不同的底物与许多不同的底物结合。小鼠人类嵌合P-GP的研究揭示了这两个相关的转运蛋白的相似结构 - 功能关系。我们为ABC转运蛋白mRNA和与培养的癌细胞耐药性相关的其他mRNA创建了高度敏感的定量测定法。我们已经详细研究了来自人类卵巢癌，肝癌（HCC）和急性骨髓性白血病（AML）的样品。在卵巢癌中，有一个11基因的MDR签名与对化学疗法的反应不佳有关。在HCC中，签名更为复杂，但准确地区分了预后不良与更好的预后癌。将基因表达的模式从不良预后变为更好的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，研究了化学疗法之前和之后的同一患者的样本。在这种情况下，在每种情况下，耐药性都显示出ABC基因和其他MDR基因的表达方式不同，这表明需要进行个性化治疗的方法。 ABCB5是ABCB1的亲密分子亲戚。它在大脑和眼睛的有色细胞以及黑色素瘤中表达。在与理查德·坎农（Richard Cannon）（新西兰奥特加大学）合作的情况下，我们表明，在酵母中表达时，ABCB5是一种多果转运蛋白。 ABCB5敲除小鼠对主要的镇静剂氟哌啶醇很敏感，与该转运蛋白在大脑中的作用一致（与斯坦福大学医学院的Gary Peltz一起）。