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: 10702284
• 负责人: Michael Gottesman
• 金额: $ 60.75万
• 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/10702284
• 关键词: ABCB1 gene; ABCC1 gene; ABCG2 gene; ATP Hydrolysis; ATP-Binding Cassette Transporters; Amino Acids; Binding; Binding Sites; Biological Models; Blood - brain barrier anatomy; Cell Line; Cells; Chemicals; Clinical; Collaborations; Cryoelectron Microscopy; Crystallography; Cytotoxic agent; Doxorubicin; Drug Efflux; Drug resistance; Failure; Family; Genes; Goals; Histone Deacetylase Inhibitor; Human; Laboratories; Malignant Neoplasms; Modeling; Molecular; Molecular Conformation; Molecular Profiling; Multi-Drug Resistance; Multidrug Resistance Gene; Mus; Natural Product Drug; Natural Products; Paclitaxel; Patients; Pattern; Pharmaceutical Preparations; Pharmacopoeias; Phenotype; Physiological; Population; Process; Property; Proteins; Refractory; Research Personnel; Resistance; Rhodamine; Role; Sampling; Site; Solubility; Specificity; Structure; Structure-Activity Relationship; System; Thiosemicarbazones; Transmembrane Domain; Vinca Alkaloids; Work; analog; cancer cell; chemotherapeutic agent; chemotherapy; genetic analysis; high throughput screening; human tissue; improved; inhibitor; insight; kinase inhibitor; member; multi drug transporter; multidrug resistant cancer; neoplastic cell; novel strategies; personalized approach; predictive signature; resistance mechanism; therapy resistant; tool; treatment response; 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. 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. In collaboration with the group of Suresh Ambudkar, we have examined the basis of directional transport of compounds out of cells by P-glycoprotein. These studies have revealed a set of amino acid residues in the transmembrane regions of P-glycoprotein which can be altered to change the direction of transport of certain rhodamine compounds from out of the cell to into the cell. This process is concentration- and ATP-dependent, and gives important insight into how directionality of transport is determined in P-glycoprotein. We have used AML as one model system to determine the clinical role of ABC transporters in drug resistance. In one study, samples from the same patients before and after chemotherapy were analyzed. 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. A more detailed analysis of a large population of primary refractory AMLs has shown that there are 3 molecular signatures that predict poor response to therapy. One of these is associated with increased expression of ABCG2. These results argue that clinical samples must be stratified to facilitate effective targeting of inhibitors of ABC transporters to circumvent drug resistance.

癌细胞对化疗的耐药性是由于内在的或获得性的 特定蛋白质表达的变化。我们研究了对天然产物的抗性 化学治疗剂如阿霉素、西兰花生物碱和紫杉醇，最近， 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， 由于细胞内药物减少， 浓度的对于天然产物药物，这种交叉耐药性通常是由于 表达能量依赖性药物外排系统（ABC转运蛋白），称为P-糖蛋白 （P-gp），MDR 1或ABCB 1基因的产物，或ABC转运蛋白的其他成员 家族，包括ABCG 2和ABCB 5。我们实验室和其他机构的研究表明， 药物对P-gp表达细胞的毒性比对非表达细胞的毒性更大，这提示了一种新的 MDR癌症的治疗方法。几种不同的化学类别都有这种特性， 包括缩氨基硫脲（例如，NSC 73306），已被鉴定。数量结构 NSC 73306类似物的活性分析，在NCI-60细胞中的进一步相关性分析 线，并在美国药典中高通量筛选化合物， 表达P-gp的细胞产生了许多额外的化合物，具有提高的杀伤能力 选择性地表达P-gp的细胞，而且还具有改善的溶解性。了解 P-gp的结构如何决定其多特异性，以及特异性如何随 折叠的变化，我们与LCB的其他高级研究人员合作，包括 迪夏，苏雷什·安布卡，斯里拉姆·苏布拉马尼亚姆。冷冻电镜研究表明，载脂蛋白 P-gp具有两个ATP结合位点分离或靠近的动态结构 一起ATP的结合将P-gp的构象固定在后一种状态和ATP水解 导致ATP位点的分离。使用小鼠P-gp作为模型的晶体学研究 表明ATP位点之间的间隔决定了跨膜（TM）的间距 底物结合的螺旋，这表明当ATP位点一起移动时， 除此之外，TM螺旋暴露出不同的残基，这些残基能够结合许多不同的 印刷受体.对鼠-人嵌合P-GPS的研究揭示了相似的结构-功能 这两个进化上相关的转运蛋白的关系。联同 小组的Suresh Ambudkar，我们已经研究了化合物的定向运输的基础， 细胞的P-糖蛋白。这些研究已经揭示了一组氨基酸残基在 P-糖蛋白的跨膜区域，其可以被改变以改变P-糖蛋白的方向。 某些罗丹明化合物从细胞外转运到细胞内。这个过程 是浓度和ATP依赖的，并提供了重要的洞察如何方向性 在P-糖蛋白中确定转运。我们使用AML作为一个模型系统来确定 ABC转运蛋白在耐药性中的临床作用。在一项研究中， 对化疗前后的患者进行分析。在这种情况下， 显示ABC基因和其他MDR基因的不同表达模式，表明 将需要个体化的方法来抵抗治疗。更详细地分析 大量的原发性难治性AML显示有3种分子标记 这预示着对治疗的反应很差。其中之一与增加的 ABCG 2.这些结果表明，临床样本必须分层，以促进有效的 靶向ABC转运蛋白的抑制剂以规避耐药性。