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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

基本信息

批准号：
7732888
负责人：
MICHAEL M GOTTESMAN
金额：
$ 101.09万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/7732888
关键词：
ABCB1 gene ABCC1 gene ABCG2 gene ATP-Binding Cassette Transporters Affect Amino Acids Anthracycline Antibiotics Anthracyclines Biological Factors Cancer cell line Capsid Proteins Cells Chemicals Class Code Complementary DNA Correlation Studies Cytotoxic agent DNA Packaging Detection Development Doxorubicin Drug Efflux Drug Transport Drug resistance Exotoxins Failure Family Genes Genetic Polymorphism Goals Haplotypes In Vitro Malignant Neoplasms Messenger RNA Modeling Molecular Molecular Conformation Multi-Drug Resistance Multidrug Resistance Associated Protein 1 Mus Natural Product Drug New Agents P-Glycoprotein P-Glycoproteins Paclitaxel Pharmaceutical Preparations Phase Phenotype Polymerase Chain Reaction Property Proteins Pseudomonas Pump RNA Recombinants Resistance Simian virus 40 Small Interfering RNA Solubility Specificity Structure System Techniques Technology Time Transfection Variant Vinca Alkaloids Work analog cancer cell chemotherapeutic agent chemotherapy gene therapy genetic analysis high throughput screening improved inhibitor/antagonist killings lipofection member neoplastic cell novel novel strategies protein folding thiosemicarbazide tool uptake vector

项目摘要

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 MDR 1 or ABCB 1 gene, or to other members of the ABC transporter family. 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 polymerase chain reaction (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 with resistance to specific cytotoxic drugs. Transfection of several of these transporters has confirmed that they confer resistance to the drugs detected in the correlation studies. 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 multi-drug resistant (MDR) cancers. Several different chemical classes with this property, including thiosemicarbazides, have been identified. One compound, NSC73306, has been studied in detail and shown to kill P-gp-expressing cells with high specificity by blocking these cells in S phase. Surviving cells do not express P-gp and are sensitive to chemotherapy with natural product drugs such as anthracyclines, paclitaxel and Vinca alkaloids. A quantitative structure activity analysis of NSC73306 analogs has yielded several additional compounds with a similar ability to kill P-gp-expressing cells, but improved solubility properties. Technology enabling a high-throughput screen for new agents that are substrates, inhibitors or specifically kill P-gp-expressing cells has been developed. Studies on the normal function of P-gp suggest that it is involved in normal uptake and distribution of many drugs. Common polymorphic variants of P-gp have been detected, but coding polymorphisms do not appear to alter the drug transport functions of P-gp. However, a synonymous polymorphism (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. This haplotype appears to change mRNA folding, and cause a major translational delay which results in altered conformation of P-gp. Use of the MDR 1 gene as a dominant selectable marker in gene therapy has focused on the development of SV40 as a vector for delivery of MDR 1. Using recombinant SV40 capsid proteins, it is possible to package DNA and RNA in vitro . In particular, siRNA and chemically modified siRNAs can be delivered with high efficiency and at much lower concentrations than are needed for lipofection. Delivery of toxic DNAs, such as Pseudomonas exotoxin cDNA, can be used to target cancers in vitro and in mouse xenoplant models.

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。我们研究了天然产物化疗药物如阿霉素、长春花碱和紫杉醇的抗药性。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-糖蛋白是MDR 1或ABCB 1基因的产物，或者是ABC转运体家族的其他成员。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，我们开发了实时聚合酶链式反应(PCR)来检测48个已知的ABC转运蛋白中的大多数；这些技术已经被用来关联新的ABC转运蛋白在已知耐药的癌细胞系中的表达。大约30个ABC转运蛋白的表达已被证明与特定细胞毒药物的耐药性有关。对其中几种转运蛋白的转染已经证实，它们对相关性研究中检测到的药物具有耐药性。此外，这一分析表明，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，这为治疗多药耐药(MDR)癌症提供了一种新的方法。具有这种性质的几种不同的化学类别，包括氨基硫脲，已经被确定。一种名为NSC73306的化合物已被详细研究，并表明通过将P-gp表达的细胞阻断在S期，可以高度特异性地杀死这些细胞。存活的细胞不表达P-gp，对天然产物药物化疗敏感，如蒽环类药物、紫杉醇和长春花碱。对NSC73306类似物的定量结构活性分析已经产生了几个额外的化合物，它们具有类似的杀死P-gp表达细胞的能力，但改善了溶解性。能够高通量筛选作为底物、抑制剂或专门杀死P-gp表达细胞的新试剂的技术已经开发出来。对P-gp正常功能的研究表明，它与许多药物的正常摄取和分布有关。已检测到P-gp的常见多态变异，但编码多态似乎不会改变P-gp的药物转运功能。然而，特定P-gp单倍型设置中的同义多态(C3435T，不改变氨基酸)可以通过改变蛋白质折叠的节奏以及改变底物和抑制物与P-gp的相互作用来影响P-gp的泵送效率。这种单倍型似乎改变了mRNA的折叠，并导致了主要的翻译延迟，从而导致P-gp的构象改变。MDR-1基因作为显性选择标记在基因治疗中的应用主要集中在发展SV40作为MDR-1的载体。利用重组的SV40衣壳蛋白，在体外包装DNA和RNA是可能的。特别是，siRNA和化学修饰的siRNA可以高效地传递，并且浓度比脂质体所需的浓度低得多。有毒DNA的传递，如假单胞菌外毒素基因，可用于在体外和小鼠异种移植模型中靶向癌症。

项目成果

期刊论文数量（42）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Mislocalization of membrane proteins associated with multidrug resistance in cisplatin-resistant cancer cell lines.

DOI：
发表时间：
2003-09
期刊：
Cancer research
影响因子：
11.2
作者：
Xing-jie Liang;D. Shen;S. Garfield;M. Gottesman
通讯作者：
Xing-jie Liang;D. Shen;S. Garfield;M. Gottesman

Drug-selected co-expression of P-glycoprotein and gp91 in vivo from an MDR1-bicistronic retrovirus vector Ha-MDR-IRES-gp91.

MDR1-双顺反子逆转录病毒载体 Ha-MDR-IRES-gp91 体内 P-糖蛋白和 gp91 的药物选择共表达。

DOI：
10.1002/jgm.362
发表时间：
2003
期刊：
The journal of gene medicine
影响因子：
0
作者：
Sugimoto,Yoshikazu;Tsukahara,Satomi;Sato,Shigeo;Suzuki,Mutsumi;Nunoi,Hiroyuki;Malech,HarryL;Gottesman,MichaelM;Tsuruo,Takashi
通讯作者：
Tsuruo,Takashi

Modulation by the ATP/GTP ratio of the phosphorylation level of P-glycoprotein and of various plasma membrane proteins of KB-V1 multidrug resistant cells.

通过 ATP/GTP 比率调节 KB-V1 多药耐药细胞的 P-糖蛋白和各种质膜蛋白的磷酸化水平。

DOI：
发表时间：
2003
期刊：
Anticancer research
影响因子：
2
作者：
Lelong-Rebel,IsabelleH;Rebel,Gerard;Cardarelli,CarolO;Pastan,Ira;Gottesman,MichaelM
通讯作者：
Gottesman,MichaelM

Therapeutic strategies involving the multidrug resistance phenotype: the MDR1 gene as target, chemoprotectant, and selectable marker in gene therapy.

涉及多药耐药表型的治疗策略：MDR1 基因作为基因治疗中的靶点、化学保护剂和选择标记。

DOI：
10.1016/s1054-3589(08)60468-8
发表时间：
1999
期刊：
Advances in pharmacology (San Diego, Calif.)
影响因子：
0
作者：
Aran,JM;Pastan,I;Gottesman,MM
通讯作者：
Gottesman,MM

Topical colchicine selection of keratinocytes transduced with the multidrug resistance gene (MDR1) can sustain and enhance transgene expression in vivo.

对转导多药耐药基因 (MDR1) 的角质形成细胞进行局部秋水仙碱选择可以维持和增强体内转基因表达。

DOI：
10.1073/pnas.192247899
发表时间：
2002
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Pfutzner,W;Terunuma,A;Tock,CL;Snead,EK;Kolodka,TM;Gottesman,MM;Taichman,L;Vogel,JC
通讯作者：
Vogel,JC