Genetic Analysis of the Multidrug Resistance Phenotype i

多药耐药表型的遗传分析i

• 批准号: 6761572
• 负责人: MICHAEL M GOTTESMAN
• 金额: --
• 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/6761572
• 关键词: P glycoprotein; adenocarcinoma; adenosinetriphosphatase; antineoplastics; biological transport; chemical binding; cis platinum compound; drug delivery systems; drug receptors; enzyme activity; enzyme structure; fungal genetics; gene expression; gene therapy; genetic markers; green fluorescent proteins; human genetic material tag; multidrug resistance; neoplastic cell; phenotype; transfection /expression vector; vaccinia virus

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 to the synthetic drug cisplatin. In both cases, cells become simultaneously resistant to multiple drugs because of reductions in intracellular drug concentrations. For the natural product drugs this cross-resistance is due to expression of an energy-dependent drug efflux system (ABC transporter) known as P-glycoprotein (P-gp), the product of the MDR1 or ABC B1 gene. For cisplatin, cross-resistance to methotrexate, some nucleoside analogs, heavy metals, and toxins is due to a reduction in drug influx resulting from a pleiotropic defect in uptake systems. Recent evidence suggests a global defect in endocytosis in these cisplatin resistant cells and defects in intracellular protein trafficking and the cytoskeleton. Single-step cisplatin resistant mutants show a defect in protein trafficking which results in accumulation of cell surface receptors/transporters/channels in the cytoplasm; a putative cisplatin carrier/channel is presumed to be among these mislocalized proteins resulting in decreased cisplatin uptake. At higher levels of resistance, after multiple steps of selection in cisplatin, there is increased methylation of genes for binding proteins (e.g., folate binding protein) and cytoskeletal proteins, among others. This hypermethylation, reversible by treatment with deoxyazacytidine, results in decreased RNA transcription of genes responsible, at least in part, for the cisplatin resistance phenotype. Studies on mechanism of action of P-gp have focused on the manner in which many different substrates and inhibitors are recognized by the transporter, how substrate interaction results in activation of ATPase, and how ATPase results in drug translocation and efflux. These studies and others have led to the conclusion that there are multiple, probably overlapping sites for interaction of substrates and inhibitors primarily formed by TM segments from both the amino-terminal (TM5,6) and carboxy-terminal (TM11,12) halves of P-gp and that activation of ATPase results in a reduction of substrate binding to P-gp. 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. 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 and microarray technology for detection of most of the 48 known ABC transporters; these techniques will be used to correlate expression of novel ABC transporters in cancer cell lines of known drug resistance. Use of the MDR1 gene as a dominant selectable marker in gene therapy has focused on the development of SV40 as a vector for delivery of MDR1. Using recombinant SV40 capsid proteins, it is possible to package DNA in vitro, including P-gp and green fluorescent protein (GFP) containing vectors. Transduction of P-gp and GFP using in vitro packaged DNA is highly efficient in many different cell types including lymphoid cells, liver cells, and keratinocytes, and allows transfer of up to 15 kb of DNA without the need for SV40 sequences in the packaged DNA. This approach offers promise for transfer of P-gp into hematopoietic and other cells for gene therapy. We have also shown in a canine model that transduction of bone marrow stem cells with a chimeric vector encoding P-gp and the human common gamma chain of interleukin receptors results in taxol-resistant bone marrow in which most circulating blood cells express the gamma chain and P-gp, and we have demonstrated in a pig model that keratinocytes transduced with MDR1-expressing vectors can be selected using colchicine ointment.

由于特定蛋白质表达的内在或获得性变化，癌细胞对化疗产生抗性。我们已经研究了对天然产物化疗药物如阿霉素、紫杉醇和合成药物顺铂的耐药性。在这两种情况下，由于细胞内药物浓度的降低，细胞同时对多种药物产生耐药性。对于天然产物药物，这种交叉耐药性是由于被称为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，其是MDR 1或ABC B1基因的产物。对于顺铂，对甲氨蝶呤、一些核苷类似物、重金属和毒素的交叉耐药性是由于摄取系统中的多效性缺陷导致的药物流入减少。最近的证据表明，在这些顺铂耐药细胞的内吞作用和细胞内蛋白运输和细胞骨架的缺陷的全球性缺陷。单步顺铂耐药突变体显示蛋白质运输缺陷，导致细胞表面受体/转运蛋白/通道在细胞质中蓄积;推测这些错误定位的蛋白质中存在推定的顺铂载体/通道，导致顺铂摄取降低。在更高水平的耐药性下，在顺铂中进行多步选择后，结合蛋白的基因甲基化增加（例如，叶酸结合蛋白）和细胞骨架蛋白等。这种超甲基化可通过脱氧氮杂胞苷治疗逆转，导致至少部分负责顺铂耐药表型的基因的RNA转录降低。对P-gp作用机制的研究主要集中在转运蛋白识别许多不同底物和抑制剂的方式、底物相互作用如何导致ATP酶激活以及ATP酶如何导致药物转运和外排。这些研究和其他研究得出的结论是，底物和抑制剂的相互作用有多个可能重叠的位点，主要由来自P-gp氨基末端（TM 5，6）和羧基末端（TM 11，12）的TM片段形成，ATP酶的激活导致底物与P-gp结合的减少。对P-gp正常功能的研究表明，P-gp参与多种药物的正常摄取和分布。已检测到P-gp的常见多态性变体，但编码多态性似乎不会改变P-gp的药物转运功能。为了探索ABC转运蛋白家族的其他成员可能参与癌症耐药性的可能性，我们开发了实时PCR和微阵列技术，用于检测48种已知ABC转运蛋白中的大多数;这些技术将用于关联已知耐药性的癌细胞系中新型ABC转运蛋白的表达。在基因治疗中使用MDR 1基因作为显性选择性标记已经集中于开发SV 40作为递送MDR 1的载体。使用重组SV 40衣壳蛋白，可以在体外包装DNA，包括含有P-gp和绿色荧光蛋白（GFP）的载体。使用体外包装的DNA转导P-gp和GFP在许多不同的细胞类型（包括淋巴样细胞、肝细胞和角质形成细胞）中是高效的，并且允许转移高达15 kb的DNA，而不需要包装的DNA中的SV 40序列。这种方法为将P-gp转移到造血细胞和其他细胞中进行基因治疗提供了希望。我们还在犬模型中显示，用编码P-gp和人类白细胞介素受体的共同γ链的嵌合载体转导骨髓干细胞导致紫杉醇抗性骨髓，其中大多数循环血细胞表达γ链和P-gp，并且我们在猪模型中证明，可以使用秋水仙碱软膏选择用MDR 1表达载体转导的角质形成细胞。