Multidrug Resistance Phenotype in Tumor Cells

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、长春花碱和紫杉醇以及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抗药性。对于天然产物药物，这种交叉耐药性是由于一种被称为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MDR1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的多效性缺陷导致药物流入减少。最近的证据表明，这些顺铂耐药细胞的内吞作用存在全球性缺陷，细胞内蛋白运输和细胞骨架也存在缺陷。单步顺铂耐药突变体表现出蛋白转运缺陷，导致细胞表面受体/转运体/通道在细胞质中聚集；推测顺铂载体/通道可能是这些错位蛋白中的一种，导致顺铂摄取减少。在较高水平的耐药性中，在顺铂的多步骤选择之后，结合蛋白(如叶酸结合蛋白)和细胞骨架蛋白等基因的甲基化增加。这种高甲基化可通过脱氧氮胞苷处理而逆转，导致至少部分与顺铂耐药表型相关的基因的RNA转录减少。对P-gp作用机制的研究主要集中在转运蛋白识别多种不同底物和抑制剂的方式，底物相互作用如何导致ATPase的激活，以及ATPase如何导致药物的转运和外排。这些研究和其他研究得出的结论是，P-gp的氨基末端(TM5，6)和羧基末端(TM11，12)的TM片段主要形成底物和抑制剂相互作用的多个可能重叠的部位，ATPase的激活导致底物与P-gp的结合减少。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。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，我们开发了实时荧光聚合酶链式反应和微阵列技术来检测48个已知的ABC转运蛋白中的大多数；这些技术已经被用于关联新的ABC转运蛋白在已知耐药的癌细胞系中的表达。在基因治疗中，MDR1基因作为显性可选择标记的应用主要集中在发展SV40作为MDR1载体的研究。利用重组的SV40衣壳蛋白可以在体外包装DNA，包括P-gp和绿色荧光蛋白(GFP)载体。使用体外包装的DNA转导P-gp和GFP在包括淋巴样细胞、肝细胞和角质形成细胞在内的许多不同类型的细胞中都是高效的，并且允许转移多达15kb的DNA，而不需要在包装的DNA中包含SV40序列。这种方法为将P-gp转移到造血细胞和其他细胞进行基因治疗提供了希望。

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 have been 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.