Multidrug Resistance Phenotype in Tumor Cells

化疗耐药发生在癌细胞中，是因为特定蛋白质表达的内在或获得性变化。我们研究了对天然产物化疗药物的耐药性，如阿霉素、长春花生物碱和紫杉醇，以及对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，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是由于摄取系统的多效性缺陷导致药物内流减少。最近的证据表明，这些顺铂耐药细胞的内吞作用和细胞内蛋白质运输和细胞骨架的缺陷存在全球性缺陷。单步顺铂耐药突变体显示蛋白质运输缺陷，导致细胞质中细胞表面受体/转运体/通道的积累；假定的顺铂载体/通道在这些错定位的蛋白质中，导致顺铂摄取减少。在耐药水平较高的情况下，经过顺铂的多个选择步骤后，结合蛋白（例如叶酸结合蛋白）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治疗逆转，导致至少部分导致顺铂耐药表型的基因RNA转录降低。对P-gp作用机制的研究主要集中在转运体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酶的激活，以及atp酶如何导致药物易位和外排。这些研究和其他研究得出的结论是，主要由P-gp的氨基端（TM5,6）和羧基端（TM11,12）一半的TM片段形成的底物和抑制剂相互作用存在多个可能重叠的位点，并且atp酶的激活导致底物与P-gp的结合减少。对P-gp正常功能的研究表明，它参与许多药物的正常摄取和分布。已经检测到P-gp的常见多态性变异，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的可能性，我们开发了实时PCR和微阵列技术，用于检测已知的48种ABC转运蛋白中的大多数；这些技术已被用于在已知耐药的癌细胞系中关联新的ABC转运蛋白的表达。在基因治疗中，将MDR1基因作为显性选择标记物的重点是将SV40作为MDR1的载体。利用重组SV40衣壳蛋白，可以在体外包装DNA，包括P-gp和含有绿色荧光蛋白（GFP）的载体。使用体外包装DNA转导P-gp和GFP在许多不同的细胞类型（包括淋巴细胞、肝细胞和角化细胞）中都是高效的，并且可以在不需要包装DNA中的SV40序列的情况下转移多达15 kb的DNA。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。

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.