Genetic Analysis of the Multidrug Resistance Phenotype i

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，这种交叉耐药性通常是由于称为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，该系统是MDR 1或ABCB 1基因或ABC转运蛋白家族其他成员的产物。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。我们已经开发了一个tet抑制的P-gp细胞系，并证明，虽然减少药物积累是P-gp表达的直接后果，许多其他表型经常归因于P-gp表达，包括改变膜流动性和膜电位，是不是直接由于P-gp。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。然而，在特定P-gp单倍型的背景下的同义多态性（无氨基酸变化）可影响P-gp泵送的效率，原因仍在研究中。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;这些技术已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。已显示约30种ABC转运蛋白的表达与对特定细胞毒性药物的抗性相关。此外，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，这提示了治疗MDR癌症的新方法。此外，与NIEHS微阵列中心合作创建了ABC ToxChip，并用于分析选择用于MDR的各种细胞系。利用该芯片鉴定了与ABCB 6、ABCA 12和ABCC 2表达相关的新的抗性表型。我们还发现了黑色素瘤细胞中ABC转运蛋白的独特特征。其中一种转运蛋白ABCB 5与P-gp（MDR 1）密切相关，可能参与黑色素瘤细胞的MDR。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，并且在体内允许转移高达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。

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 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 other members of the ABC transporter family. 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 the normal function of P-gp suggest that it is involved in normal uptake and distribution of many drugs. We have developed a tet-repressible P-gp cell line and demonstrated that although reduced drug accumulation is a direct consequence of P-gp expression, many other phenotypes frequently attributed to P-gp expression, including altered membrane fluidity and membrane potential, are not due directly to P-gp. 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 (no amino acid change) in the setting of a specific P-gp haplotype can affect efficiency of P-gp pumping for reasons still under investigation. 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. Expression of approximately 30 ABC transporters has been shown to correlate with resistance to specific cytotoxic drugs. Furthermore, some drugs are more toxic to P-gp expressing cells than to non-expressors, suggesting a novel approach to treatment of MDR cancers. In addition, an ABC ToxChip has been created in collaboration with the NIEHS microarray center and used to analyze various cell lines selected for MDR. New resistance phenotypes associated with expression of ABCB6, ABCA12 and ABCC2 have been identified with this chip. We have also found a unique signature of ABC transporters in melanoma cells. One of these transporters, ABCB5, is closely related to P-gp (MDR1) and may contribute to MDR in melanoma cells. 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 in vivo 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.