Genetic Analysis of the Multidrug Resistance Phenotype i

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，这种交叉耐药通常是由于被称为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，该转运蛋白是MDR1或ABCB1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普遍存在缺陷，细胞内蛋白质运输、细胞骨架和葡萄糖代谢存在缺陷。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。在较高的耐药水平下，经过顺铂的多个选择步骤后，结合蛋白（例如叶酸结合蛋白）的基因甲基化增加，细胞骨架蛋白（包括丝蛋白、肌动蛋白、微管蛋白和g-连环蛋白）的调控改变，以及参与糖代谢调控的基因。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。我们已经开发了一种可抑制P-gp的细胞系，并证明了尽管P-gp的表达直接导致了药物积累的减少，但许多其他通常归因于P-gp表达的表型，包括膜流动性和膜电位的改变，并不是由P-gp直接引起的。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。然而，在特定P-gp单倍型的设置中，同义多态性（没有氨基酸变化）可能影响P-gp泵送效率，其原因仍在研究中。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。大约30种ABC转运蛋白的表达与对特定细胞毒性药物的耐药性有关。此外，一些药物对P-gp表达细胞的毒性比对非P-gp表达细胞的毒性更大。多药耐药细胞暴露于这些化合物导致幸存者不再具有多药耐药，这提示了一种治疗多药耐药癌症的新方法。此外，与NIEHS微阵列中心合作创建了ABC毒理hip，用于分析选择的多种耐多药细胞系。该芯片已鉴定出与ABCB6、ABCA12和ABCC2表达相关的新抗性表型。我们还在黑色素瘤细胞中发现了ABC转运蛋白的独特特征。其中一种转运蛋白ABCB5与P-gp （MDR1）密切相关，似乎有助于黑色素瘤细胞的多药耐药。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。

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, the cytoskeleton, and in glucose metabolism. 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), altered regulation of cytoskeletal proteins including filamin, actin, tubulin and g-catenin, and genes involved in regulation of glucose metabolism. 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. Exposure of multidrug-resistant cells to these compounds results in survivors that are no longer multidrug-resistant, suggesting a novel approach to treatment of multidrug-resistant cancers. In addition, an ABC ToxiChip has been created in collaboration with the NIEHS microarray center and used to analyze various cell lines selected for multidrug resistance. 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 appears to contribute to multidrug resistance 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.