GENETIC ANALYSIS OF THE MULTIDRUG RESISTANCE PHENOTYPE IN TUMOR CELLS

肿瘤细胞多药耐药表型的遗传分析

• 批准号: 6289127
• 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/6289127
• 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; human genetic material tag; multidrug resistance; neoplastic cell; transfection /expression vector; vaccinia virus

Cancer cells elude chemotherapy 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 known as P-glycoprotein (P-gp), the product of the MDR1 gene. For cisplatin, cross-resistance to methotrexate, some nucleoside analogs, and heavy metals is due to a reduction in drug influx resulting from a pleiotropic defect in uptake systems. 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. We have made molecular alterations in various domains of the 12 transmembrane (TM), 2 ATP-site MDR1 transporter and characterize the effects of these mutations on transport function after high-level transient expression of P-gp in a vaccinia virus-based system in cultured cells. Functional chimeras of MDR1 and MDR2 have revealed that minor alterations in TM6 of MDR2 allow the N-half of MDR2 to support multidrug transport in an MDR1 backbone, demonstrating the importance of TM6 in determining substrate specificity. In addition, we have shown that a specific mutation in TM12 alters ability of MDR to recognize a specific class of substrates including flupentixol. Mutational and biochemical analysis of the two ATP sites demonstrates that both are essential, but their ATP binding and catalytic activities differ. These studies and others have led to the following major conclusions: (1) 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; (2) substrate interaction sites include a high affinity on site, a lower affinity off site, and an allosteric site which affects ability of substrates to bind to the on site; (3) both amino- and carboxy- terminal ATP sites are essential for function of P-gp and the sites are partially interchangeable, but not identical; (4) both ATP sites are not utilized simultaneously, supporting a model of alternating activation of ATPase during substrate transport; and (5) activation of ATPase results in a reduction of substrate binding to P-gp, consistent with translocation of substrate from the on site to the off site. Use of the MDR1 gene as a dominant selectable marker in gene therapy has recently focused on the development of SV40 as a vector for delivery of MDR1. MDR1 can be efficiently packaged into SV40 vectors and delivered into hematopoietic cells. - cisplatin, gene therapy, multidrug resistance, phosphorylation, vaccinia virus, vectors, - Neither Human Subjects nor Human Tissues

癌细胞由于特定蛋白质表达的内在或后天变化而逃避化疗。我们研究了对天然产物化疗药物（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称为 P-糖蛋白 (P-gp) 的能量依赖性药物流出系统（MDR1 基因的产物）的表达所致。对于顺铂，与甲氨蝶呤、某些核苷类似物和重金属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。 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 ATP 酶激活以及 ATP 酶如何导致药物易位和外排。我们对 12 次跨膜 (TM)、2 个 ATP 位点 MDR1 转运蛋白的各个域进行了分子改变，并在培养细胞中基于痘苗病毒的系统中高水平瞬时表达 P-gp 后，表征了这些突变对转运功能的影响。 MDR1 和 MDR2 的功能嵌合体表明，MDR2 TM6 的微小改变使得 MDR2 的 N 半部分能够支持 MDR1 主链中的多药物转运，这证明了 TM6 在确定底物特异性方面的重要性。此外，我们还发现 TM12 中的特定突变会改变 MDR 识别包括氟哌噻吨在内的特定类别底物的能力。两个 ATP 位点的突变和生化分析表明，两者都是必需的，但它们的 ATP 结合和催化活性不同。这些研究和其他研究得出了以下主要结论：（1）底物和抑制剂的相互作用存在多个可能重叠的位点，这些位点主要由来自 P-gp 氨基端（TM5,6）和羧基端（TM11,12）半部的 TM 片段形成； (2)底物相互作用位点包括高亲和力的位点、较低亲和力的场外以及影响底物与位点结合能力的变构位点； (3) 氨基端和羧基端 ATP 位点对于 P-gp 的功能都是必需的，并且这些位点可以部分互换，但不相同； (4) 两个 ATP 位点不同时被利用，支持底物运输过程中 ATP 酶交替激活的模型； (5) ATP酶的激活导致底物与P-gp的结合减少，这与底物从位点到非位点的易位一致。使用 MDR1 基因作为基因治疗中的显性选择标记最近集中于开发 SV40 作为 MDR1 递送载体。 MDR1可以有效地包装到SV40载体中并递送到造血细胞中。 - 顺铂、基因治疗、多重耐药性、磷酸化、痘苗病毒、载体， - 既不是人类受试者，也不是人类组织