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: 6160929
• 负责人: 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/6160929
• 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

We have been interested in defining the major mechanisms of simultaneous resistance of cancer cells to multiple chemotherapeutic agents. One major mechanism is expression of an energy-dependent efflux pump, termed P-glycoprotein (P-gp), or the multidrug transporter, encoded in humans by the MDR1 gene. The sequence of the MDR1 cDNA led to a model of the transporter as a pump with 12 transmembrane domains and 2 adenosine 5'- triphosphate (ATP) sites; determination of the domains of P-gp responsible for substrate binding and coupling of ATPase activity to substrate transport are the major goals of our work. Model systems based on stable expression or transient expression of mutated P-gps by a vaccinia virus expression system or a baculovirus system have been developed to assay functional effects of these mutations on drug binding, drug-dependent ATPase, drug resistance and drug transport. Mutations in either or both ATP sites eliminate the ability of P-gp to pump fluorescent substrates or confer drug resistance. These ATP sites are not fully functionally interchangeable as demonstrated by creation of P-gp chimeras and by labeling experiments with 32P-azido-ATP, supporting a model of alternating use of ATP sites in which the N- terminal site is utilized first. Evidence for the interaction of the C- terminal ATP sites with an N-terminal substrate binding site has been obtained by analysis of a mutation in the TM6 which affects substrate binding, but also allows a deletion in the "C" region of the C-terminal ATP site to be expressed on the cell surface. We have constructed bicistronic retroviral expression vectors carrying MDR1 and several other genes for treatment of immunodeficiency, X-linked severe combined, adenosine deaminase deficiency, Fabry disease, Gaucher disease, and chronoic granulomatous disease, as well as a ribozyme directed against the long terminal repeat (LTR) in human immunodeficiency virus (HIV), luciferase and beta-galactosidase as marker genes, and other drug- resistance genes, dihydrofolate reductase (DHFR), and methylguanine methyltransferase (MGMT). These vectors may be delivered to bone marrow stem cells grown ex vivo or complexed to liposomes in vivo. We have analyzed the mechanism of multidrug resistance resulting from selection in cisplatin of hepatoma cells and KB adenocarcinoma cells. Cisplatin- resistant hepatoma and KB cells are cross-resistant to methotrexate, arsenite and antimonite and show reduced accumulation of these toxic agents due to the pleiotropic absence of specific uptake systems for these toxic agents.

我们一直有兴趣在定义的主要机制，同时 癌细胞对多种化疗剂的抗性。一 主要机制是表达能量依赖性外排泵，称为 P-糖蛋白（P-gp），或多药转运蛋白，在人体内编码 MDR 1基因。MDR 1 cDNA序列导致了一个模型， 作为泵的转运蛋白，具有12个跨膜结构域和2个腺苷5 '- 三磷酸（ATP）位点; P-gp结构域的测定 负责底物结合和ATP酶活性与 衬底传输是我们工作的主要目标。基于模型系统 稳定表达或瞬时表达突变的P-gps的研究 牛痘病毒表达系统或杆状病毒系统已经被 开发用于测定这些突变对药物的功能影响， 结合、药物依赖性ATP酶、耐药性和药物转运。 ATP位点之一或两个位点的突变消除了P-gp的能力， 泵浦荧光底物或赋予耐药性。这些ATP位点 在功能上不能完全互换， 的P-gp嵌合体，并通过标记实验与32 P-叠氮-ATP， 支持交替使用ATP位点的模型，其中N- 首先使用终端站点。证据表明C- 末端ATP位点与N-末端底物结合位点的结合， 通过分析影响底物的TM 6中的突变获得 结合，但也允许在C-末端的“C”区域中缺失 细胞表面上要表达的ATP位点。我们已经构建 携带MDR 1和几种MDR 2的双顺反子逆转录病毒表达载体 用于治疗免疫缺陷的其他基因，X连锁严重联合， 腺苷脱氨酶缺乏症、法布里病、戈谢病和 慢性肉芽肿病，以及针对 人类免疫缺陷病毒（HIV）中的长末端重复序列（LTR）， 荧光素酶和β-半乳糖苷酶作为标记基因，以及其它药物- 耐药基因、二氢叶酸还原酶（DHFR）和甲基鸟嘌呤 甲基转移酶（MGMT）。这些载体可以被递送到骨髓 离体生长的干细胞或与体内脂质体复合的干细胞。我们有 分析了选择导致多药耐药的机制 顺铂对人肝癌细胞和KB腺癌细胞的作用。顺铂- 抗性肝癌和KB细胞对甲氨蝶呤交叉抗性， 砷和锑，并显示这些有毒物质的积累减少 由于缺乏特异性摄取系统， 这些有毒物质。