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: 2463749
• 负责人: I PASTAN
• 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/2463749
• 关键词: P glycoprotein; adenosinetriphosphatase; antineoplastics; complementary DNA; drug receptors; enzyme activity; gene expression; gene therapy; genetic disorder; genetic markers; membrane transport proteins; multidrug resistance; neoplasm /cancer genetics; neoplastic cell; nucleic acid sequence; phenotype; protein structure function; 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 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 have been developed to assay functional effects of these mutations on drug binding, drug-dependent ATPase, drug resistance and drug transport. Substitution of all known phosphorylation sites in P-gp with either Ala or Asp does not affect ability of P-gp to confer multidrug resistance. The creation of bicistronic retroviral expression vectors able to confer multidrug resistance has enabled the development of vectors for treatment of other genetic diseases such as Fabry disease, Gaucher disease, chronic granulomatous disease, X-linked severe combined immunodeficiency and adenosine deaminase deficiency. In these vectors, P-gp serves as a dominant selectable marker. These vectors may be delivered to bone marrow stem cells grown ex vivo or complexed to liposomes in vivo. We have also begun to explore 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 accumulate reduced amounts of these three toxic agents.

我们一直有兴趣定义以下主要机制： 肿瘤细胞对多种化疗药物的同时耐药 探员们。一个主要的机制是表达一种能量依赖的 外排泵，称为P-糖蛋白(P-gp)，或多药 转运蛋白，在人类中由mdr1基因编码。按顺序排列的 Mdr1基因的cdna导致了一个具有12个转运体的模型 跨膜结构域和2个三磷酸腺苷位点.结构域的测定 负责底物结合和偶联ATPase活性的P-gp 到衬底的运输是我们工作的主要目标。模型系统 基于突变的P-GPS的稳定表达或瞬时表达 一种痘苗病毒表达系统已经被开发出来用于检测 这些突变对药物结合的功能影响，药物依赖 ATPase、耐药性和药物转运。已知的全部替换 丙氨酸或天冬氨酸对P-gp的磷酸化位点无影响 P-gp诱导多药耐药的能力。创造了一个 可赋予多药的双顺反子逆转录病毒表达载体 抗药性使治疗该病的媒介得以发展 其他遗传性疾病，如法布里病、高谢病、慢性 肉芽肿性疾病，X连锁严重联合免疫缺陷和 腺苷脱氨酶缺乏症。在这些载体中，P-gp作为一种 显性可选标记。这些载体可能会被输送到骨骼中 骨髓干细胞在体外生长或在体内与脂质体复合。我们 也开始探索多药耐药的机制 肝癌细胞和KB顺铂筛选的结果 腺癌细胞。耐顺铂的肝癌和KB细胞 对甲氨蝶呤、亚砷酸盐和亚硫酸盐交叉抗药性并积累 减少了这三种毒剂的用量。