GENETIC ANALYSIS OF THE MULTIDRUG RESISTANCE PHENOTYPE IN TUMOR CELLS

我们一直有兴趣确定的主要机制， 癌细胞对多种化疗药物同时耐药 剂. 一个主要的机制是表达一种能量依赖性的 外排泵，称为P-糖蛋白（P-gp），或多药 转运蛋白，在人类中由MDR 1基因编码。 的序列 MDR 1 cDNA导致转运蛋白作为泵的模型，具有12 跨膜结构域和2个ATP位点; 负责底物结合和ATP酶活性偶联的P-gp 基板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， 已经开发了牛痘病毒表达系统来测定 这些突变对药物结合、药物依赖性 ATP酶、耐药性和药物转运。 替代所有已知的 P-gp中具有Ala或Asp的磷酸化位点不影响 P-gp赋予多药耐药性的能力。 建立 能够赋予多药的双顺反子逆转录病毒表达载体 耐药性使得能够开发用于治疗 其他遗传性疾病如法布里病、戈谢病、慢性 肉芽肿病，X连锁严重联合免疫缺陷， 腺苷脱氨酶缺乏症 在这些载体中，P-gp充当 显性选择标记 这些载体可以被递送到骨 骨髓干细胞离体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 由肝癌细胞和KB的顺铂选择产生 腺癌细胞 顺铂耐药肝癌和KB细胞是 对甲氨蝶呤、亚砷酸盐和亚锑酸盐交叉耐药， 减少这三种有毒物质的含量。

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.