GENETIC ANALYSIS OF THE MULTIDRUG RESISTANCE PHENOTYPE IN TUMOR CELLS

我们一直对定义同步的主要机制感兴趣 癌细胞对多种化疗药物的耐药性。一 主要机制是表达一种能量依赖的外排泵，称为 P-糖蛋白(P-gp)，或人类编码的多药转运蛋白 由mdr1基因决定。Mdr1基因的序列导致了一种模型 转运蛋白作为泵具有12个跨膜结构域和2个腺苷5‘- 三磷酸(ATP)部位.P-gp结构域的测定 负责底物结合和ATPase活性偶联 基质传输是我们工作的主要目标。基于模型系统的 突变的P-GPS基因的稳定表达或瞬时表达 痘苗病毒表达系统或杆状病毒系统已经 用来测试这些突变对药物的功能影响 结合，药物依赖的ATPase，耐药和药物转运。 其中一个或两个ATP位点的突变会消除P-gp的 泵送荧光底物或产生抗药性。这些ATP站点 在功能上不能完全互换，如创作所示 通过~(32)P-叠氮-三磷酸腺苷的标记实验， 支持交替使用ATP站点的模型，其中N- 首先利用终端站点。C-分子相互作用的证据 具有N-末端底物结合位点的末端ATP位点 通过分析TM6中影响底物的突变而获得 结合，但也允许在C-末端的“C”区缺失 三磷酸腺苷要在细胞表面表达。我们已经建造了 双顺反子逆转录病毒表达载体携带mdr1和几种 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、高谢病和 慢性肉芽肿性疾病，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，以及其他药物- 耐药基因二氢叶酸还原酶(DHFR)和甲基鸟嘌呤 甲基转移酶(MGMT)。这些载体可能被输送到骨髓。 干细胞在体外生长或在体内与脂质体复合。我们有 多药耐药的机制分析 在顺铂的肝癌细胞和KB腺癌细胞中。顺铂- 耐药肝癌和KB细胞对甲氨蝶呤交叉耐药， 亚砷酸盐和亚硫酸盐，并显示这些有毒物质的积累减少 由于缺乏多效性的特定摄取系统而导致的药物 这些有毒物质。

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.