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 治疗免疫缺陷和腺苷脱氨酶的其他基因 缺乏，以及针对长末端的核酶 人类免疫缺陷病毒(HIV)、荧光素酶和β-内切酶中的重复(LTR) 以半乳糖苷酶为标记基因，等耐药基因， 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培养的骨髓干细胞。 体外或与脂质体在体内络合。我们已经分析了 顺铂多药耐药机制的研究进展 肝癌细胞和KB腺癌细胞。对顺铂耐药 肝癌和KB细胞对氨甲喋呤、亚砷酸盐和 并显示这些有毒物质的积累减少是由于 这些毒素的多效性缺乏特定的摄取系统 探员们。

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 and adenosine deaminase deficiency, 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.