The mechanism of regulation of P-glycoprotein (Analysis of multidrug-resistant mutants that do not express P-glycoprotein)

1)我们已经分离出对高水平阿霉素耐药的人表皮样KB细胞系C-A90、C-A120、C-A500和C-A1000。它们在含有递增浓度的阿霉素、1 μ g/ml千金藤素（一种多药耐药（MDR）逆转剂）和100 nM美泽瑞因（一种蛋白激酶C激活剂）的选择培养基中分离。阿霉素耐药KB细胞系之一C-A500对典型的多药耐药表型的药物具有交叉耐药性，如长春新碱、放线菌素D，2）与KB-3-1细胞相比，阿霉素和长春新碱在C-A 500细胞中的蓄积减少，阿霉素从C-A 500细胞中的外排增加。耐药KB细胞不含可检测水平的P-糖蛋白或过表达MDR 1。多药耐药相关蛋白（MRP）和MRP mRNA在阿霉素耐药KB细胞C-A120、C-A500和C-A1000中表达，而在亲代KB-3-1和rev-1000中不表达。 ...更多信息 突变C-AR细胞。4）C-A500和C-A1000细胞DNA拓扑异构酶II水平明显降低，而C-A120细胞仅轻微降低。以上结果表明，MRP在耐药细胞中的过表达可能是导致阿霉素和长春新碱积累减少的原因，MRP表达增加和拓扑异构酶II水平降低是C-A120、C-A500和C-A1000细胞耐药的基础（KB-8-5、KB-C2、C-A40和C-A120）、人非小细胞肺癌（NSCLC）、胃癌和结直肠癌细胞系中的表达，并与药物敏感的人KB细胞中的表达进行比较。MRP基因在9例肺鳞癌中有8例（89%）表达阳性。4例肺鳞癌（L13、18、19和20）MRP表达均高于KB-3-1细胞3.6倍以上，且肺鳞癌MRP mRNA平均表达水平明显高于肺腺癌、结直肠癌和胃癌。少

1) We have isolated human epidermoid KB cell lines resistant to high levels of adriamycin, C-A90, C-A120, C-A500 and C-A1000. They were isolated in selection medium containing increasing concentrations of adriamycin, 1mug/ml of cepharanthine, a multidrug-resistance (MDR) reversing agent, and 100nM mezerein, a protein kinas C activating agent. One of the adriamycin-resistant KB cell lines, C-A500, was cross-resistant to drugs that typify the classical multidrug resistance phenotype, such as vincristine, actinomycin D,VP-16 and colchicine.2) The accumulation of adriamycine and vincristine was decreased in C-A500 cells and the efflux of adriamycine from C-A 500 was enhanced compared with parental KB-3-1 cells.These adriamycin-resistant KB cells did not contain detectable levels of P-glycoprotein or overexpress MDR1. Multidrug-resistance-associated protein (MRP) and MRP mRNA were expressed in the adriamycin-resistant KB-cells, C-A120, C-A500 and C-A1000, but not in parental KB-3-1 and reve … More rtant C-AR cells. The MRP gene was amplified in these cells that overexpressed MRP mRNA.4) DNA topoisomerase II levels were markedly decreased in C-A500 and C-A1000 cells but only slightly decreased in C-A120 cells. These results indicate that MRP overexpressed in the resistant cells may be responsible for the reduced accumulation of adriamycin and vincrisrine and that both the increased expression of MRP and decreasd levels of topoisomerase II underlie te drug resistance in C-A120, C-A500 and C-A1000 cell lines.5) We investigated the expression of MRP mRNA in multidrug-resistant KB cell line (KB-8-5, KB-C2, C-A40 and C-A120), human non-small-cell lung carcinomas (NSCLC), gastric and colorectal carcinomas and compared it with that in drug-sensitive human KB cells. MRP gene expression was elavated in 8 of 9 (89%) squamous-cell carcinomas of the lung. Furthermore, MRP expression in 4 squamous-cell carcinomas (L13,18,19 and 20) was more than 3.6 times higher than in KB-3-1 cells, and the average MRP mRNA expression level of all squamous-cell carcinomas was significantly higher than that of adenocarcinoma of the lung and of colorectal and gastric carcinomas. Less