Fardel O, Loyer P, Lecureur V, Glaise D, Guillouzo A
Unité de Recherches Hépatologiques U 49 de l'Institut National de la Santé et de la Recherche Médicale, Hôpital Pontchaillou, Rennes, France.
Eur J Biochem. 1994 Jan 15;219(1-2):521-8. doi: 10.1111/j.1432-1033.1994.tb19967.x.
P-glycoprotein is a plasma-membrane glycoprotein involved in multidrug resistance. P-glycoprotein overexpression has been demonstrated to occur in tumor cells after cytotoxic drug exposure, but also in some cancers including hepatocellular carcinomas before any chemotherapeutic treatment. In order to better analyze this constitutive type of tumoral drug resistance, we have investigated P-glycoprotein expression and function in rat liver tumors induced experimentally by administration of diethylnitrosamine and in two cell clones derived from one of these tumors designated as RHC1 and RHC2. High levels of P-glycoprotein mRNAs were found in both liver tumor samples and the two hepatoma cell clones as assessed by Northern blotting; both RHC1 and RHC2 cells displayed altered liver functions commonly observed in rat hepatoma cells, particularly the decreased expression of albumin and overexpression of the fetal glutathione S-transferase 7-7. The use of specific multidrug resistance (mdr) probes revealed a major induction of the mdr1 gene in liver tumor samples while RHC1 and RHC2 cells expressed both mdr1 and mdr3 genes without displaying a major alteration in the number of mdr gene copies as assessed by Southern blotting. High amounts of P-glycoprotein were also demonstrated in RHC1 and RHC2 cells by Western blotting. These cells were strongly resistant to doxorubicin and vinblastine, two anticancer drugs transported by P-glycoprotein. Doxorubicin intracellular retention was low in RHC1 and RHC2 cells, but was strongly enhanced in the presence of verapamil, a known modulator agent of P-glycoprotein; low retention appeared to occur via a drug efflux mechanism, indicating that P-glycoprotein was fully active. These results show that rat hepatoma cells can display elevated levels of functional P-glycoprotein without any prior cytotoxic drug selection and suggest that these cells represent a useful model for analyzing P-glycoprotein regulation in intrinsically clinical drug-resistant cancers.
P-糖蛋白是一种参与多药耐药的质膜糖蛋白。已证明，细胞毒性药物暴露后肿瘤细胞中会出现P-糖蛋白过表达，但在一些癌症中，包括肝细胞癌，在任何化疗治疗之前也会出现这种情况。为了更好地分析这种肿瘤药物耐药的组成型类型，我们研究了通过给予二乙基亚硝胺实验诱导的大鼠肝肿瘤以及源自其中一个肿瘤的两个细胞克隆（命名为RHC1和RHC2）中P-糖蛋白的表达和功能。通过Northern印迹法评估，在肝肿瘤样本和两个肝癌细胞克隆中均发现了高水平的P-糖蛋白mRNA；RHC1和RHC2细胞均表现出大鼠肝癌细胞中常见的肝功能改变，特别是白蛋白表达降低和胎儿谷胱甘肽S-转移酶7-7过表达。使用特异性多药耐药（mdr）探针显示肝肿瘤样本中mdr1基因有主要诱导，而RHC1和RHC2细胞表达mdr1和mdr3基因，通过Southern印迹法评估，mdr基因拷贝数没有显示出主要改变。通过Western印迹法也证明RHC1和RHC2细胞中有大量的P-糖蛋白。这些细胞对阿霉素和长春碱这两种由P-糖蛋白转运的抗癌药物具有很强的抗性。阿霉素在RHC1和RHC2细胞中的细胞内滞留率较低，但在维拉帕米（一种已知的P-糖蛋白调节剂）存在下会强烈增强；低滞留似乎是通过药物外排机制发生的，表明P-糖蛋白是完全活跃的。这些结果表明，大鼠肝癌细胞在没有任何先前细胞毒性药物选择的情况下可以显示出功能性P-糖蛋白的升高水平，并表明这些细胞代表了一个有用的模型，用于分析内在临床耐药癌症中P-糖蛋白的调节。
