Pirity M, Hevér-Szabó A, Venetianer A
Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.
Cytotechnology. 1996;19(3):207-14. doi: 10.1007/BF00744214.
We have earlier isolated a glucocorticoid-resistant, dedifferentiated rat hepatoma variant, the clone 2, which exhibited deficient stress activation of the major stress-inducible heat-shock protein hsp68. Multidrug-resistant variants were isolated from clone 2 cells using increasing concentrations of colchicine. The induction deficiency of hsp68 was maintained in the colchicine-resistant clone 2 cells grown for several months in the presence of 1 microgram/ml colchicine (termed as highly multidrug-resistant variant) indicating that this heat-shock protein is not involved in the multidrug resistance. No alteration of the protein synthesis pattern was observed except the strong increase of the P-glycoprotein, which correlated with high level of corresponding mRNA. Stable heat-resistant variants of clone 2 were also isolated, which showed increased drug resistance to several drugs, i.e. they became moderately multidrug-resistant. This moderate multidrug resistance of the heat-resistant variants was further increased by stepwise selection with colchicine (highly multidrug-resistant heat-resistant variants). The levels of P-glycoprotein mRNA and protein were elevated both in the heat-resistant, non drug selected, moderately drug-resistant and in heat-resistant, colchicine selected, highly drug-resistant variants. Decreased retention of antitumor drugs was observed in all multidrug-resistant variants indicating that P-glycoprotein was functional. Verapamil increased doxorubicin retention and cytotoxicity significantly. Our results showing that severely stressed hepatoma cells overexpressed the multidrug resistance gene(s) raise the possibility that the P-glycoprotein may participate in protection against environmental stress such as heat.
我们之前分离出了一种糖皮质激素抵抗性、去分化的大鼠肝癌变异体，即克隆2，它表现出主要应激诱导型热休克蛋白hsp68的应激激活缺陷。使用浓度递增的秋水仙碱从克隆2细胞中分离出多药耐药变异体。在1微克/毫升秋水仙碱存在下培养数月的秋水仙碱抗性克隆2细胞中，hsp68的诱导缺陷得以维持（称为高度多药耐药变异体），这表明这种热休克蛋白不参与多药耐药。除了P-糖蛋白强烈增加外，未观察到蛋白质合成模式的改变，这与相应mRNA的高水平相关。还分离出了克隆2的稳定耐热变异体，它们对几种药物的耐药性增加，即它们变成了中度多药耐药。通过用秋水仙碱逐步选择（高度多药耐药耐热变异体），耐热变异体的这种中度多药耐药性进一步增加。在耐热、未选择药物的中度耐药变异体以及耐热、经秋水仙碱选择的高度耐药变异体中，P-糖蛋白mRNA和蛋白质水平均升高。在所有多药耐药变异体中均观察到抗肿瘤药物的滞留减少，表明P-糖蛋白具有功能。维拉帕米显著增加了阿霉素的滞留和细胞毒性。我们的结果表明，受到严重应激的肝癌细胞过度表达多药耐药基因，这增加了P-糖蛋白可能参与抵御热等环境应激的可能性。
