Slapak C A, Fracasso P M, Martell R L, Toppmeyer D L, Lecerf J M, Levy S B
Center for Adaptation Genetics and Drug Resistance, Tufts University School of Medicine, Boston, MA 02111.
Cancer Res. 1994 Nov 1;54(21):5607-13.
Multidrug-resistant sublines of the murine erythroleukemia cell line PC4 were sequentially selected in increasing vincristine concentrations (5-160 ng/ml). The low- and intermediate-level resistant cell lines, selected in < or = 40 ng/ml of vincristine, demonstrated resistance to Vinca alkaloids and to an epipodophyllotoxin but little or none to an anthracycline. The expression of murine mdr genes, as analyzed by Northern blotting, revealed a baseline expression of murine mdr2 in parental cells that was unchanged in the drug-resistant cell lines. Overexpression of mdr3 was observed only in the highest-level resistant cell line, PC-V160, whereas mdr1 mRNA was not detected in any of the cell lines. The polymerase chain reaction, using mdr3-specific primers, excluded the possibility that low levels of P-glycoprotein expression contributed to the resistance phenotype in the low and intermediate-level resistant cell lines. Northern blot analysis using a human complementary DNA probe for the multidrug resistance-associated protein (MRP) demonstrated overexpression of murine mrp in each of the vincristine-selected sublines. Genomic amplification of the mrp gene was coincident with mrp overexpression. The expression of mrp was also examined in two series of previously characterized doxorubicin-selected cell lines derived from parental PC4 and C7D murine erythroleukemia cells. In contrast to the vincristine-selected cell lines, overexpression of mrp was not detected. These studies demonstrate that, in murine erythroleukemia cells selected for vincristine resistance, overexpression of murine mrp occurred prior to that for murine mdr. In contrast to human MRP, selection for vincristine, but not doxorubicin resistance, resulted in the overexpression of murine mrp.
在不断增加的长春新碱浓度（5 - 160 ng/ml）下，对小鼠红白血病细胞系PC4的多药耐药亚系进行了连续选择。在≤40 ng/ml长春新碱中选择的低水平和中等水平耐药细胞系，对长春花生物碱和一种鬼臼毒素表现出抗性，但对蒽环类药物几乎没有抗性。通过Northern印迹分析的小鼠mdr基因表达显示，亲本细胞中存在小鼠mdr2的基础表达，在耐药细胞系中未发生变化。仅在最高水平耐药细胞系PC - V160中观察到mdr3的过表达，而在任何细胞系中均未检测到mdr1 mRNA。使用mdr3特异性引物的聚合酶链反应排除了低水平P - 糖蛋白表达导致低水平和中等水平耐药细胞系中耐药表型的可能性。使用针对多药耐药相关蛋白（MRP）的人互补DNA探针进行的Northern印迹分析表明，在每个长春新碱选择的亚系中均存在小鼠mrp的过表达。mrp基因的基因组扩增与mrp过表达一致。还在源自亲本PC4和C7D小鼠红白血病细胞的两系列先前表征的阿霉素选择的细胞系中检查了mrp的表达。与长春新碱选择的细胞系相反，未检测到mrp的过表达。这些研究表明，在选择长春新碱耐药的小鼠红白血病细胞中，小鼠mrp的过表达先于小鼠mdr。与人类MRP相反，选择长春新碱耐药而非阿霉素耐药导致小鼠mrp的过表达。
