Kosztyu Petr, Dolezel Petr, Vaclavikova Radka, Mlejnek Petr
Department of Anatomy, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic.
Department of Biology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic.
Eur J Haematol. 2015 Aug;95(2):150-9. doi: 10.1111/ejh.12470. Epub 2015 Jan 22.
Increased expression of the ABCB1 gene in cancer cells is usually connected with occurrence of multidrug resistance (MDR) and poor prognosis. However, the correlation between ABCB1 expression and MDR phenotype is difficult to prove in clinical samples. Most of the researchers believe that these difficulties are due to the poor reliability and sensitivity of assays for detection of ABCB1 expression in clinical samples. However, the complexity of P-gp mediated resistance cannot be reduced to the methodical difficulties only. Here, we addressed the question how widely used methods for detection of ABCB1 expression levels could predict its functional activity and thus its contribution to drug resistance in defined conditions in vitro. The ABCB1 expression was assessed at the mRNA level by quantitative real-time polymerase chain reaction (qRT-PCR), and at the protein level by flow cytometry using UIC2 antibody. The ABCB1 function was monitored using a calcein AM accumulation assay. We observed that K562 cells have approximately 320 times higher level of ABCB1 mRNA than HL-60 cells without detectable function. In addition, resistant K562/Dox cells exhibited significantly higher ABCB1 mRNA expression than resistant K562/HHT cells. However, the functional tests clearly indicated opposite results. Flow cytometric assessment of P-gp, although suggested as a reliable method, contradicted the functional test in K562/Dox and K562/HHT cells. We further used a set of MDR cells expressing various levels of P-gp. Similarly here, flow cytometry not always corresponded to the functional analysis. Our results strongly suggest that an approach which exclusively relies on a simple correlation between ABCB1 expression, either at the mRNA level or protein level, and overall resistance may fail to predict actual contribution of P-gp to overall resistance as the data indicating transporter expression reflect its function only roughly even in well-defined in vitro conditions.
ABCB1基因在癌细胞中的表达增加通常与多药耐药性（MDR）的发生及不良预后相关。然而，在临床样本中很难证明ABCB1表达与MDR表型之间的相关性。大多数研究人员认为，这些困难是由于临床样本中检测ABCB1表达的检测方法可靠性和敏感性较差所致。然而，P-糖蛋白介导的耐药性的复杂性不能仅仅归结为方法上的困难。在此，我们探讨了广泛使用的检测ABCB1表达水平的方法在体外特定条件下预测其功能活性以及对耐药性贡献的能力。通过定量实时聚合酶链反应（qRT-PCR）在mRNA水平评估ABCB1表达，使用UIC2抗体通过流式细胞术在蛋白质水平评估ABCB1表达。使用钙黄绿素AM积累试验监测ABCB1功能。我们观察到，K562细胞的ABCB1 mRNA水平比HL-60细胞高约320倍，但未检测到功能。此外，耐药的K562/Dox细胞的ABCB1 mRNA表达明显高于耐药的K562/HHT细胞。然而，功能测试结果却恰恰相反。虽然流式细胞术评估P-糖蛋白被认为是一种可靠的方法，但在K562/Dox和K562/HHT细胞中，其结果与功能测试相矛盾。我们进一步使用了一组表达不同水平P-糖蛋白的MDR细胞。同样，在这里流式细胞术并不总是与功能分析一致。我们的结果强烈表明，仅依赖于mRNA水平或蛋白质水平的ABCB1表达与总体耐药性之间简单相关性的方法可能无法预测P-糖蛋白对总体耐药性的实际贡献，因为即使在明确的体外条件下，表明转运蛋白表达的数据也只能大致反映其功能。
