Can the assessment of ABCB1 gene expression predict its function in vitro?
Can the assessment of ABCB1 gene expression predict its function in vitro?
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DOI：
10.1111/ejh.12470
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发表时间：
2015-08-01
影响因子：
3.1
通讯作者：
Mlejnek, Petr
中科院分区：
文献类型：
--
作者：
Kosztyu, Petr;Dolezel, Petr;Mlejnek, Petr
ABCB 1基因在癌细胞中的表达增加通常与多药耐药（MDR）的发生和不良预后有关。然而，ABCB 1表达与MDR表型之间的相关性难以在临床样本中证明。大多数研究人员认为，这些困难是由于检测临床样本中ABCB 1表达的检测方法的可靠性和灵敏度较差。然而，P-gp介导的耐药的复杂性不能仅仅归结为方法上的困难。在这里，我们解决了一个问题，即广泛使用的检测ABCB 1表达水平的方法如何预测其功能活性，从而预测其在体外特定条件下对耐药性的贡献。通过定量实时聚合酶链反应（qRT-PCR）在mRNA水平评估ABCB 1表达，并使用UIC 2抗体通过流式细胞术在蛋白水平评估ABCB 1表达。使用钙黄绿素AM积累测定来监测ABCB 1功能。我们观察到K562细胞的ABCB 1 mRNA水平比HL-60细胞高约320倍，但没有检测到功能。K562/Dox耐药细胞ABCB 1 mRNA的表达明显高于K562/HHT耐药细胞。然而，功能测试清楚地表明了相反的结果。流式细胞术评估P-gp，虽然建议作为一种可靠的方法，矛盾的K562/Dox和K562/HHT细胞的功能测试。我们进一步使用了一组表达不同水平P-gp的MDR细胞。类似地，流式细胞术并不总是与功能分析相对应。我们的研究结果强烈表明，一种方法，它完全依赖于一个简单的ABCB 1表达之间的相关性，无论是在mRNA水平或蛋白质水平，和整体电阻可能无法预测的实际贡献的P-gp的整体电阻的数据表明，转运蛋白的表达反映其功能，即使在明确的体外条件下，只有粗略的。
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 ABCB1mRNA than HL-60 cells without detectable function. In addition, resistant K562/Dox cells exhibited significantly higher ABCB1mRNA 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.