Gromicho Marta, Rueff José, Rodrigues António Sebastião
Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Rua Câmara Pestana 6, 1150-008, Lisbon, Portugal.
Methods Mol Biol. 2016;1395:75-85. doi: 10.1007/978-1-4939-3347-1_6.
Cellular drug resistance remains a major concern in cancer therapy and usually results from increased expression of ABC drug transporters. Imatinib mesylate (IM), a competitive inhibitor of BCR/ABL1 tyrosine kinase activity, is the current standard therapy for chronic myeloid leukaemia (CML) which is caused by the BCR/ABL1 gene fusion encoding a constitutively active tyrosine kinase. However, up to 33 % of CML patients do not respond to therapy either initially or due to acquired resistance. Usually, IM resistance is due to the presence of BCR-ABL1 mutations but in many cases resistance is far from being completely understood or from being satisfactorily addressed from a therapeutic standpoint. Although second- and third-generation TKIs (e.g., dasatinib (DA), nilotinib, and bosutinib) were developed to override this phenomenon, resistance remains an unsolved problem. Above all, as more patients are treated with TKIs, more cases of resistance are expected and the discovery of biomarkers of resistance acquires a crucial clinical significance. We established a valuable in vitro experimental system that mimics the acquired resistance in the absence of mutations. It was developed by the continuous exposure of K562, a human CML-derived cell line expressing BCR-ABL gene, to increasing concentrations of IM and DA (over 36 and 24 weeks, respectively) allowing us to obtain several cell lines with different resistance levels, and therefore to evaluate drug transporters' role in the dynamic cellular responses allied with resistance evolution. The development of such cell models is fundamental to understand the role of drug transporters in resistance since the majority of previous studies were performed on cell lines engineered to over-express a single transporter. Drug transporters were overexpressed in the majority of resistant cell lines and cell lines from all levels of resistance had increased expression of more than one drug transporter. However, the transporters that attain higher mRNA overexpression (e.g., ABCB1 and ABCG2) did not substantiate a linear relation with the level of resistance. Also, variation in expression of these genes occurs over time of exposure to the same concentration of IM while maintaining resistance, suggesting that resistance mechanisms could vary dynamically in patients as disease progresses. Indeed, we observed that while responding patients demonstrated stable transporters' expression signatures in consecutive samples, in IM-resistant patients they vary significantly over time, advising caution when comparing single-point samples from responsive and resistant patients.
细胞耐药性仍然是癌症治疗中的一个主要问题，通常是由ABC药物转运蛋白的表达增加所致。甲磺酸伊马替尼（IM）是BCR/ABL1酪氨酸激酶活性的竞争性抑制剂，是目前治疗慢性髓性白血病（CML）的标准疗法，CML由编码组成型活性酪氨酸激酶的BCR/ABL1基因融合引起。然而，高达33%的CML患者最初对治疗无反应，或因获得性耐药而无反应。通常，IM耐药是由于存在BCR-ABL1突变，但在许多情况下，耐药的原因远未完全明了，从治疗角度来看也未得到令人满意的解决。尽管开发了第二代和第三代酪氨酸激酶抑制剂（TKIs，如达沙替尼（DA）、尼洛替尼和博舒替尼）来克服这一现象，但耐药性仍然是一个未解决的问题。最重要的是，随着越来越多的患者接受TKIs治疗，预计会出现更多的耐药病例，因此耐药生物标志物的发现具有至关重要的临床意义。我们建立了一个有价值的体外实验系统，该系统可模拟无突变情况下的获得性耐药。它是通过将表达BCR-ABL基因的人CML衍生细胞系K562分别连续暴露于浓度不断增加的IM和DA（分别超过36周和24周）而建立的，这使我们能够获得几种具有不同耐药水平的细胞系，从而评估药物转运蛋白在与耐药演变相关的动态细胞反应中的作用。这种细胞模型的建立对于理解药物转运蛋白在耐药中的作用至关重要，因为以前的大多数研究都是在经过工程改造以过度表达单一转运蛋白的细胞系上进行的。在大多数耐药细胞系中药物转运蛋白过度表达，并且来自所有耐药水平的细胞系中不止一种药物转运蛋白的表达增加。然而，mRNA过度表达较高的转运蛋白（如ABCB1和ABCG2）与耐药水平之间并未证实存在线性关系。此外，在暴露于相同浓度的IM并维持耐药性的过程中，这些基因的表达会随时间发生变化，这表明随着疾病进展，患者的耐药机制可能会动态变化。事实上，我们观察到，有反应的患者在连续样本中表现出稳定的转运蛋白表达特征，而在IM耐药患者中，它们随时间变化显著，这建议在比较有反应和耐药患者的单点样本时要谨慎。
