Divisão de Laboratórios CEMO, INCA, Rio de Janeiro, Brazil.
BMC Cancer. 2012 Jul 23;12:303. doi: 10.1186/1471-2407-12-303.
The advanced phases of chronic myeloid leukemia (CML) are known to be more resistant to therapy. This resistance has been associated with the overexpression of ABCB1, which gives rise to the multidrug resistance (MDR) phenomenon. MDR is characterized by resistance to nonrelated drugs, and P-glycoprotein (encoded by ABCB1) has been implicated as the major cause of its emergence. Wnt signaling has been demonstrated to be important in several aspects of CML. Recently, Wnt signaling was linked to ABCB1 regulation through its canonical pathway, which is mediated by β-catenin, in other types of cancer. In this study, we investigated the involvement of the Wnt/β-catenin pathway in the regulation of ABCB1 transcription in CML, as the basal promoter of ABCB1 has several β-catenin binding sites. β-catenin is the mediator of canonical Wnt signaling, which is important for CML progression.
In this work we used the K562 cell line and its derived MDR-resistant cell line Lucena (K562/VCR) as CML study models. Real time PCR (RT-qPCR), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), flow cytometry (FACS), western blot, immunofluorescence, RNA knockdown (siRNA) and Luciferase reporter approaches were used.
β-catenin was present in the protein complex on the basal promoter of ABCB1 in both cell lines in vitro, but its binding was more pronounced in the resistant cell line in vivo. Lucena cells also exhibited higher β-catenin levels compared to its parental cell line. Wnt1 and β-catenin depletion and overexpression of nuclear β-catenin, together with TCF binding sites activation demonstrated that ABCB1 is positively regulated by the canonical pathway of Wnt signaling.
These results suggest, for the first time, that the Wnt/β-catenin pathway regulates ABCB1 in CML.
慢性髓性白血病（CML）的晚期阶段已知对治疗更具抵抗力。这种耐药性与 ABCB1 的过度表达有关，这导致了多药耐药（MDR）现象。MDR 的特征是对非相关药物的耐药性，并且 P-糖蛋白（由 ABCB1 编码）已被牵连为其出现的主要原因。Wnt 信号在 CML 的几个方面都被证明是重要的。最近，Wnt 信号通过其经典途径与 ABCB1 的调节有关，该途径由β-连环蛋白介导，在其他类型的癌症中也是如此。在这项研究中，我们研究了 Wnt/β-连环蛋白途径在 CML 中 ABCB1 转录调节中的作用，因为 ABCB1 的基本启动子有几个β-连环蛋白结合位点。β-连环蛋白是经典 Wnt 信号的介质，对 CML 的进展很重要。
在这项工作中，我们使用了 K562 细胞系及其衍生的多药耐药性细胞系 Lucena（K562/VCR）作为 CML 研究模型。实时 PCR（RT-qPCR）、电泳迁移率变动分析（EMSA）、染色质免疫沉淀（ChIP）、流式细胞术（FACS）、western blot、免疫荧光、RNA 敲低（siRNA）和荧光素酶报告基因方法被使用。
β-连环蛋白存在于两种细胞系的 ABCB1 基本启动子的蛋白质复合物中，但在体内耐药细胞系中的结合更为明显。与亲本细胞系相比，Lucena 细胞也表现出更高的β-连环蛋白水平。Wnt1 和β-连环蛋白耗竭以及核β-连环蛋白的过表达以及 TCF 结合位点的激活表明，ABCB1 受 Wnt 信号的经典途径正向调节。
这些结果首次表明，Wnt/β-连环蛋白途径在 CML 中调节 ABCB1。
