Hekmatshoar Yalda, Ozkan Tulin, Altinok Gunes Buket, Bozkurt Sureyya, Karadag Aynur, Karabay Arzu Zeynep, Sunguroglu Asuman
Department of Medical Biology, School of Medicine, Ankara University, Ankara, Turkey.
Department of Medical Biology, School of Medicine, Istinye University, Istanbul, Turkey.
Cell Mol Biol (Noisy-le-grand). 2018 May 15;64(6):23-30.
Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by the t(9; 22) and the related oncogene, BCR-ABL. Tyrosine kinase activity of fusion protein BCR-ABL is the main cause of CML. Even if imatinib is used as a tyrosine kinase inhibitor (TKI) for CML therapy, drug resistance may occur in patients and the clinical failure of imatinib treatment in resistant patients had resulted with the use of another alternative TKIs. BCR-ABL dependent and independent molecular mechanisms have crucial roles in drug resistance. To reveal the underlying molecular mechanisms which play significant roles in imatinib resistance in CML, we established K562 imatinib-resistant cell line (K562r5) which was continuously exposed to (5µM) imatinib to investigate molecular mechanisms which play significant roles in drug resistance. First of all, we analyzed T315I, M351T, F315L and F359C/L/V mutations with DNA sequencing as a BCR-ABL dependent mechanism in our cell lines. Moreover, we investigated BCR-ABL independent mechanisms such as apoptosis, autophagy, drug transport and DNA repair which affect drug resistance in these cell lines. In vitro cell viability was determined by MTT assay. DNA sequencing analysis was performed to detect BCR-ABL mutations. The apoptotic effect of imatinib on CML cell lines was tested by flow cytometric Annexin V-PE staining and caspase activation assays. Apoptotic, autophagic, drug transporter and DNA repair genes expression levels were determined by RT-PCR. The conventional cytogenetic analysis was performed on K562s and K562r cells. Our results indicate that inhibition of apoptosis, induction of autophagy, overexpression of efflux gene MDR1 and down-regulation of influx gene OCT1 play crucial roles in the progression of imatinib resistance.
慢性髓性白血病（CML）是一种造血系统恶性肿瘤，其特征为t(9; 22)以及相关致癌基因BCR-ABL。融合蛋白BCR-ABL的酪氨酸激酶活性是CML的主要病因。即便伊马替尼被用作CML治疗的酪氨酸激酶抑制剂（TKI），患者仍可能出现耐药性，而耐药患者使用伊马替尼治疗的临床失败促使了其他替代TKI的使用。BCR-ABL依赖性和非依赖性分子机制在耐药性中起着关键作用。为揭示在CML伊马替尼耐药中起重要作用的潜在分子机制，我们建立了K562伊马替尼耐药细胞系（K562r5），该细胞系持续暴露于（5µM）伊马替尼以研究在耐药中起重要作用的分子机制。首先，我们通过DNA测序分析了T315I、M351T、F315L和F359C/L/V突变，作为我们细胞系中BCR-ABL依赖性机制。此外，我们研究了BCR-ABL非依赖性机制，如凋亡、自噬、药物转运和DNA修复，这些机制影响这些细胞系中的耐药性。通过MTT法测定体外细胞活力。进行DNA测序分析以检测BCR-ABL突变。通过流式细胞术Annexin V-PE染色和半胱天冬酶激活试验检测伊马替尼对CML细胞系的凋亡作用。通过RT-PCR测定凋亡、自噬、药物转运体和DNA修复基因的表达水平。对K562s和K562r细胞进行常规细胞遗传学分析。我们的结果表明，凋亡抑制、自噬诱导、外排基因MDR1的过表达和内流基因OCT1的下调在伊马替尼耐药进展中起关键作用。
