K562多药耐药细胞系中肿瘤干细胞样细胞对伊马替尼耐药机制的初步研究

 目的 进一步阐明一些高表达P-糖蛋白（P-gp）的慢性粒细胞白血病细胞对伊马替尼耐药的机制。方法 经过对K562细胞系长期的足叶乙苷（VP16）诱导和克隆筛选，建立一株耐药细胞系K562／VP16；利用干细胞高效能将Hoechst 33342 荧光染料泵出细胞的特性，采用流式细胞术，从K562／VP16细胞系中分选出一小群细胞，即边缘细胞（SP），称为K562／VP16 SP细胞，并初步探讨其抗伊马替尼的机制。结果 bcr／abl和abl 蛋白在K562细胞、K562／VP16 SP细胞及非K562／VP16 SP细胞（non-SP K562／VP16）中的表达水平差异无统计学意义；P-gp在K562细胞中不表达，在K562／VP16 SP及non-SP K562／VP16细胞中均高表达且表达水平一致；与non-SP K562／VP16细胞比较，K562／VP16 SP细胞对伊马替尼的耐药性更强，并且这种抗性几乎不能被多种多药耐药逆转剂逆转；另外，体内外实验显示，K562／VP16细胞的致瘤性几乎全部来源于K562／VP16 SP细胞。结论 bcr／abl基因的扩增、过度表达和多药耐药基因及其蛋白表达产物P-gp的高表达，可能不是白血病细胞产生对伊马替尼临床耐药的重要机制；白血病细胞对伊马替尼具有一定的抗性，可能与数量极少的白血病干细胞有直接的关系。因此，这类数量极少的干细胞样的肿瘤细胞应当成为有效治疗肿瘤的靶细胞。

Study on the mechanisms of cancer stem-like cells in multidrug-resistant K562 cells of insensitive to imatinib

Objective To further elucidate the mechanisms involved in some chronic myeloid leukemia (CML) cell overexpressing exclusively P-glycoprotein (P-gp) resistance to imatinib. Methods Generation of resistant K562 cell line (K562/VP16) which overexpressed P-gp was achieved after exposure of the K562 cells to stepwise increase of concentrations of VP16. A small side population (SP) with the characteristics of stem cells being capable of efflux fluorescent dye Hoechst 33342 in K562/VP16 cell line was isolated by flow cytometry. The mechanisms involved in K562/VP16 SP cells resistance to imatinib were studied. Results The levels of bcr/abl and abl proteins in K562 cells were similar to those in non-SP K562/VP16 and K562/VP16 SP cells. The 170 KDa P-gp was detected in K562/VP16 and K562/VP16 SP cells but not in K562 cells and the expression levels of P-gp in these two cell lines were similar. Compared with non-SP K562/VP16, K562/VP16 SP cells were more resistant to imatinib, and many multidrug resistance inhibitors could hardly reverse this resistance. In addition, in vivo study showed that the malignancy of K562/VP16 cells in vivo is largely dependent on the SP cells. Conclusions bcr/abl gene amplification and multidrug-resistant gene 1 (MDR1) overexpression might not be an important clinical mechanism in the diversity of resistance development to imatinib treatment, and the development of drug resistance by leukemia cells may be at least partly due to a rare SP of tumor stem-like cells which drives leukemia occurrence and maintenance. So, these SP cells need to be targeted for effective cancer therapy. <