Gleevec耐药白血病细胞中P-糖蛋白功能失调及其调节

P-glycoprotein Dysfunction and Modulation in Gleevec-resistant Leukemia Cells

目的:明确Pgp在Gleevec耐药白血病细胞中的表达和功能,为临床合理用药提供理论指导。方法:四甲基偶氮唑盐(MTT)法检测K562/G01细胞对阿霉素(Doxorubicin,DOX)和伊马替尼(Gleevec,STI571)细胞毒作用的敏感性以及Pgp抑制剂维拉帕米(Verapamil,VPL)对K562/G01细胞药物敏感性的调节作用;RT-PCR和Western-blotting方法检测细胞中MDR基因mdr1及Pgp的表达;流式细胞仪(Flow cytometry,FCM)检测细胞内DOX及罗丹明123(Rhodamin123,Rho123)水平以及Gleevec和ATP对Pgp功能的调节作用。结果:人白血病Gleevec耐药K562/G01细胞对Gleevec耐药约40倍,但对MDR类化疗药物DOX却保持敏感,而且,Pgp抑制剂VPL不改变K562/G01细胞对DOX的敏感性。与MDR表型的K562/A02细胞一样,K562/G01细胞表达mdr1及其编码的Pgp蛋白,但Pgp却没有药物外排泵功能,K562/G01细胞中DOX或Rh123浓度与敏感细胞K562相当。加入Gleevec或ATP可明显降低K562/G01细胞中DOX水平。结论:人白血病Gleevec耐药细胞K562/G01表达mdr1/Pgp,但Pgp功能明显受阻,Bcr/Abl抑制剂Gleevec或ATP可部分恢复K562/G01细胞中Pgp的药物外排泵功能。

Objective：To assess the expression and function of P-glycoprotein（Pgp）in Gleevec-resistant leukemia cells.Methods：MTT assay was performed to evaluate the sensitivity of K562/G01 cells to doxorubicin（DOX）and Gleevec.The effect of verapamil（VPL）on the sensitivity of K562/G01 cells to drugs was also determined.RT-PCR and Western blotting were used to determine the expression of the multidrug resistance gene mdr1 and its protein product Pgp.Flow cytometry（FCM）was used to analyze intracellular levels of DOX and Rhodamine 123（Rho123）,as well as the modulation of Pgp function by Gleevec and ATP.Results：Like K562/A02 MDR cells,K562/G01 cells expressed mdr1/Pgp.However,the drug efflux function of Pgp failed to be detected in K562/G01 cells.The intracellular concentrations of DOX and Rho123 were similar to those found in K562 cells.Adding either Gleevec or ATP to K562/G01 cells resulted in a markedly decreased level of intracellular DOX.Conclusion：Human leukemia Gleevec-resistant K562/G01 cells express dysfunctional mdr1/Pgp.The drug efflux pump activity of Pgp can be partially restored by adding Bcr/Abl inhibitor,Gleevec,or ATP.