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Reversal of mdr1 gene-dependent multidrug resistance in multidrug resistance human leukemia cell line K562/ADM using short hairpin RNA expression vectors

GAN Hui-zhu ZHANG Gui-zhen LU Zhen-xia PU Li-sha YANG Shao-juan GAO Shen ZHENG De-ming Department of Hematology,China-Japan Union Hospital,Jilin University,Changchun 130031,China  
Objective To explore the role of reversal muhidrug resistance(MDR)using short hairpin RNA(shRNA)expression vectors in multidrug resistance human leukemia cell line K562/ADM.Methods The oligonucleotides with 19-mer hairpin structure were synthesized.The shRNA expression vectors were con- structed and introduced into K562/ADM cells.Expression of mdr1 mRNA was assessed by RT-PCR,and P- gp expression was determined by Western blot.The apoptosis and sensitivity of the K562/ADM cells to doxo- rubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium(MTT)assays,respectively.Cellu- lar daunorubicin accumulation was assayed by laser confocol scanning microscope(LCSM).Results In positive clones of K562/ADM cells stably transfected with pSilencer~(TM)3.1-H1 neo mdr1-A and mdr1-B shRNA expression vectors,RT-PCR showed that mdr1 mRNA expression was significantly reduced to 35.9% (P0.05),27.5%(P0.01),respectively.Western blot showed that P-gp expression was significantly and specifically inhibited.Resistance against doxorubicin was decreased from 79-fold to 38-fold(P0.05), 30-fold(P0.01)respectively.Furthermore,the fluorescence intensity of K562/ADM cells was increased significantly compared with the control,shRNA vectors significantly enhanced the cellular daunorubicin accu- mulation.The percent of the apoptosis cell was significantly enhanced to 18.1%(P0.05),54.4%(P 0.01)respectively.Conclusions shRNA expression vectors can effectively reverse MDR,and restore the sensitivity of drug-resistance K562/ADM cells to conventional chemotherapeutic agents.
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