Reversal of mdrl gene-dependent multidrug resistance in multidrug resistance human leukemia cell line K562/ADM using short hairpin RNA expression vectors.
- Author:
Hui-zhu GAN
1
;
Gui-zhen ZHANG
;
Zhen-xia LU
;
Li-sha PU
;
Shao-juan YANG
;
Shen GAO
;
De-ming ZHENG
Author Information
- Publication Type:Journal Article
- MeSH: ATP Binding Cassette Transporter, Sub-Family B; ATP-Binding Cassette, Sub-Family B, Member 1; genetics; metabolism; Apoptosis; Doxorubicin; pharmacology; Drug Resistance, Multiple; genetics; Drug Resistance, Neoplasm; genetics; Genetic Vectors; Humans; K562 Cells; RNA Interference; RNA, Messenger; genetics; Transfection
- From: Chinese Journal of Hematology 2007;28(6):383-387
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo explore the role of reversal multidrug resistance (MDR) using short hairpin RNA (shRNA) expression vectors in multidrug resistance human leukemia cell line K562/ADM.
METHODSThe oligonucleotides with 19-mer hairpin structure were synthesized. The shRNA expression vectors were constructed 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 doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscope (LCSM).
RESULTSIn positive clones of K562/ADM cells stably transfected with pSilencer 3.1-HI neo mdr1-A and mdr1-B shRNA expression vectors, RT-PCR showed that mdr1 mRNA expression was significantly reduced to 35.9% (P < 0.05), 27.5% (P < 0.01), respectively. Western blot showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 79-fold to 38-fold (P < 0.05), 30-fold (P < 0.01) respectively. Furthermore, the fluorescence intensity of K562/ADM cells was increased significantly compared with the control. shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The percent of the apoptosis cell was significantly enhanced to 18.1% (P < 0.05) , 54.4% (P < 0.01) respectively.
CONCLUSIONSshRNA expression vectors can effectively reverse MDR, and restore the sensitivity of drug-resistance K562/ADM cells to conventional chemotherapeutic agents.
