Stable RNAi-Mediated Knock-Down of P-Glycoprotein by a Transposon-Based Vector System Restores Imatinib Sensitivity.

Abstract

Overexpression of P-glycoprotein (PgP) is critical for resistance of malignant cells against cytotoxic agents. However, the role of PgP overexpression for the sensitivity of chronic myeloid leukemia (CML) to imatinib remains controversial. We therefore constructed a transposon-based (Sleeping beauty) RNAi system for the stable down-regulation of PgP in imatinib/doxorubicin resistant K562/Dox cell lines. RT-PCR demonstrated the stable integration of the transposon into the genome. Real-time PCR revealed a rapid and almost complete degradation of MDR1 mRNA 72 h after nucleofection. PgP knockdown on protein level was much more efficient in the stable setting as compared to the transient approach. This observation points out to the requirement of stable knockdown approaches for siRNA-mediated downregulation of proteins with long half-life time. The functional impact of stable PgP knock-down was demonstrated by a reduced efflux of rhodamine and doxorubicin. In parallel, MDR1 knockdown cells were re-sensitized to doxorubicin as well as to imatinib-induced cell death. This observation supports the notion that up-regulation of MDR1 in CML might contribute to imatinib resistance in vivo.In summary we describe a very efficient tool for the stable knock-down of PgP by transposon-mediated siRNA delivery in CML cells and propose that the down-regulation of PgP in CML can restore sensitivity to imatinib.