Phosphorylation of RNA helicase A by DNA-dependent protein kinase is indispensable for expression of the MDR1 gene product P-glycoprotein in multidrug-resistant human leukemia cells.
Development of multidrug resistance (MDR) in cancer frequently involves overexpression of the MDR1 gene product P-glycoprotein (P-gp), a drug transporter which severely impedes the efficacy of chemotherapy. Because intensive efforts to identify therapeutics that reverse MDR by inhibiting the drug transport activity of P-gp have not yet met with success, we have focused on the alternative strategy of targeting MDR1 promoter activation to knockdown P-gp expression in cancer cells. We recently identified RNA helicase A (RHA) inhibition as a rational strategy to downregulate P-gp in leukemia cells by showing that RHA RNAi knockdown abrogated P-gp expression in MDR variants of human leukemia HL-60 cells. In that report, we also demonstrated that RHA activated the MDR1 promoter in the MDR variant cells but not in the drug-sensitive counterpart. This led us to hypothesize that P-gp induction by RHA required cooperation with another factor present only in the MDR variants. Here, we identify the RHA cooperating factor as DNA-PK catalytic subunit (cs), and we show that DNA-PKcs resides with RHA at the MDR1 promoter in a multiprotein complex. Furthermore, targeted DNA-PKcs inhibition abrogated P-gp expression in the MDR variant cells. We demonstrate that constitutive multisite RHA phosphorylation producing retarded migration in SDS-PAGE is catalyzed by DNA-PKcs in the MDR variants, and does not occur in the parental cells, which are DNA-PKcs deficient. The indispensable role played by DNA-PK in P-gp overexpression in MDR leukemia cells in this report identifies targeted DNA-PK inhibition as a rational strategy to reverse drug resistance in cancer.
Zhong
Xiaoling
X
Department of Pharmacology and Toxicology and Indiana University Cancer Center, 1044 West Walnut Street, Indianapolis, Indiana 46202, USA.
Safa
Ahmad R
AR
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis*; D000818:Animals; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D004926:Escherichia coli / Q000378:metabolism; D019450:Genes, MDR; D018922:HL-60 Cells; D006801:Humans; D009363:Neoplasm Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D051747:DNA-Activated Protein Kinase; C098487:DHX9 protein, human; D053487:DEAD-box RNA Helicases