Annals of oncology : official journal of the European Society for Medical Oncology · 2009
Down-regulation of mitochondrial ATPase by hypermethylation mechanism in chronic myeloid leukemia is associated with multidrug resistance.
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Li R J, Zhang G S, Chen Y H, Zhu J F, Lu Q J, Gong F J, Kuang W Y
Аннотация
BACKGROUND: To identify novel proteins involved in multidrug resistance in chronic myeloid leukemia (CML).
MATERIALS AND METHODS: Comparative proteomics was used to screen multidrug resistance-related proteins from K562 and K562/A02; the differently expressed proteins were further confirmed by western blot and real-time PCR. short hairpin RNA (shRNA) assay was applied to determine the relationship between candidate protein and adriamycin resistance. Bisulfite sequencing was carried out to assess methylation status of candidate multidrug resistance-related gene promoter. K562/A02 was treated with 5-azacytidine or trichostatin A (TSA); multidrug resistance phenotype and corresponding protein or gene changes were detected.
RESULTS: Seventeen proteins with altered abundances of more than twofold were detected, among which mitochondrial ATPase in K562/A02 was significantly down-regulated. Suppressing mitochondrial ATPase by shRNA could enhance adriamycin resistance and antiapoptosis activity of K562. The promoter hypermethylation in mitochondrial ATPase was found to be attributed to the adriamycin-resistant phenotype of both K562/A02 (methylated frequency 18.18%) and CML primary cells in accelerated phase (methylated frequency 7.95%) or blast crisis (methylated frequency 26.59%). Inhibition of hypermethylation increased adriamycin sensitivity of K562/A02. A synergistic effect on reversing adriamycin-resistant phenotype was obtained when 5-azacytidine was combined with TSA.
CONCLUSION: Down-regulation of mitochondrial ATPase can lead to adriamycin resistance in CML and the mechanism is associated with DNA methylation regulation.
MATERIALS AND METHODS: Comparative proteomics was used to screen multidrug resistance-related proteins from K562 and K562/A02; the differently expressed proteins were further confirmed by western blot and real-time PCR. short hairpin RNA (shRNA) assay was applied to determine the relationship between candidate protein and adriamycin resistance. Bisulfite sequencing was carried out to assess methylation status of candidate multidrug resistance-related gene promoter. K562/A02 was treated with 5-azacytidine or trichostatin A (TSA); multidrug resistance phenotype and corresponding protein or gene changes were detected.
RESULTS: Seventeen proteins with altered abundances of more than twofold were detected, among which mitochondrial ATPase in K562/A02 was significantly down-regulated. Suppressing mitochondrial ATPase by shRNA could enhance adriamycin resistance and antiapoptosis activity of K562. The promoter hypermethylation in mitochondrial ATPase was found to be attributed to the adriamycin-resistant phenotype of both K562/A02 (methylated frequency 18.18%) and CML primary cells in accelerated phase (methylated frequency 7.95%) or blast crisis (methylated frequency 26.59%). Inhibition of hypermethylation increased adriamycin sensitivity of K562/A02. A synergistic effect on reversing adriamycin-resistant phenotype was obtained when 5-azacytidine was combined with TSA.
CONCLUSION: Down-regulation of mitochondrial ATPase can lead to adriamycin resistance in CML and the mechanism is associated with DNA methylation regulation.
Предметные рубрики MeSH
ATP Binding Cassette Transporter, Subfamily B, Member 1Antibiotics, AntineoplasticAntimetabolites, AntineoplasticApoptosisAzacitidineBase SequenceBlotting, WesternDNA MethylationDown-RegulationDoxorubicinDrug Resistance, MultipleDrug Resistance, NeoplasmElectrophoresis, Gel, Two-DimensionalFlow CytometryGene Expression Regulation, EnzymologicHistone Deacetylase InhibitorsHumansHydroxamic AcidsLeukemia, Myelogenous, Chronic, BCR-ABL PositiveMitochondrial Proton-Translocating ATPasesMolecular Sequence DataRNA, MessengerReverse Transcriptase Polymerase Chain ReactionSpectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationTumor Cells, Cultured