Down-regulation of mitochondrial ATPase by hypermethylation mechanism in chronic myeloid leukemia is associated with multidrug resistance.
BACKGROUND
To identify novel proteins involved in multidrug resistance in chronic myeloid leukemia (CML).


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.


CONCLUSIONS
Down-regulation of mitochondrial ATPase can lead to adriamycin resistance in CML and the mechanism is associated with DNA methylation regulation.
Li
R J
RJ
Department of Hematology/Institute of Molecular Hematology, Second Xiang-Ya Hospital, Central South University, Changsha, Hunan, China.
Zhang
G S
GS
Department of Hematology/Institute of Molecular Hematology, Second Xiang-Ya Hospital, Central South University, Changsha, Hunan, China. Electronic address: zgsllzy@163.com.
Chen
Y H
YH
Department of Molecular and Computational Biology, University of Southern California, CA, USA.
Zhu
J F
JF
Department of Hematology/Institute of Molecular Hematology, Second Xiang-Ya Hospital, Central South University, Changsha, Hunan, China.
Lu
Q J
QJ
Department of Dermatology and Epigenetic Research Center, Second Xiang-Ya Hospital, Central South University, Changsha, Hunan, China.
Gong
F J
FJ
Department of Hematology/Institute of Molecular Hematology, Second Xiang-Ya Hospital, Central South University, Changsha, Hunan, China.
Kuang
W Y
WY
Department of Hematology/Institute of Molecular Hematology, Second Xiang-Ya Hospital, Central South University, Changsha, Hunan, China.
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D019175:DNA Methylation; D015536:Down-Regulation; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D015180:Electrophoresis, Gel, Two-Dimensional; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics*; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D000964:Antimetabolites, Antineoplastic; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D012333:RNA, Messenger; C012589:trichostatin A; D004317:Doxorubicin; C465022:ATP5ME protein, human; D025261:Mitochondrial Proton-Translocating ATPases; D001374:Azacitidine