Decitabine (5-Aza-2'-deoxycytidine) decreased DNA methylation and expression of MDR-1 gene in K562/ADM cells.
Multidrug resistance (MDR) is a major problem in patients with hematological malignancies. Although drug-resistance is known to be induced by the expression of P-glycoprotein (P-gp) encoded by the MDR-1 gene, little is known about the mechanisms regulating this gene. Herein, we studied the DNA methylation patterns at the enhancer and repressor binding sites of the MDR-1 gene using the human erythroleukemia cell line K562 and its multidrug resistant derivative K562/ADM (adriamycin). Direct DNA sequence analysis demonstrated methylation to be present at the repressor site (minus 110 GC-box) of the MDR-1 gene in K562/ADM cells, but not in parental K562 cells. Methylation-specific PCR (MSP) analysis yielded similar results. Treatment of K562/ADM cells with 5-Aza-2'-deoxycytidine (decitabine; DAC), an inhibitor of DNA methyltransferase, caused demethylation of the repressor binding site of MDR-1 gene, as assessed by MSP, and also decreased P-gp expression, as assessed by flow cytometric and Northern blot analysis. Although it is generally accepted that DAC upregulates gene expression by demethylating the activator binding sites, our present results suggest that DAC induces down-regulation of P-gp expression as a result of demethylation at the repressor binding site in K562/ADM cells. In this regard, methylation-dependent regulation of the MDR-1 gene in K562/ADM cells is unique.
Ando
T
T
The Third Department of Internal Medicine, Yamaguchi University School of Medicine, Japan.
Nishimura
M
M
Oka
Y
Y
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis*; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D018899:CpG Islands; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors*; D019175:DNA Methylation / Q000187:drug effects*; D004273:DNA, Neoplasm / Q000737:chemistry; D000077209:Decitabine; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects*; D019450:Genes, MDR; D006801:Humans; D020014:K562 Cells / Q000187:drug effects* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D000964:Antimetabolites, Antineoplastic; D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D000077209:Decitabine; D004317:Doxorubicin; D004248:DNA (Cytosine-5-)-Methyltransferases; D001374:Azacitidine