Association of 5' CpG demethylation and altered chromatin structure in the promoter region with transcriptional activation of the multidrug resistance 1 gene in human cancer cells.
Selection of human cells for resistance to vincristine or doxorubicin often induces overexpression of the multidrug resistance 1 gene (MDR1), which encodes the cell surface P-glycoprotein, as a result of gene amplification or transcriptional activation. However, the precise mechanism underlying such transcriptional activation of MDR1 remains unclear. The relation between methylation status of CpG sites in the MDR1 promoter region and transcriptional activation of MDR1 has now been investigated. The P-glycoprotein-overexpressing, multidrug-resistant KB/VJ300 and KB-C1 cells, which were established from human cancer KB3-1 cells, were examined; MDR1 is transcriptionally activated but not amplified in KB/VJ300 cells, whereas it is amplified in KB-C1 cells. Determination of the methylation status revealed that the MDR1 promoter region was hypomethylated in KB/VJ300 and KB-C1 cells, but hypermethylated in KB3-1 cells. Prior treatment of KB3-1 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine resulted in a 90-fold increase in the frequency of vincristine-resistance. Of three lines, KB/CdR-1, KB/CdR-2, and KB/CdR-3, established from KB3-1 cells after exposure to 5-aza-2'-deoxycytidine, MspI/HpaII sites in the MDR1 promoter region were hypomethylated in KB/CdR-1 and KB/CdR-2 cells, but not in KB/CdR-3 cells. MDR1 mRNA expression was detected in KB/CdR-1 and KB/CdR-2 cells, but not in KB/CdR-3 cells. The binding of YB-1 and Sp1, transcription factors implicated in MDR1 expression, in the MDR1 promoter was not affected by the methylation status of a neighboring CpG sites. The MDR1 promoter region in KB/VJ300 cells showed an increased sensitivity to DNase I compared with that in KB3-1 cells, suggesting an altered chromatin structure. The methylation status of the promoter region may plays an important role in MDR1 overexpression and in acquisition of the P-glycoprotein-mediated multidrug resistance phenotype.
Kusaba
H
H
Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan.
Nakayama
M
M
Harada
T
T
Nomoto
M
M
Kohno
K
K
Kuwano
M
M
Wada
M
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism; D002999:Clone Cells / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D015226:Dinucleoside Phosphates / Q000235:genetics / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D005784:Gene Amplification; D019450:Genes, MDR / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000502:physiology; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002843:Chromatin; D015226:Dinucleoside Phosphates; D000077209:Decitabine; D001374:Azacitidine