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 en codes 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 m ethylation status of CpG sites in the MDR1 promoter region and transcriptio nal activation of MDR1 has now been investigated. The P-glycoprotein-overex pressing, multidrug-resistant KB/VJ300 and KB-C1 cells, which were establis hed 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-CI cells, but hyperm ethylated in KB3-1 cells. Prior treatment of KB3-1 cells with the DNA methy ltransferase inhibitor 5-aza-2'-deoxycytidine resulted in a 90-fold increas e 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 hypometh ylated 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-I and Spl, transcription factors implicated in M DR1 expression, in the MDR1 promoter was not affected by the methylation st atus 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 cel ls, suggesting an altered chromatin structure. The methylation status of th e promoter region may plays an important role in MDR1 overexpression and in acquisition of the P-glycoprotein-mediated multidrug resistance phenotype.