The effect of DNA methylation on CXCR4 expression has been demonstrated in 
pancreatic cancer and melanoma cells, but little is known about the effect of 
DNA methyltransferases 1 and 3 (DNMT1 and DNMT3B) on CXCR4 expression. Employing 
lentiviral vectors, we created stable RNA interference-mediated knockdown of 
DNMT1 and DNMT3B in AsPC1 pancreatic cancer cells. Using reverse transcription 
real-time quantitative PCR and flow cytometric analysis, we evaluated the 
increase in the expression of CXCR4 transcript and protein levels in these 
cells. Bisulfite sequencing analysis showed that the level of promoter 
demethylation appeared more effective in cells with knockdown of DNMT1 than in 
those with DNMT3B knockdown. Furthermore, the combined RNA interference 
knockdown of both DNMT1 and DNMT3B increased promoter demethylation, leading to 
a slight increase in CXCR4 expression. However, the demethylating agent 
5-Aza-2'-deoxycytidine exhibited the strongest effect on promoter demethylation, 
which correlated with the highest production of CXCR4 transcript and protein in 
AsPC1 cells. Our results indicate that DNMT1 plays the main role in maintenance 
of methylation of CXCR4 promoter, while DNMT3B may function as an accessory DNA 
methyltransferase to modulate CXCR4 expression in AsPC1 cells.