Objective: To study the regulation of methylation inhibitor 5-aza-2'-deoxycytidine on transcription of EphB4 gene and effects on the proliferation and apoptosis of human acute lymphocyte leukemia cell line CEM.
Methods: Bisulfite sequencing PCR was used to detect CpG island methylation density in EphB4 promoter. The expression of EphB4 mRNA and protein was determined by Q-PCR and Western blot. MTS assay and flow cytometry were used to detect the apoptosis of CEM cells after treatment with different concentrations of 5-aza-2'-deoxycytidine (1.0, 2.5 and 5 μmol/L).
Results: Methylation of EphB4 gene promoter was detected in CEM cells (31.4%). The methylation level of EphB4 gene was down-regulated after treatment with various concentrations of 5-aza-2'-deoxycytidine. The EphB4 mRNA and protein expression in CEM cells increased after 5-aza-2'-deoxycytidine treatment. 5-Aza-2'-deoxycytidine significantly inhibited the cell growth in dose and time dependent manners. Early apoptosis rates of CEM cells increased from 4.1% to 24.8% 96 hrs after 5-aza-2'-deoxycytidine treatment. CEM cells in G1 phase decreased from 62.4% to 46.8%, cells in G2 phase increased from 2.1% to 16.2%, and CEM cells were arrested in G2 phase after treatment with 5 μmol/L 5-aza-2'-deoxycytidine for 96 hrs.
Conclusions: 5-Aza-2'-deoxycytidine, an inhibitor of specific methylation transferase, can induce expression of the silent EphB4 gene in CEM cells, inhibit the proliferation of leukemia cells and induce cell apoptosis.