Effect Of 5-Aza-CdR On The Biological Activity And Inhibitor Of DNA Binding 4 (ID4) Gene Expression Of Human Erythromyeloblastoid Leukemia Cell Line K562

Objective: To study the effect of 5-Aza-CdR on the biological activity of human erythromyeloblastoid leukemia cell line K562 and the expression of inhibitor of DNA binding 4 (ID4), and to search the new target of gene therapy.Methods: 1. To detect the situation of ID4 methylation in K562 cell line by methylation specific PCR (MS-PCR). 2. To analyze the expression levels of ID4 mRNA in K562 cell line with the treatment of different concentrations of 5-Aza-CdR (0.5, 2, 5, 10μM) after 48h by RQ-PCR. 3. After treated by different concentrations of 5-Aza-CdR,K562 cells were stained with FITC labeled annexin V/propidium iodide (Annexin V-FITC/PI). The change of apoptosis rate and cell cycle of were analyzed by Flow Cytometry.Results: The K562 cells exist ID4 gene methylation. After 5-Aza-CdR is added to the medium, there is a dose dependent upregulation of the gene ID4 mRNA expression. The differences of the gene ID4 mRNA level among experimental groups are obvious (P < 0.01). 5-Aza-CdR can also induce the apoptosis of K562. The apoptosis rates of k562 cells are of time and dose dependent. 5μM 5-Aza-CdR results in 15.3%, 17.6% and 21.3% K562 cells apoptosis at 24h, 48h and 72h respectively. The apoptosis rates of K562 cells are 6.9%, 12.6%, 17.6% and 29.3% respectively after treated with different concentrations of 5-Aza-CdR (0.5, 2, 5, 10μM) for 48 hours. Compared with the control group (0μM), there is a significant difference (P <0.05). The change of cell cycle of K562 cells also are of dose dependent. Treatment of K562 cells with 5-Aza-CdR (2, 5μM) for 48h caused a 1.27-fold and 1.54-fold increase in K562 cells that arrest in G0/G1 phase in comparison with control cells. The treatment also caused a decease K562 cells that entered in G2/M phase. The treated K562 cells in G2/M phase were 0.48-fold and 0.37-fold of control group correspondingly. There is a significant statistical difference between treated groups and control group (P <0.01).Conclusions: Methyltransferase inhibitor 5-Aza-CdR can result in the re-expressions of the silent ID4 gene in K562 cells. The upregulation of ID4 maybe is key factor which is related with cell apoptosis and cell-cycle arrest of K562 cells treated by 5-Aza-CdR.