Effect of 5-Aza-2′-Deoxycytidine in High-Risk Myelodysplasia Cells in Vitro.

5-Aza-2′-deoxycytidine (5-Aza-CdR; Decitabine) is an active antineoplastic agent in patients with leukemia. Due to its unique mechanism of action of demethylating DNA, 5-Aza-CdR has the potential to activate tumor suppressor and differentiation genes that have been accidentally silenced by DNA methylation in leukemic cells. Since5-Aza-CdR can induce clinical remissions in patients with high-risk myelodysplastic syndromes (MDS), we therefore attempt to investigate the mechanisms of the effect of 5-Aza-CdR by using MUTZ-1 cells in vitro, which are confirmed as a high-risk myelodysplasia Cell line that derived from a MDS patient (FAB subtype refractory anemia with excess of blasts). Our results indicated that 5-Aza-CdR showed inhibition of the growth of MUTZ-1 cells. The IC₅₀ values of 24 hours, 48 hours and 72 hours were 6.75mmol/L, 2.82mmol/L and 5.45mol/L respectively. Characteristic changes of apoptosis emerged in MUTZ-1 cells after being exposed to 5-Aza-CdR in the different concentration from 0.8 mmol/L to 3.2 mmol/L, and the positive cells of Annexin V^FITC on the membrane of MUTZ-1 cells were analyzed by flow cytometry. The percentage of apoptosis cells treated by 5-Aza-CdR at a dose of 3.2mmol/L for 48 hours was higher than that in untreated cells (16.7%±5.3% vs 2.9%±1.2%, p<0.05). The CpG islands in the promoter regions of p15INK4B gene were found to be hypermethylated in MUTZ-1 cells, resulting in a significant reduced expression of p15INK4B gene in the cells. However, the expression of p15INK4B gene was recovered and the methylation level of p15INK4B gene was also decreased (densitometry readings: 253.994±26.536 vs 369.641±28.915, p=0.003), after the cells exposed to 5-Aza-CdR at a dose of 3.2 mmol/L for 48 hours. Furthermore, 5-Aza-CdR could significantly down-regulate the expressions of DNA methyltransferase genes DNMT_3A, which encodes a deno methyltransferase, at mRNA level in a dose dependent manner(r=−0.879,p=0.03). However, it had no effects on DNMT_3B gene and DNMT₁ gene that codes a maintenance methylase. The expression level of DNMT_3A mRNA in MUTZ-1 cells treated with 5-Aza-CdR at a concentration 3.2 mmol/L for 48 hours was lower than that in untreated cells (densitometry readings:0. 385±0.086 vs 0.654±0.074 p<0.05). These observations suggest that 5-Aza-CdR can inhibit the growth and induce the apoptosis of MUTZ-1 cells within the range of concentration from 0.8 mmol/L to 3.2 mmol/L, which might be one of the mechanisms of antitumor effects of 5-Aza-CdR. The drug can activate the expression of p15INK4B gene in MUTZ-1 cells by demethylation of the p15INK4B gene through inhibiting the expression of DNMT_3A gene. That might be the mechanisms of 5-Aza-CdR in the treatments of MDS.