Effects of uroacitides on the methylation of PTEN gene in myelodysplastic syndrome cells and its mechanism

Abstract

To investigate the methylation patterns of PTEN gene in myelodysplastic syndrome (MDS) cell line MUTZ-1 cells and the primary cells, to explore the effects of uroacitides (CDA- II) on the methylation patterns, and to provide novel target therapy for MDS. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were used to determine the expression of PTEN and DNA methyltransferases (DNMTs) as well as the effects of CDA-II on them. Methylation specific PCR (MSP) was used to identify the methylation patterns of PTEN and the effects of CDA-II on them. PTEN was completely methylated in high-risk MDS and acute myeloid leukemia (AML) transformed from MDS. High expressions of DNMTs proteins, especially DNMT3b protein were found in high-risk MDS and AML cells transformed from MDS. PTEN expression level was increased from 0.02 ± 0.01 at control group to 0.23 ± 0.11, 0.54 ± 0.11 and 0.92 ± 0.13 after treatment with CDA-II at 2, 4 and 8 mg/ml (P<0.01). If the cells were treated with CDA-II at 4 mg/ml for 12, 24 and 48 hours, PTEN expression level was up- regulated to 0.15 ± 0.06, 0.52 ± 0.12 and 0.89 ± 0.13, which were significantly higher than that of control group 0.02 ± 0.01 (P<0.01). CDA-II could significantly down-regulate the expression of DNMTs in MUTZ-1 cells in a dose-(r=0.999, 0.992, 0.995, P<0.01) and time-dependent (r=0.989, 0.981, 0.985, P<0.05) manner, which led to the demethylation of PTEN. Among them, DNMT1 was the most downregulated (F=21.256, P<0.05). PTEN gene was hypermethylated in high-risk MDS, and target therapy of PTEN demethylation might be a new strategy for MDS therapy. CDA-II could down regulate the expression of DNMTs and lead to PTEN demethylation which induce MUTZ-1 cells apoptosis.