Huang Jian, Yang Min, Jin Jie
Department of Hematology, the First Affiliated Hospital of the Medical College of Zhejiang University, Hangzhou 310003, China.
Zhonghua Xue Ye Xue Za Zhi. 2013 Jul;34(7):600-5. doi: 10.3760/cma.j.issn.0253-2727.2013.07.009.
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.
研究髓系发育异常综合征（MDS）细胞系MUTZ-1细胞及原代细胞中PTEN基因的甲基化模式，探讨尿嘧啶阿糖胞苷（CDA-II）对甲基化模式的影响，为MDS提供新的靶向治疗方法。
采用逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测PTEN及DNA甲基转移酶（DNMTs）的表达以及CDA-II对它们的影响。采用甲基化特异性PCR（MSP）鉴定PTEN的甲基化模式及CDA-II对其的影响。
高危MDS及由MDS转化而来的急性髓系白血病（AML）中PTEN完全甲基化。高危MDS及由MDS转化而来的AML细胞中发现DNMTs蛋白高表达，尤其是DNMT3b蛋白。PTEN表达水平从对照组的0.02±0.01分别提高到2、4和8 mg/ml CDA-II处理后的0.23±0.11、0.54±0.11和0.92±0.13（P<0.01）。若细胞用4 mg/ml CDA-II处理12、24和48小时，PTEN表达水平上调至0.15±0.06、0.52±0.12和0.89±0.13，显著高于对照组的0.02±0.01（P<0.01）。CDA-II能以剂量依赖（r=0.999、0.992、0.995，P<0.01）和时间依赖（r=0.989、0.981、0.985，P<0.05）的方式显著下调MUTZ-1细胞中DNMTs的表达，导致PTEN去甲基化。其中，DNMT1下调最为明显（F=21.256，P<0.05）。
高危MDS中PTEN基因高度甲基化，PTEN去甲基化靶向治疗可能是MDS治疗的新策略。CDA-II可下调DNMTs表达并导致PTEN去甲基化，诱导MUTZ-1细胞凋亡。
