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目的:研究白血病患者和白血病细胞株中p21CIP1和p57KIP2基因启动子区域甲基化水平,应用地西他滨(DAC)或联合ATRA对白血病细胞生长抑制及甲基化水平改变的影响。方法:选取白血病细胞株HL60和Molt4及ALL患者骨髓细胞,测定p21CIP1和p57KIP2基因启动子区域甲基化,检测使用DAC或联合ATRA对白血病细胞株生存的影响、p21CIP1和p57KIP2基因甲基化改变和细胞周期改变。结果:不同浓度的DAC和ATRA培养,表现出对细胞的生长抑制作用;ALL患者p57KIP2治疗前后均为高甲基化状态,而p21CIP1治疗前后均为低甲基化。Molt4和HL60的p57KIP2为高甲基化状态,DAC处理使2种细胞p57KIP2甲基化明显降低(P<0.01或P<0.05);DAC联合ATRA处理2种细胞株,p57KIP2甲基化状态均明显减低(P<0.01)。Molt4细胞对照组的p21CIP1为低甲基化状态,以DAC或DAC加ATRA处理可以使甲基化水平降低(P<0.05);HL60细胞对照组p21CIP1为高甲基化状态,以DAC或DAC加ATRA处理使p21CIP1低甲基化(P<0.01)。联合上述2种药物处理,使细胞凋亡率比单独使用DAC时明显增加。结论:DAC联合ATRA,在较小的剂量下即可实现对白血病细胞明显的生长抑制作用,细胞S期阻滞,凋亡率明显增加。p57KIP2在ALL患者和白血病细胞株中均表现高甲基化,提示其可能与白血病发生相关,而DAC联合ATRA应用可以进一步降低p57KIP2和p21CIP1启动子区域的甲基化水平。
Aims: To investigate the methylation status of p21CIP1 and p57KIP2 promoter in leukemia and leukemia cell lines, and the effect of decitabine (DAC) or combined with ATRA on the growth inhibition and the methylation level of leukemia cells. Methods: Bone marrow cells of leukemia cell line HL60, Molt4 and ALL were selected to detect the promoter methylation of p21CIP1 and p57KIP2 gene. The effects of DAC or ATRA on the survival of leukemia cell lines were examined. The methylation changes of p21CIP1 and p57KIP2 Cell cycle changes. RESULTS: Different concentrations of DAC and ATRA showed inhibitory effect on cell growth. ALL patients had hypermethylation status before and after p57KIP2 treatment, whereas p21CIP1 was hypomethylated before and after treatment. The p57KIP2 methylation status of Molt4 and HL60 was hypermethylated, and the methylation of p57KIP2 was significantly decreased in both cell lines (P <0.01 or P <0.05) by DAC treatment. The methylation status of p57KIP2 was significantly reduced by both DAC and ATRA treatment P <0.01). The methylation status of p21CIP1 in Molt4 cells was hypomethylated, and the methylation level was decreased by DAC or DAC plus ATRA (P <0.05). The p21CIP1 in HL60 cells was hypermethylated with DAC or DAC plus ATRA P21CIP1 was hypomethylated (P <0.01). Combined with the above two kinds of drug treatment, the apoptosis rate was significantly increased compared with DAC alone. Conclusion: DAC combined with ATRA can inhibit the growth of leukemia cells in a small dose, and arrest the cells in S phase and increase the apoptosis rate obviously. p57KIP2 showed hypermethylation in ALL patients and leukemia cell lines, suggesting that it may be associated with leukemia. DAC combined with ATRA could further reduce the methylation level of p57KIP2 and p21CIP1 promoter regions.