联合应用地西他滨和ATRA对白血病细胞p21^(CIP1)和p57^(KIP2)基因启动子区域甲基化的影响

Effect of combination of decitabine and ATRA on the methylation of promotor regions at p21^(CIPI) and p57^(KIP2) in leukemia cells

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。

Objective：To explore the methylations of promotor regions at p21CIP1 and p57KIP2 in leukemia cell lines and cells from leukemia patients,and their change after treated by Decitabine（DAC） with or without ATRA.Method：Methylations of promotor regions at p21CIP1 and p57KIP2 were analyzed in HL60,Molt4 cells and cells from leukemia patients.After treated by DAC with or without ATRA,cell survival rate,cell cycles and methylation of genes above mentioned of these cells were evaluated.Result：Cell survival of leukemia cells were inhibited after cultured with different concentrations of DAC with or without ATRA.In ALL patients,p57KIP2 was hypermethylation before and after therapy,whereas p21CIP1 was hypomethylation.p57KIP2 in HL60 and Molt4 was hypermethylation,which decreased obviously after DAC treatment（P0.01 or P0.05）.When combining DAC and ATRA to treat these 2 cell lines,p57KIP2 was hypomethalation in both cell lines（P0.01）.p21CIP1 was hypomethylation in Molt4 cells untreated group,but its methylation level decreased furtherly（P0.05） after treated by DAC or DAC＋ATRA.In HL60 cells,p21CIP1 was hypermethylation in control group,but its methylation level was turned into hypomethylation after DAC or DAC＋ATRA treatment（P0.01）.Combining these 2 reagents to treat cells,apoptosis rate increased obviously.Conclusion：Combining DAC and ATRA to treat leukemia cells at low dosage resulted in cell growth inhibition,S stage blockage and apoptsis rate increase.p57KIP2 was hypermethylation in both cells from leukemia patients and cell lines,which indicated that it was related with leukemia genesis.Combined use of DAC and ATRA could furtherly reduce methylation level of promoter regions at p57KIP2and p21CIP1.