脱乙酰化酶抑制剂与去甲基化制剂联合诱导U266细胞凋亡和p16基因重新表达

Apoptosis and Re-expression of p16 Gene in the Myeloma Cell Line U266 Induced by Synergy of Histone Deacetylase Inhibitor and Demethylating Agent

背景与目的:DNA甲基化调节基因表达与组蛋白脱乙酰化的作用密切相关,针对这两大途径用药,观察对肿瘤细胞的影响是有意义的。本文探讨脱乙酰化酶抑制剂苯丁酸钠(sodiumphenylbutyrate,SPB)联合去甲基化制剂5-氮杂-2'-脱氧胞苷(5-Aza-CdR)处理人骨髓瘤细胞系U266诱导高甲基化失活的p16基因重新表达的可能性及其对细胞生长的影响。方法:通过透射电镜、DNA梯形条带及流式细胞术分析细胞凋亡及细胞周期的变化;RT-PCR和Westernblot检测p16表达水平。结果:单用5-Aza-CdR(1μmol/L)或SPB(1mmol/L)及两药联合诱导的细胞凋亡率分别是15.09%,89.19%和85.18%。单用5-Aza-CdR或SPB对细胞G1期无影响,单用SPB使G2/M期细胞比例增高,联合用药发生明显的G1期阻滞,且出现亚G1期峰达50%。单用PB不能诱导U266细胞p16的表达,单用5-Aza-CdR可诱导p16重新表达,两药联合明显增强p16表达水平。结论:PB与5-Aza-CdR协同可显著诱导U266细胞p16重新表达,细胞阻滞在G1期,并使细胞凋亡发生的时相不同于单独用药组。

Background & Objective: Histone deacetylation is associated with transcriptional activation controlled by DNA methylation. It is important to investigate changes of tumor cells treated with agent through two kinds of mechanisms. This study was designed to investigate the synergic effect of histone deacetylase inhibitor,sodium phenylbutyrate(SPB),and demethylating agent,5 Aza 2′ deoxycytidine(5 Aza CdR),on cell growth and explore the possibility of re expression of the hypermethylated and silenced p16 gene in the myeloma cell line U266. Methods:The cell cycle was analyzed by flow cytometry. Apoptosis was observed by transmission electron microscopy, DNA ladder, fluorescence activated cell sorter(FACS). The expression level of p16 was detected by RT PCR and Western blot analysis. Results: The apoptotic rates of U266 cells induced by 5 Aza CdR(1 μmol/L), SPB(1 mmol/L) alone and combination of 5 Aza CdR and SPB were 15.09%,89.19%, and 85.18%, respectively. The G1 phase was arrested and sub G1 phase(50%) was induced by combination of 5 Aza CdR and SPB. There was no G1 phase arrested when SPB or 5 Aza CdR was used alone. The proportion of cells in G2 phase was increased with SPB alone. SPB was not able to induce the expression of p16. The expression level of p16 was induced with 5 Aza CdR. The expression level of both mRNA and protein of p16 was increased significantly by synergy of SPB and 5 Aza CdR. Conclusions: p16 gene in U266 cell line could be reactivated markedly with synergy of 5 Aza CdR and SPB with cell cycle arresting in G1 phase. Meanwhile, the cell cycle phase occurring apoptosis that induced by combination of 5 Aza CdR with SPB is different from that induced by each alone.