Du Hong-ling, Qi Yu, Shi Yong-jin, Bu Ding-fang, Wu Shu-lan
Department of Hematology, 2. Central Lab, First Hospital, Peking University, Beijing 100034, P. R. China.
Ai Zheng. 2002 Oct;21(10):1057-61.
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.
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.
The apoptotic rates of U266 cells induced by 5-Aza-CdR(1 mumol/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.
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.
组蛋白去乙酰化与受DNA甲基化控制的转录激活相关。研究通过两种机制处理的肿瘤细胞的变化具有重要意义。本研究旨在探讨组蛋白脱乙酰酶抑制剂苯丁酸钠（SPB）与去甲基化剂5-氮杂-2'-脱氧胞苷（5-氮杂-CdR）对细胞生长的协同作用，并探索骨髓瘤细胞系U266中高甲基化且沉默的p16基因重新表达的可能性。
采用流式细胞术分析细胞周期。通过透射电子显微镜、DNA梯状条带、荧光激活细胞分选仪（FACS）观察细胞凋亡情况。采用逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析检测p16的表达水平。
单独使用5-氮杂-CdR（1 μmol/L）、SPB（1 mmol/L）以及5-氮杂-CdR与SPB联合处理U266细胞后的凋亡率分别为15.09%、89.19%和85.18%。5-氮杂-CdR与SPB联合处理使细胞G1期阻滞并诱导亚G1期（50%）出现，单独使用SPB或5-氮杂-CdR时未出现G1期阻滞。单独使用SPB使G2期细胞比例增加。SPB不能诱导p16表达，5-氮杂-CdR可诱导p16表达，SPB与5-氮杂-CdR协同作用显著增加p16的mRNA和蛋白表达水平。
5-氮杂-CdR与SPB协同作用可显著重新激活U266细胞系中的p16基因，并使细胞周期阻滞于G1期。同时，5-氮杂-CdR与SPB联合诱导凋亡的细胞周期时相与各自单独诱导的不同。
