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目的研究组蛋白脱乙酰化酶(HDAC)抑制剂苯丁酸钠(PB)对白血病细胞系细胞周期的影响,探讨其分子机制。方法 PB 处理白血病细胞系 Kasumi-1、U937和 NB4细胞,分别于处理后24,48和72h 收集细胞。碘化丙锭 DNA 染色,流式细胞术分析细胞周期的变化。半定量逆转录-聚合酶链反应(RT-PCR)分析细胞周期相关基因 p21WAF1/CIP1表达的变化。在人肾上皮细胞系293T 细胞用荧光素酶报告基因分析 PB 对 p21WAF1/CIP1基因启动子活性的影响。结果 PB 可以抑制 Kasu-mi-1、U937和 NB4细胞的细胞周期,作用呈时间和剂量依赖关系。3 mmol/L PB 作用72 h,分别使 Ka-sumi-1、U937和 NB4细胞的 G_0/G_1期细胞比例增加42.03%、44.36%和26.82%,S 期细胞比例减少31.86%、38.91%和26.77%。PB 使 Kasumi-1、U937和 NB4细胞 p21WAF1/CIP1表达增高。PB 处理后,p21WAF1/CIP1的表达水平较处理前增高(2.06±0.27),(2.78±0.40)和(1.78±0.20)倍。PB 可以上调 p21 WAF1/CIP1启动子的转录活性,且呈剂量依赖关系。3 mmol/L PB 处理48 h 使转录活性增高(5.74±0.93)倍。PB 上调 p21WAF1/CIP1启动子转录活性主要是依赖于转录起始位点上游101 bp的序列。结论 PB 可以抑制白血病细胞系的细胞周期,这种作用可能是通过上调细胞周期相关基因p21 WAF1/CIP1的表达实现的。
Objective To study the effect of sodium phenylbutyrate (HDAC), a histone deacetylase (HDAC) inhibitor, on the cell cycle of leukemia cell lines and to explore its molecular mechanism. Methods PB cells were treated with Kasumi-1, U937 and NB4 cells and the cells were harvested at 24, 48 and 72 hours after treatment. Propidium iodide DNA staining, flow cytometry analysis of cell cycle changes. Semi - quantitative reverse transcription - polymerase chain reaction (RT - PCR) analysis of cell cycle related gene p21WAF1 / CIP1 expression changes. The effect of PB on p21WAF1 / CIP1 gene promoter activity was analyzed using a luciferase reporter gene in human renal epithelial cell line 293T. Results PB could inhibit the cell cycle of Kasu-mi-1, U937 and NB4 cells in a time-and dose-dependent manner. 3 mmol / L PB for 72 h, respectively, increased the percentage of G 0 / G 1 phase cells by 42.03%, 44.36% and 26.82% in Ka-sumi-1, U937 and NB4 cells respectively, and decreased the proportion of S phase cells by 31.86%, 38.91% and 26.77 %. PB increased p21WAF1 / CIP1 expression in Kasumi-1, U937 and NB4 cells. After PB treatment, the expression of p21WAF1 / CIP1 was increased (2.06 ± 0.27), (2.78 ± 0.40) and (1.78 ± 0.20) times higher than that before treatment. PB can up-regulate the transcriptional activity of p21 WAF1 / CIP1 promoter in a dose-dependent manner. Treatment with 3 mmol / L PB for 48 h increased the transcriptional activity (5.74 ± 0.93) fold. PB up-regulates p21WAF1 / CIP1 promoter transcriptional activity mainly by a sequence of 101 bp upstream of the transcriptional start site. Conclusion PB can inhibit the cell cycle of leukemia cell lines, and this effect may be through up-regulating the expression of cell cycle-related gene p21 WAF1 / CIP1.