摘要
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 elucidate effects of histone deacetylase inhibitors on cell cycle of leukemia cell lines and investigate its molecular mechanisms. Methods Kasumi-1, U937 and NB4 cell lines were exposed to a histone deacetylase inhibitor, phenyl butyrate(PB), for 24, 48 and 72 hrs. Cells were harvested for cell cycle analysis by flow cytometry. Gene expression of p21WAF1/CIP1, a cyclin-dependent kinase inhibitor, was determined by semi-quantitative reverse transcriptase polymerase chain reaction (semi-quantitative RT-PCR). Promoter activity of p21WAF1/CIP1 was determined by luciferase-reporter assay in 293T cell line. Results PB inhibited cell cycle of Kasumi-1, U937 and NB4 cell lines, showing G0/G1 phase arrest and S-phase fraction reduction with a dose and time dependent manner. After Kasumi-1, U937 or NB4 cells exposed to 3 mmoL/L PB for 72 hrs, G0/G1-phase fraction was increased by 42. 03% , 44. 36% and 26.82%, and S-phase fraction was decreased by 31.86%, 38.9%and 26.77%, respectively. After Kasumi-1, U937 and NB4 cell lines exposed to PB, the expression of p21WAF1/CIP1 gene was increased by (2.06±0.27), (2.78±0.40) and ( 1.78±0.20) times at its maximum, respectively. PB could stimulate p21WAF1/CIP1 promoter activity ( by luciferase-reporter assay) and the effect was dose dependent. The promoter activity was increased by 5.74 times after the cells exposed to 3 mmoL/L PB for 48 hrs. PB stimulating p21WAF1/CIP1 promoter activity was mainly mediated by a 101 base pairs fragment upstream of transcription start site. Conclusion PB could inhibit cell cycle of leukemia cell lines. The effects were mainly through up-regulation of p21WAF1/CIP1 expression.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2006年第5期294-297,共4页
Chinese Journal of Hematology
基金
国家自然科学基金资助项目(30370593)
天津市科技发展计划资助项目(05YESZSF02400)