TSA抑制NB4细胞去乙酰化酶活性并促进细胞周期素激酶抑制剂表达

目的　研究曲古菌素A(TrichostatinA,TSA)对NB4细胞组蛋白去乙酰化酶HDAC1的作用及细胞周期依赖激酶抑制剂P21WAF1 /CIP1的表达,探讨TSA抗白血病的作用机制。方法　培养人的急性早幼粒细胞白血病细胞株NB4,应用TSA在不同浓度和不同时间点处理细胞,提取细胞的总蛋白和mRNA,用Westernblot蛋白质印迹技术检测HDAC1和P21WAF1 /CIP1 蛋白表达, 并用RT PCR技术同时检测P21WAF1 /CIP1 mRNA的表达水平。结果　①TSA明显抑制HDAC1的活性和表达,在IC50浓度时作用 4hHDAC1已经降低,持续 48 h;在较低浓度 ( 37 5 nmol·L-1 )时就对HDAC1有明显的抑制作用,但在 75 ~ 300 nmol·L-1时HDAC1降低水平未见明显区别;②TSA明显促进P21WAF1 /CIP1蛋白和mRNA的表达,在 8h时mRNA已增高, 12h后可见到蛋白增加,在浓度大于 150nmol·L-1时呈明显时间和剂量依赖性。结论　TSA明显抑制HDAC1活性和表达,促进细胞周期依赖性激酶抑制剂P21WAF1 /CIP1蛋白和mRNA水平升高,有明显的抗白血病作用。抑制HDAC1活性和促进P21WAF1 /CIP1增加可能是TSA抗白血病的机制之一。

The inhibition of histone deacetylase and expression promotion of cyclin dependent kinase inhibitor in raji cells by trichostatin A

Aim To investigate the effect of Trichostatin A(T SA)on histone deacetylase (HDAC1) and P21 WAF1/CIP1,cyclin dependent ki nase inhibitor,in NB4 cells for exploring the underlying mechanism of TSA in an ti-leukemia.Methods The total proteins and P21 WAF1/CIP1 mRN A were extracted from acute promyelocytic leukemia NB4 cells treated with or wit hout TSA of different concentrations at different time points,Western blot anal ysis was performed to determine the level of HDAC1 and P21 WAF1/CIP1 protei n,respectively,and RT-PCR was performed to detect the level of P21 WAF1/CI P1 mRNA.Results (1)The level of HDAC1 was obviously inhibited by TSA,and had decreased at 4 hours at IC 50 and lasted for 48 h. The inhibi tion of HDAC1 was significant at the TSA concentration of 37.5 nmol·L -1 , but there was no difference between 75~300 nmol·L -1.(2)The levels of P21 WAF1/CIP1 mRNA and protein were up-regulated by TSA in dose-and ti me-dependent manner,and mRNA increased in 8 h,but the P21 WAF1/CIP1 prot ein was detected at 12 hours and lasted for 48 hours.Conclusion TSA could inhibit the level of HDAC1 and enhance the expression of P21 WAF1/CIP1 protein and mRNA,and the results suggest that inhibiting HDAC1 and increasin g P21 WAF1/CIP1 may be one of the possible mechanisms of anti-leukemia by TSA.