p53在二乙基亚硝胺诱导肝细胞转录因子AP-1活化中的作用

The role of p53 on the diethylnitrosamine-induced activation of activator protein 1 in hepatic cells

目的研究p53对二乙基亚硝胺(DEN)诱导肝细胞恶变过程的影响,探讨p53功能缺失与细胞恶变的关系。方法使用p53特异性抑制剂pifithrin-α(PFT-α)和(或)人p53小RNA干扰质粒sip53抑制L02细胞p53的转录活性。采用荧光素酶报告基因法检测不同时间点、不同浓度DEN对p53功能正常和缺失的L02细胞AP-1转录激活活性的影响,检测不同DEN处理时间对p53转录激活活性的影响及PFT-α对AP-1转录激活活性的影响。转录激活活性用相对荧光强度表示。结果应用PFT-α或转染sip53质粒24h后,p53的转录活性明显下降。单纯DEN处理后AP-1的相对荧光强度轻度上调,而DEN+PFT-α和DEN+sip53处理的细胞内AP-1相对荧光强度与单纯DEN处理组比较均有上调,呈现时间和浓度双重依赖性;在作用24h时点或DEN浓度为100ng/L时作用达高峰,随后回落。DEN处理后p53相对荧光强度明显上调,且存在时间依赖性,在24h达高峰,随后回落。DEN+PFT-α处理的细胞p53相对荧光强度在各时间点的变化不明显,始终保持在较低水平。以PFT-α抑制p53功能后,L02细胞内AP-1的相对荧光强度上调,且此上调作用与PFT-α的作用时间相关。结论DEN刺激可上调L02细胞内p53的表达,后者可抑制细胞的恶性增殖。p53功能缺失可明显促进DEN诱导的L02细胞AP-1转录激活活性上调,提示p53是正常肝细胞抗恶变的重要保护机制。

Objective To explore the changes in carcinogenesis in p53-defunction hepatic cells induced by DEN,and to investigate the ralationship and mechanism between p53 and hepatic cells carcinogenesis.Methods PFT-α（pifithrin-α,an inhibitor for p53-dependent transcriptional activation） and （or）p53 small interfering RNA plasmid sip53 was used to inhibit p53 transcription activity.Luciferase reporter gene method was used to detect AP-1 transactivation activity in p53 normal or defunction L02 cells at different time points and DEN concentrations,to detect the influence of p53 transactivation activity by DEN pretreatment at different time points,and the influence of AP-1 transactivation activity by PFT-α.Relative fluorescence intensity represented transactivation activity.Results p53 transactivation activity decreased markedly after pretreatment with PFT-α or sip53 for 24 hours.DEN stimulation resulted in a slight increase in relative AP-1 activity,while treatment with DEN＋PFT-α or DEN＋sip53 resulted in a marked increase in activation of AP-1 in a dose-and time-dependent manner,peaking at the time point of 24h or with the concentration of 100ng/L,followed by a fall.Relative AP-1 activity increased significantly in DEN＋PFT-α or DEN＋sip53 group at various time points and concentrations.Relative p53 dependent transcription activity increased significantly after DEN stimulation,and it also peaked at the time point of 24h being followed by a fall.This phenomenon could be blocked by PFT-α.Pretreatment with PFT-α resulted in an up-regulation of relative AP-1 activity in L02 cells,which correlated to the action time.Conclusions DEN stimulation results in up-regulation of p53 transcription activity in L02 cells,which protects cells from carcinogenesis.Loss of p53 function enhances DEN induced activation of AP-1 in L02 cells,suggesting that p53 may play an essential protective role in normal hepatic cells from carcinogenesis.