苯并(a)芘通过活化蛋白-1非依赖性通路诱导细胞中p53蛋白过表达及高磷酸化  

Overexpression and higher phosphorylation of p53 induced by benzo(a) pyrene through activated protein 1 independent pathway

作　　者：叶萌[1] 刘秉慈[1] 贾效伟[1] 高艾[1] 焦石[1] 张凤梅[1] 

机构地区：[1]中国疾病预防控制中心职业卫生与中毒控制所,北京100050

出　　处：《卫生研究》2008年第3期255-258,共4页Journal of Hygiene Research

基　　金：国家自然科学基金项目(No.30671747;30371206);973国家重点基础研究发展规划项目(No.2002CB512905);美国Philip Morris USA Inc and Philip Morris International资助

摘　　要：目的探讨苯并(a)芘[B(a)P]诱导的人胚肺成纤维细胞(HELF)中p53蛋白、p53磷酸化水平及亚细胞分布和活化蛋白-1(AP-1)活性的改变,以及p53与AP-1的上下游关系。方法转染p53小干扰RNAp53 siRNA质粒(p53-H)、载体CMV的HELF细胞(HELF/CMV)和转染AP-1荧光报告质粒的HELF细胞(AP-H)无血清培养48h后,加入2μmol/L B(a)P作用24h,用Western blot及免疫荧光法检测细胞中p53蛋白及p53磷酸化的改变,利用细胞核、细胞浆分离技术观察其亚细胞定位,用荧光检测法检测AP-1的相对活性。用AP-1的化学抑制剂curcumin抑制其活性,用p53的化学抑制剂pifithrin-α(PFT)抑制其表达,观察了二者的上下游关系。结果2μmol/L B(a)P作用24h后细胞内p53蛋白及p53蛋白20位丝氨酸磷酸化水平增加,并且主要分布在细胞核内;AP-1的活性增加。抑制AP-1活性后,对B(a)P诱导的p53蛋白含量增加没有明显的影响;抑制p53表达后,对B(a)P诱导的AP-1活性的增加没有明显影响。结论B(a)P通过AP-1非依赖的信号通路引起人胚肺成纤维细胞p53蛋白含量的增加。Objective To investigate changes of p53 expression, p53 phosphorylation, and their subcellular localizations and activated protein 1 （AP-1） activity on human embryo lung fibroblasts （HELF） induced by benzo（a）pyrene （B（a）P） , and relationships between p53 and AP-1. Methods Cells transfected with p53 small interference RNA （p53 siRNA） plasmid （p53-H）, CMV vector （HELF/CMV） and AP- 1 luciferase reporter plasmid （AP-H） were cultured with serumfree RPMI-1640 for 48h, then treated with 2μmol/L B （a）P for 24h. Changes of p53 expression and p53 phosphorylation were checked by immunofluorescence and Western blot assay. The subcellular localizations of p53 and phosphorylated p53 were checked by cytoplasmic and nuclear extraction. AP-1 relative activities were detected by luciferase assay. Chemical inhibitors of p53 （pifithrin-α （PFT）） and AP-1 （curcumin） were used to observe relationships of p53 and AP-1. Results The cells were treated with 2vmol/L B（a） P for 24h, expression of p53 and phosphorylated p53 at 20 site were significantly increased, and they were entirely localized in the nucleus. AP-1 activities significantly increased after B （a） P treatment. When AP-1 activities were inhibited by curcumin, overexpression of p53 induced by B（a）P was not markedly changed. When p53 expression was inhibited by PFT, AP-1 activities were not significantly changed. Conclusion Overexpression of p53 in HELF induced by B（a）P could be through AP-1 independent pathways.

关 键 词：苯并(A)芘 P53 亚细胞定位 活化蛋白-1 人胚肺成纤维细胞 

分 类 号：Q51[生物学—生物化学] R994.6[医药卫生—毒理学]