化学致癌物暴露后哺乳动物细胞内及培养上清中核型dUTP焦磷酸酶的表达分析

Extracellular and intracellular expression pattern of nuclear isoform of dUTP pyrophosphatase in mammalian cells after chemical carcinogen exposure

目的:研究不同化学致癌物暴露后核型dUTP焦磷酸酶(DUT-N)在哺乳动物细胞内及培养上清中的表达情况。方法:分别用N-亚硝胺类烷化剂N-甲基-N’-硝基-N-亚硝基胍(MNNG)和多环芳烃类致癌物苯并[a]芘在体内的终致癌物7,8-二羟-9,10-环氧苯并[a]芘(BPDE)处理人羊膜上皮FL细胞、人肝癌HepG2细胞和人宫颈癌HeLa细胞。采用SDS-PAGE和免疫印迹法检测细胞培养上清中DUT-N蛋白的分泌及细胞内该蛋白的表达情况。结果:低浓度MNNG(0.25μmol/L)和BPDE(5nmol/L)暴露后都可引起DUT-N出现在FL细胞的培养上清中,而对HeLa细胞均无此影响;两者对HepG2细胞的影响不同,前者使DUT-N在细胞外出现,而后者却没有改变。同时,尽管MNNG与BPDE都可引起FL细胞胞外DUT-N的出现,但MNNG暴露后胞内DUT-N的表达dUTP下调,而BPDE暴露后却dUTP上调。结论:化学致癌物暴露后核型dUTP焦磷酸酶在细胞培养上清中出现的现象不具有化合物的特异性,但对特定细胞系(如FL细胞)可能存在化学致癌剂或DNA损伤剂的共性。同时,不同致癌物引起DUT-N出现的细胞反应可能是不同的。

AIM： To study the extracellular and inlracellular expression pattern of dUTP pyrophosphatase （ nuclear isoform, DUT - N） in mammalian cells after exposure to different chemical carcinogens. METHODS ： Human amniotic epithelial cells （FL cells）, hepatoma cells （HepG2 cells） and cervical cancer cells （HeLa cells） were treated with N - methyl - N＇ - nitro - N - nitrosoguanidine （ MNNG, N - nitroso alkylating agent） and benzo [a] pyrene - 7, 8 - di- hydroxy- 9, 10- epoxide （BPDE, the metabolic activated form of Benzo[a] pyrene）. SDS- PAGE and immunoblotting were used to examine the extracellular and intracellular protein levels of DUT - N. RESULTS ： Immunoblotting showed that DUT -N appeared in the culture medium of FL cells, but not HeLa cells, after MNNG and BPDE exposure. MNNG, not BPDE induced the appearance of this protein in the culture medium of HepG2 cells. Although MNNG and BPDE treatment both induced the appearance of DUT - N, the intracellular protein level was down - regulated in the former and up - regula- ted in the latter. CONCLUSION ： The appearance of DUT - N in the culture medium after chemical carcinogen treatment is not a phenomenon induced by specific carcinogen, but there are some common features between different carcinogens or DNA - damaging agents for given cell lines, such as FL cell. Meanwhile, the cellular mechanism of DUT - N appearance remains different from one carcinogen to another.