Baik J H, Chikhi N, Bulle F, Giuili G, Guellaën G, Siegrist S
U-99 INSERM, Hôpital Henri Mondor, Creteil, France.
J Cell Physiol. 1992 Nov;153(2):408-16. doi: 10.1002/jcp.1041530221.
The role of DNA methylation in the expression of the rat gamma-glutamyl transpeptidase (GGT) gene was assessed in the Fao cell line using a hypomethylating agent, 5-azacytidine. Ten repetitive treatments of the cells, with 8 microM 5-azacytidine for 24 h, led to 13- and 80-fold increases, respectively, in GGT activity and in GGT mRNA level. The DNA methylation patterns generated by the isoschizomeric restriction enzymes Hpa II and Msp I indicated that the GGT gene, highly methylated in Fao cells, became strongly demethylated after 5-azacytidine treatments. Thus, DNA demethylation increases the expression of the GGT gene. 5-Azacytidine treatments also increased, but to a lesser extent, mRNAs level for actin, albumin, mitochondrial aspartate aminotransferase, aldolase B mRNAs (12- to 16-fold) as well as for tubulin, gluthathione transferase, and tyrosine aminotransferase mRNAs (2- to 5-fold). The GGT gene expression was further studied in B4 cells, cloned from the demethylated Fao cell population. This clone B4 exhibited a stable and strong GGT activity and a highly demethylated GGT gene. Among the three GGT mRNA I, II, or III, transcribed from three different promoters of the single rat GGT gene, only mRNA III was detected in Fao cells and was increased in clone B4, indicating that the demethylation acts on the promoter for mRNA III. The analysis of the differentiation state of B4 cells, as compared to Fao cells, showed a loss of the regulation of GGT and aspartate aminotransferase genes by dexamethasone, as well as a loss of the gluconeogenic pathway. Interestingly, B4 cells have retained many other specific functions of hepatic differentiation and have acquired alpha-fetoprotein expression; thus this clone exhibits the characteristics of a hepatic fetal phenotype.
利用去甲基化剂5-氮杂胞苷，在Fao细胞系中评估了DNA甲基化在大鼠γ-谷氨酰转肽酶（GGT）基因表达中的作用。用8μM 5-氮杂胞苷对细胞进行十次重复处理，每次处理24小时，结果GGT活性和GGT mRNA水平分别增加了13倍和80倍。同裂酶Hpa II和Msp I产生的DNA甲基化模式表明，在Fao细胞中高度甲基化的GGT基因，在5-氮杂胞苷处理后强烈去甲基化。因此，DNA去甲基化增加了GGT基因的表达。5-氮杂胞苷处理还使肌动蛋白、白蛋白、线粒体天冬氨酸氨基转移酶、醛缩酶B的mRNA水平（增加12至16倍）以及微管蛋白、谷胱甘肽转移酶和酪氨酸氨基转移酶的mRNA水平（增加2至5倍）有所升高，但升高幅度较小。在从去甲基化的Fao细胞群体中克隆得到的B4细胞中，对GGT基因表达进行了进一步研究。该克隆B4表现出稳定且强烈的GGT活性以及高度去甲基化的GGT基因。在从大鼠单个GGT基因的三个不同启动子转录而来的三种GGT mRNA I、II或III中，Fao细胞中仅检测到mRNA III，而在克隆B4中其水平升高，这表明去甲基化作用于mRNA III的启动子。与Fao细胞相比，对B4细胞分化状态的分析表明，地塞米松对GGT和天冬氨酸氨基转移酶基因的调控丧失，同时糖异生途径也丧失。有趣的是，B4细胞保留了肝分化的许多其他特定功能，并获得了甲胎蛋白表达；因此，该克隆表现出肝胎儿表型的特征。
