Song Yin-hong, Zhang Chang-jü
Department of Immunology, Medical College of Three Gorges University, Yichang, Hubei 443002, China.
Zhonghua Bing Li Xue Za Zhi. 2007 Sep;36(9):614-8.
To investigate the correlation between methylation status and gene expression of APC (adenomatous polyposis coli) gene in HeLa, CaSki and SiHa cell lines of cervical carcinoma, and explore the effect of hydralazine on the transcription regulation of the 5'CpG island demethylation of APC gene and the proliferation and apoptosis of the cell lines.
Methylation status and the expression of APC gene were analyzed using methylated specific PCR, RT-PCR and FQ-PCR methods. The expression of beta-catenin protein which correlates closely with APC was detected by SP method after treatment with Hydralazine. MTT and FCM assays were used to observe the changes of proliferation activity and apoptosis of the cells after Hydralazine treatment.
(1) APC gene was methylated or hemimethylated respectively in HeLa and CaSki cell lines, at the same time, APC gene was not methylated in SiHa cell. (2) After having been treated by 40 micromol/L Hydralazine for 72 hours, growth inhibitory ratios of HeLa, CaSki and SiHa cell lines were (52.12 +/- 3.78)%, (44.31 +/- 2.59)% and (47.73 +/- 4.73)% respectively, on the contrary, normal cell HECV's growth inhibitory ratio was only (27.18 +/- 0.79)%. APC gene in HeLa and CaSki cell lines which were treated by 40 micromol/L Hydralazine for 72 hours was demethylated and expressed positively, the expression of APC mRNA in HeLa, CaSki and SiHa cell lines increased to 10.35, 11.40 and 0.73 times respectively. (3) Hydralazine, when used at the concentration of 40 micromol/L for 72 hours, induced S phase and G2/M phase arrest and apoptosis in HeLa and CaSki cells. beta-catenin protein can be expressed in cell membrane after treatment with Hydralazine.
APC gene methylation plays an important role in the carcinogenesis of cervical cells and can re-express after the treatment with Hydralazine which also could inhibit the growth of the cervical cancer cells.
研究宫颈癌HeLa、CaSki和SiHa细胞系中APC（腺瘤性结肠息肉病）基因甲基化状态与基因表达的相关性，探讨肼屈嗪对APC基因5'CpG岛去甲基化转录调控及细胞系增殖和凋亡的影响。
采用甲基化特异性PCR、RT-PCR和FQ-PCR方法分析APC基因的甲基化状态和表达。用肼屈嗪处理后，通过SP法检测与APC密切相关的β-连环蛋白的表达。采用MTT和FCM法观察肼屈嗪处理后细胞增殖活性和凋亡的变化。
（1）HeLa和CaSki细胞系中APC基因分别发生甲基化或半甲基化，而SiHa细胞中APC基因未甲基化。（2）用40μmol/L肼屈嗪处理72小时后，HeLa、CaSki和SiHa细胞系的生长抑制率分别为（52.12±3.78）%、（44.31±2.59）%和（47.73±4.73）%，而正常细胞HECV的生长抑制率仅为（27.18±0.79）%。用40μmol/L肼屈嗪处理72小时的HeLa和CaSki细胞系中APC基因去甲基化并呈阳性表达，HeLa、CaSki和SiHa细胞系中APC mRNA表达分别增加到10.35、11.40和0.73倍。（3）40μmol/L肼屈嗪处理72小时可诱导HeLa和CaSki细胞发生S期和G2/M期阻滞及凋亡。用肼屈嗪处理后，β-连环蛋白可在细胞膜表达。
APC基因甲基化在宫颈细胞癌变中起重要作用，经肼屈嗪处理后可重新表达，且肼屈嗪能抑制宫颈癌细胞生长。
