肺腺癌细胞 P15中凋亡相关基因甲基化状态与化疗敏感性的关系

摘要

目的：研究肺腺癌细胞P15中凋亡相关基因甲基化状态与化疗敏感性的关系。方法：采用甲基化特异性PCR法检测未处理对照组和地西他滨（ DAC）处理组P15细胞的 p73、p14ARF、p16INK4a和bax基因甲基化情况，利用RT－PCR检测p73、bcl－xL、bad、bax、p14ARF和p16INK4a的mRNA表达；采用平板克隆形成实验和细胞生长抑制实验检测DAC处理前后P15细胞对顺铂（ C－DDP）的敏感性，DAPI染色法检测DAC处理前后C－DDP对P15细胞的凋亡情况。结果：p73、p16INK4a和bax在甲基化状态下均有表达，经DAC处理后p16INK4a表达减弱，p73和bax表达消失。 p73、p16INK4a和bax在非甲基化状态下表达较弱，经DAC处理后三者均表达增强。 DAC加C－DDP处理组与未处理对照组相比，前者的克隆形成率和细胞生存显著下降（ P＜0．05）。 DAC加C－DDP组的细胞凋亡显著高于未处理对照组（ P＜0．05）。结论：由于DAC活化多个因甲基化而表达沉默的促凋亡基因，用DAC处理肺腺癌细胞P15后，P15细胞对化疗药物C－DDP的敏感性增强。 DAC和C－DDP协同作用促进了肿瘤细胞的凋亡。 DAC与C－DDP联合应用可逆转化疗耐药，恢复肺腺癌细胞对化疗药物的敏感性，两者具有明显的协同抗肿瘤作用。%AIM:Toinvestigatetheassociationbetweenmethylationstatusofapoptosis-relatedgenesandche-mosensitivity in the lung adenocarcinoma cell line P 15.METHODS: Methylation-specific PCR was applied to detect the methylation status of p73, p14ARF, p16INK4a and bax genes of P15 cells in untreated control group and decitabine (DAC) treatment group.RT-PCR was used to detect the expression of p 73, bcl-xL, bad, bax, p14ARF and p16INK4a at mRNA level. Colony formation assay and cell growth inhibition assay were used to detect the sensitivity of P 15 cells to cis-diaminedichlo-roplatinum ( C-DDP) before and after DAC treatment .DAPI staining was used to determine the apoptosis of P 15 cells ex-posed to C-DDP before and after DAC treatment .RESULTS:p73, p16INK4a and bax were expressed in the methylation sta-tus.After DAC treatment, p16INK4a expression was decreased , and the expression of p73 and bax disappeared .The expres-sion of p73, p16INK4a and bax in the unmethylated status was weak , but the enhanced expression was observed following DAC treatment.After P15 cells were treated with DAC and C-DDP, the colony formation rate of the P15 cells was signifi-cantly decreased as compared with untreated control group .The apoptotic P15 cells in DAC+C-DDP treatment group were significantly higher than those in untreated control group (P<0.05).CONCLUSION:After treated with DAC, the sensi-tivity of P15 cells to C-DDP is increased due to the activation of silenced pro-apoptotic genes .DAC and C-DDP synergisti-cally promote tumor cell apoptosis .They have significant anti-tumor effect .