Qin Sisi, Li Qiang, Zhou Jing, Liu Zhao-Jun, Su Na, Wilson James, Lu Zhe-Ming, Deng Dajun
Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Cancer Etiology, Peking University Cancer Hospital & Institute, Beijing, China.
GRU Cancer Center, Georgia Regents University, Augusta, Georgia, United States of America.
PLoS One. 2014 May 14;9(5):e97785. doi: 10.1371/journal.pone.0097785. eCollection 2014.
p16 Methylation frequently occurs in carcinogenesis. While it has been hypothesized that the p16 methylation states are dynamically maintained in cancer cells, direct evidence supporting this hypothesis has not been available until now.
A fusion cell model was established which reprogrammed the native DNA methylation pattern of the cells. The methylation status of the p16 alleles was then repeatedly quantitatively analyzed in the fusion monoclonal, parental cancer cell lines (p16-completely methylated-AGS and unmethylated-MGC803), and HCT116 non-fusion cell using DHPLC and bisulfite sequencing. Histone methylation was analyzed using chromatin immuno-precipitation (ChIP)-PCR. P16 expression status was determined using immuno-staining and RT-PCR.
The methylation status for the majority of the p16 alleles was stably maintained in the fusion monoclonal cells after up to 60 passages. Most importantly, focal de novo methylation, demethylation, and hydroxymethylation were consistently observed within about 27% of the p16 alleles in the fusion monoclones, but not the homozygously methylated or unmethylated parental cells. Furthermore, subclones of the monoclones consistently maintained the same p16 methylation pattern. A similar phenomenon was also observed using the p16 hemi-methylated HCT116 non-fusion cancer cell line. Interestingly, transcription was not observed in p16 alleles that were hydroxymethylated with an antisense-strand-specific pattern. Also, the levels of H3K9 and H3K4 trimethylation in the fusion cells were found to be slightly lower than the parental AGS and MGC803 cells, respectively.
The present study provides the first direct evidence confirming that the methylation states of p16 CpG islands is not only homeostatically maintained, but also accompanied by a dynamic process of transient focal methylation, demethylation, and hydroxymethylation in cancer cells.
p16甲基化在肿瘤发生过程中频繁出现。虽然已有假说认为p16甲基化状态在癌细胞中是动态维持的，但直到现在仍缺乏支持这一假说的直接证据。
建立了一种融合细胞模型，该模型可对细胞的天然DNA甲基化模式进行重编程。然后使用变性高效液相色谱（DHPLC）和亚硫酸氢盐测序法，对融合单克隆细胞、亲代癌细胞系（p16完全甲基化的AGS细胞系和未甲基化的MGC803细胞系）以及HCT116非融合细胞中p16等位基因的甲基化状态进行反复定量分析。使用染色质免疫沉淀（ChIP）-PCR分析组蛋白甲基化。通过免疫染色和逆转录聚合酶链反应（RT-PCR）确定p16表达状态。
在多达60次传代后，融合单克隆细胞中大多数p16等位基因的甲基化状态得以稳定维持。最重要的是，在融合单克隆细胞中约27%的p16等位基因内持续观察到局灶性从头甲基化、去甲基化和羟甲基化，但在纯合甲基化或未甲基化的亲代细胞中未观察到。此外，单克隆细胞的亚克隆始终保持相同的p16甲基化模式。使用p16半甲基化的HCT116非融合癌细胞系也观察到了类似现象。有趣的是，在以反义链特异性模式发生羟甲基化的p16等位基因中未观察到转录。此外，发现融合细胞中H3K9和H3K4三甲基化水平分别略低于亲代AGS细胞系和MGC803细胞系。
本研究提供了首个直接证据，证实p16 CpG岛的甲基化状态不仅能稳态维持，而且在癌细胞中还伴随着短暂的局灶性甲基化、去甲基化和羟甲基化的动态过程。
