Homeostatic maintenance of allele-specific p16 methylation in cancer cells accompanied by dynamic focal methylation and hydroxymethylation.
Homeostatic maintenance of allele-specific p16 methylation in cancer cells accompanied by dynamic focal methylation and hydroxymethylation.
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癌细胞中等位基因特异性 p16 甲基化的稳态维持，伴有动态局部甲基化和羟甲基化
DOI：
10.1371/journal.pone.0097785
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Deng D
中科院分区：
文献类型：
--
作者：
Qin S;Li Q;Zhou J;Liu ZJ;Su N;Wilson J;Lu ZM;Deng D
甲基化经常发生在癌变过程中。虽然人们假设p16甲基化状态在癌细胞中是动态维持的，但直到现在还没有支持这一假设的直接证据。方法建立融合细胞模型，对细胞原有的DNA甲基化模式进行重编程。然后使用DHPLC和亚硫酸酯测序方法，在融合的单克隆、亲本癌细胞系（p16-完全甲基化- ags和未甲基化- mgc803）和未融合的HCT116细胞中反复定量分析p16等位基因的甲基化状态。采用染色质免疫沉淀(ChIP)-PCR分析组蛋白甲基化。采用免疫染色和RT-PCR检测P16表达情况。结果大多数p16等位基因的甲基化状态在融合单克隆细胞中稳定维持了60代。最重要的是，在融合单克隆中约27%的p16等位基因中一致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本细胞中却没有。此外，单克隆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类似的现象。有趣的是，在以反义链特异性模式羟甲基化的p16等位基因中未观察到转录。融合细胞中H3K9和H3K4三甲基化水平分别略低于亲本AGS和MGC803细胞。结论本研究提供了第一个直接证据，证实p16 CpG岛的甲基化状态在癌细胞中不仅是稳态维持的，而且伴随着短暂的局灶甲基化、去甲基化和羟甲基化的动态过程。
Aim 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. Methods 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. Results 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. Conclusion 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.
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影响因子：
64.8
作者：
通讯作者：
--
影响因子：
14.9
作者：
Bhattacharyya S;Yu Y;Suzuki M;Campbell N;Mazdo J;Vasanthakumar A;Bhagat TD;Nischal S;Christopeit M;Parekh S;Steidl U;Godley L;Maitra A;Greally JM;Verma A
通讯作者：
Verma A
DOI：
10.1016/s1383-5718(02)00288-7
发表时间：
2003-02-05
影响因子：
1.9
作者：
Bai, H;Gu, LK;Deng, DJ
通讯作者：
Deng, DJ
DOI：
10.1073/pnas.95.20.11891
发表时间：
1998-09-29
影响因子：
11.1
作者：
Belinsky, SA;Nikula, KJ;Herman, JG
通讯作者：
Herman, JG
影响因子：
11.2
作者：
Egger, Gerda;Aparicio, Ana M.;Jones, Peter A.
通讯作者：
Jones, Peter A.