Zhang Xuehong, Li Paiyun, Gan Ying, Xiang Shengyan, Gu Liankun, Zhou Jing, Zhou Xiaorui, Wu Peihuang, Zhang Baozhen, Deng Dajun
Key Laboratory of Carcinogenesis and Translational Research (MOE/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Beijing 100142, China.
Division of Etiology, Beijing Cancer Hospital, Beijing 100142, China.
Chin Med J (Engl). 2025 Feb 5;138(3):332-342. doi: 10.1097/CM9.0000000000003004. Epub 2024 Feb 29.
P16 inactivation is frequently accompanied by telomerase reverse transcriptase ( TERT ) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT . However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development.
A zinc finger protein-based P16 -specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution.
Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population.
P16 methylation drives TERT -mediated immortalization and transformation of normal human cells that may contribute to cancer development.
在人类癌症基因组中，p16基因失活常伴有端粒酶逆转录酶（TERT）扩增。在正常细胞通过TERT永生化过程中，DNA甲基化导致的p16基因失活常常自动发生。然而，仍需获得直接证据来支持表观遗传变化（如p16甲基化）对癌症发展的因果效应。本研究旨在提供实验证据，证明p16甲基化直接驱动癌症发展。
使用基于锌指蛋白的p16特异性DNA甲基转移酶（P16-Dnmt）载体，其包含“Tet-On”开关，以诱导正常人成纤维细胞CCD-18Co细胞中p16基因启动子区域的广泛甲基化。通过一系列实验评估细胞在其整个生命周期中的永生化和转化情况。使用细胞亚克隆和DNA条形码技术追踪细胞进化的多样性。
渗漏的P16-Dnmt表达（无强力霉素诱导）可通过DNA甲基化特异性地使p16表达失活。p16甲基化仅促进了CCD-18Co细胞的增殖并延长了其寿命，但未诱导其永生化。值得注意的是，在P16-Dnmt&TERT细胞中，细胞永生化、接触抑制丧失和不依赖贴壁生长总是普遍存在，表明细胞发生了转化。相比之下，几乎所有TERT细胞在复制危机中死亡。只有少数TERT细胞从危机中恢复，其中发生了DNA甲基化导致的p16自发失活。此外，P16-Dnmt&TERT细胞的亚克隆形成能力是TERT细胞的两倍。DNA条形码分析表明，P16-Dnmt&TERT细胞群体的多样性远大于TERT细胞群体。
p16甲基化驱动TERT介导的正常人细胞永生化和转化，这可能有助于癌症发展。
