Driving effect of P16 methylation on telomerase reverse transcriptase-mediated immortalization and transformation of normal human fibroblasts

被引：0

作者：

Zhang Xuehong ^{[1
]}

Li Paiyun ^{[2
,3
]}

Gan Ying ^{[1
]}

Xiang Shengyan ^{[1
]}

Gu Liankun ^{[1
]}

Zhou Jing ^{[1
]}

Zhou Xiaorui ^{[4
]}

Wu Peihuang ^{[4
]}

Zhang Baozhen ^{[1
,2
]}

Deng Dajun ^{[1
]}

机构：

[1] Key Laboratory of Carcinogenesis and Translational Research (MOE/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Beijing, China

[2] Division of Etiology, Beijing Cancer Hospital, Beijing, China

[3] Radiation Oncology Department, Tianjin Medical University General Hospital, Tianjin, China

[4] Department of Biomedical Engineering, Peking University Cancer Hospital and Institute, Beijing, China

来源：

中华医学杂志英文版 | 2025年 / 138卷 / 03期

关键词：

P16 methylation; Telomerase reverse transcriptase; Immortalization; Transformation; Barcode lineage tracking; Human fibroblasts;

D O I：

暂无

中图分类号：

R730.2 [肿瘤病理学、病因学];

学科分类号：

100214 ;

摘要：

Background: 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 byTERT. However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such asP16 methylation, on cancer development. This study aimed to provide experimental evidence thatP16 methylation directly drives cancer development.Methods: A zinc finger protein-basedP16-specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation ofP16 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.Results: Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivateP16 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 spontaneousP16 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.Conclusion: P16 methylation drivesTERT-mediated immortalization and transformation of normal human cells that may contribute to cancer development.

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