Vitamin C delays mouse embryonic cells immortalization by maintaining enhancer hypomethylation of senescence-related genes

Immortalization is the first step necessary for cancer cells to overcome replicative senescence and proliferate indefinitely, and it is known to be influenced by DNA cytosine methylation. Vitamin C (vitC) is a known cofactor for the Ten-eleven-translocation (Tet) enzymes, which are involved in active DNA demethylation. However, its role during this key process remains unclear. In this study, we evaluated vitC´s potential to prevent cell immortalization and its associated DNA methylation changes in a mouse embryonic cell culture model. Our findings demonstrate that vitC at physiological concentrations delays embryonic cell immortalization, inducing beta-Galactosidase positive senescence, and increasing the global levels of 5-hydroxymethylcytosine in DNA. DNA methylation arrays revealed that immortalization induces a substantial reprogramming of the DNA methylation landscape, characterized by hypomethylation of Partially Methylated Domains (PMDs) and hypermethylation of enhancers, while vitC treatment prevented the immortalization-acquired DNA methylation. Gene expression analysis confirmed the upregulation of senescence and adipogenesis-related genes in vitC-treated cultures. Additionally, DNA hypomethylation was detected in key regulatory regions associated with these upregulated genes, particularly in enhancers. Interestingly, treated cultures sometimes acquired deletions of the Cdkn2a gene, suggesting that additional genetic insults are necessary for cells to overcome vitC-induced senescence. Collectively, our findings provide insight into how vitC modifies the epigenetic landscape of embryonic cells, inducing hypomethylation of DNA regulatory elements, thus preventing cells from overcoming replicative senescence and becoming immortal.