Two mechanisms underlying the loss of p16(Ink4a) function are associated with distinct tumorigenic consequences for WS MEFs escaping from senescence.

Werner syndrome (WS) mouse embryonic fibroblasts (MEFs) can spontaneously escape from senescence and become immortalized, either tumorigenic or non-tumorigenic. Our data revealed a single p53(N236S) point mutation in the tumorigenic cell lines, which was correlated with the down-regulation of p21(Waf1/Cip1). p16(Ink4a) expression was significantly decreased in all immortalized cell lines. Bisulfate sequencing indicated that the p16(Ink4a) gene was methylated in the tumorigenic cells. Exogenous overexpression of p21(Waf1/Cip1) demethylated p16(Ink4a) and restored its expression, which induced cell growth arrest and senescence. While in non-tumorigenic immortalized cells, the Ink4a loci and adjacent genomic DNA were found to be deleted. These data suggest that the loss of p16(Ink4a) function by either genomic DNA deletion or methylation have been adopted by senescent WS MEFs escaping from senescence, with distinct tumorigenic consequences. The fact that cells that had escaped senescence via the spontaneous biallelic deletion of the Ink4a loci could not form tumors suggests that the functional loss of p16(Ink4a)per se might not be sufficient for tumorigenesis; most likely, it is a byproduct and passenger mutation. The mutations in factors regulating p16(Ink4a) methylation might be the driver mutation. These findings shed light on the strategy of anti-aging by regulating p16(Ink4a) expression.