5-Azacytidine-induced demethyelation of DNA to senescent level does not block proliferation of human fibroblasts

Abstract

IMR-90 human diploid fibroblasts (HDF) lose from 30-50% of their genomic 5-methyldeoxycytidine (5mdC) during the cellular aging process. In contrast, immortal SV40-transformed IMR-90 maintain a constant level of 5mdC in culture. Precrisis SV40-transformed HDF (AG3204) represent a stage in between normal cell aging and immortalization because these cells still have a finite proliferative lifespan, but it is longer than that of normal HDF and ends in cell death rather than in G1-arrest. We find that AG3204 cells continue to lose from 12-33% of their 5mdC after a population has become 99% positive for SV40 T-antigen. Both IMR-90 cells and AG3204 cells have similar levels of 5mdC (average of 2.25%) at the end of lifespan. We investigated whether this level of 5mdC is an absolute block to further proliferation by treating IMR-90 and AG3204 cells with 5-azacytidine (5azaC) to reduce their 5mdC levels below the terminal level normally achieved at end of lifespan. We find that both IMR-90 and AG3204 cells undergo extensive proliferation with subterminal levels of 5mdC and that the lifespans of both cell types are shortened by 5azaC treatment. These studies indicate that random genomic DNA demethylation to a specific level of 5mdC is not a direct cause of finite proliferative lifespan. However, the correlation between accelerated DNA demethylation and accelerated aging still suggests that these two phenomena are related. Two ways to explain this relationship are: (1) DNA demethylation during aging is not random, and/or (2) both DNA demethylation and other independent aging processes cooperate to produce finite lifespan. In both cases, accelerated random DNA demethylation could accelerate aging, but not necessarily in direct relationship to the final genomic level of 5mdC achieved during the normal aging process.