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Development of agents that suppress aging (aging suppressants) requires quantification of cellular senescence. Cellular senescence in vitro is characterized by a large cell morphology and permanent loss of proliferative potential. When HT-1080 cells were arrested by p21, they continued to grow exponentially in size and became hypertrophic with a 15-fold increase in the protein content per cell. These changes were mirrored by accumulation of GFP (driven by CMV promoter) per cell, which also served as a marker of cellular hypertrophy. Preservation of proliferative potential (competence) was measured by an increase in live cell number, when p21 was switched off. While modestly decreasing hypertrophy in p21-arresrted cells, rapamycin considerably preserved competence, converting senescence into quiescence. Preservation of proliferative potential (competence) correlated with inhibition of S6 phosphorylation by rapamycin. When p21 was switched off, competent cells, by resuming proliferation, became progressively less hypertrophic. Preservation of proliferative potential is a sensitive and quantitative measure of suppression of mTOR-driven senescence.

Quantifying pharmacologic suppression of cellular senescence: prevention of cellular hypertrophy versus preservation of proliferative potential