Quantifying pharmacologic suppression of cellular senescence: prevention of cellular hypertrophy versus preservation of proliferative potential
Abstract
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.
Keywords: cellular senescence, cellular hypertrophy, aging-suppression, rapamycin, mTOR
Introduction
In cell culture, cellular senescence is usually defined as a state of irreversible cell cycle arrest [1,2]. Hence, cellular senescence is sometimes confused with growth inhibition. Here we will use the term ‘growth' as an increase in cellular mass, regardless of whether cells proliferate or not. Intriguingly, Ras, MEKeIF-4E and serum, which stimulate growth-promoting pathways, contribute to and facilitate cellular senescence [3-6]. In theory, cellular senescence is caused by inappropriate activation of growth-promoting pathways, when actual growth is impossible [7,8]. In proliferating cells, growth-promoting mTOR (Target of Rapamycin) and MAPK (Mitogen-activated Protein Kinase) pathways drive both cellular mass growth and cell cycle progression. When the cell cycle is blocked by either p21 or p16, growth-stimulation via mTOR leads to cellular senescence [9]. Serum withdrawal, PI-3K, mTOR and MEK inhibitors, all decreased mTOR activity and prevented permanent loss of proliferative potential [10,11]. The term "permanent loss of proliferative potential" means that, even when p21 and p16 were shut off, cells cannot resume proliferation [12]. Inhibitors of mTOR such as rapamycin preserved proliferative potential [9-11]. To avoid confusions, we stress that rapamycin does not stimulate proliferation, does not abrogate cell cycle arrest caused by p21 and does not force cells to by-pass cell cycle arrest. Rapamycin converts senescence (an irreversible condition) into quiescence (a reversible condition). It is still unknown whether rapamycin suppresses senescence in a dose-dependent manner and whether this suppression correlates with the degree of mTOR inhibition.
Another common marker of cell senescence is a large cell morphology (hypertrophy). Cellular hypertrophy is usually measured as a cell diameter. Given that volume (or cell mass) is proportional to the cube of diameter, then the amount of protein per cell (cell mass) may be a more sensitive parameter than cell diameter. For example if diameter is increased 2-fold, cell mass is increased 8-fold. In theory, cell mass could be estimated as an amount of any fluorescent protein such as green fluorescent protein (GFP), expressed by a constitutive viral promoter such as CMV promoter. If the cell cycle is blocked but cells continue to grow in size, then GFP should accumulate. Here we tested this prediction. Independently from our study, a clone of HT-p21 cells, known as p21-9, had been stably transfected with CMV-EGFP [13,14,15] and thus expresses enhanced GFP. We predict that induction of p21 by IPTG should increase GFP per cell, as a marker of cellular hypertrophy. Given cell-doubling time of 20 hours, there should be a 10-14 fold increase in GFP/cell in 3 days. Here, we confirmed this prediction. We further investigated the link between mTOR activity, cellular hypertrophy and loss of proliferative potential. We found that preservation of proliferative (competence) was the most sensitive marker of mTOR inhibition, easily detectable even at concentrations of rapamycin when inhibition of mTOR was marginal. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2815749/
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