Revert Senescent-Like Rat Embryonic Fibroblasts by Lowering the E2F-Switching Threshold

Cellular senescence is a cell dormancy state irreversible to proliferation at physiological conditions induced by cellular stress, DNA damage, and aging. Quiescent cells similarly reside in a non-proliferative state but can distinctly reenter the cell cycle upon growth signals which is essential for tissue repair and regeneration. While quiescence and senescence are considered to possess distinguishable phenotypes and mechanisms, identical molecular components of the RB-E2F-CDK network facilitate quiescence and senescence induction2, 3, 4. We hypothesize that quiescence and senescence are linked through the E2F-switching threshold: this is referred to as the lowest concentration of serum required to turn on E2F for S phase entry. This RB-E2F-CDK network switch is known to regulate quiescence depth; deep quiescent cells have a higher switching threshold as they require stronger and longer growth stimulation than shallower ones. Quiescence deepening likely exhibits reduced proliferative tendencies with an increased switching threshold and a transitional path to permanently arrested senescence; cellular dormancy is a continuum. Here we established a senescence inducing protocol in rat embryonic fibroblasts with the DNA damaging agent hydroxyurea (HU) and measured cell cycle reentry using 5-ethynyl-2´-deoxyuridine (EdU) bromodeoxyuridine (BrdU). We found that greater than 85% of senescence induced were irreversibly dormant 2 days after induction and 72% remained stably arrested 12 days after induction. Senescence induced cells expressed known biomarkers associated with senescence, namely increased cell size and beta-galactosidase (β-gal) activity, further characterizing them as senescent-like (S-L). We lowered the E2F-switching threshold in S-L cells by using lentiviruses to express cMyc and mutant cyclin-dependent kinase 4 (CDK4), known modulators of the RB-E2F-CDK network that activate E2F and thus promote cell cycle reentry. Quiescence and senescence are hypothesized to be connected by an increased E2F-switching threshold and a corresponding reduced proliferation capacity. We found that by reducing the E2F-switching threshold, a portion of S-L cells were converted to quiescence and proliferation