Quiescence-Origin Senescence Induced by Oxidative Stress

Abstract

Cellular quiescence is defined as a state in which cells have entered the “G0” phase of the cell cycle and are no longer actively dividing due to the lack of growth signals or non-permissive growth conditions1. However, quiescent cells can be restimulated back into the cell cycle with the appropriate growth signals and actively start dividing again. Senescence is also part of the “G0” phase of the cell cycle, however, cells in senescence are not able to be restimulated back into the cell cycle and are in a permanently arrested state of the cell cycle. Quiescence and senescence contain different markers and phenotypes; however, it is hypothesized that the governance of their entrance into these states is facilitated by the RB-E2F pathway2,3. Our hypothesis is that quiescence and senescence is linked to the RB-E2F pathway and that the E2F switching threshold is what governs the cell’s ability to turn E2F on for S phase entry which is determined by the lowest serum concentration4. This network regulates quiescence depth where deeply quiescent cells require a higher serum concentration for a longer period in comparison to shallow quiescent cells. Deeply quiescent cells are characterized by a reduced proliferative potential and an increased E2F switching threshold. However, quiescence is a heterogenous state in which a population of cells can exist in different levels of quiescence, where some are shallow, and some are deeply quiescent4. We established a senescence inducing protocol from quiescent Retinal Pigment Epithelial (RPE WT) cells using Nitric Oxide (NO) and tert-Butyl-hydroperoxide (tBHP) which create oxidative stress in the cell. We measured cell cycle reentry over a 10-day period using 5-ethynyl-2’-deoxyuridine (EdU)5. We found that 96% of cells treated with NO for 7 days remained senescent-like after 10 days of stimulation and 80% of cells treated with tBHP for 3 hours and 74% of cells treated with tBHP for 3.5 hours remained senescent-like after 10 days of stimulation. Proliferation-origin senescence express known biomarkers such as increased cell size and Beta-Galactosidase (B-gal) activity6, however, with quiescent-origin senescence, alternative biomarkers may be prevalent.