After 4 d, cells started to form colonies (Fig.?3, encircled), suggesting either successful replication or conglomeration or both. decreased replicative stress. When p21 was switched off, cells successfully progressed through both S phase and mitosis. Also, senescent mouse embryonic fibroblasts (MEFs) overexpressed cyclin D1. After release HPGDS inhibitor 2 from cell cycle arrest, senescent MEFs joined S phase but could not undergo mitosis and did not proliferate. We conclude that cellular senescence is characterized by futile hyper-mitogenic drive associated with mTOR-dependent mitotic incompetence. strong class=”kwd-title” Keywords: MTOR, rapamycin, aging, cyclins, cell cycle, regenerative/proliferative potential Introduction In cell culture, senescence is characterized by cellular hypertrophy, SA–Gal staining, hyper-secretory phenotype and permanent loss HPGDS inhibitor 2 of regenerative or replicative potential (RP), meaning that cells cannot restart proliferation after release from cell cycle arrest.1-3 These hallmarks of senescence are promoted by growth-promoting pathways such as mTOR (target of rapamycin), when the cell cycle is usually arrested.3 For example, while arresting cell cycle, p21 and p16 do not inhibit mTOR, which, in turn, converts p21/p16-induced arrest into irreversible senescence.4-6 Thus, active growth-promoting pathways in resting cells drive a senescent program, a process named gerogenic conversion or geroconversion.3,6 Rapamycin and other inhibitors of mTOR as well as serum starvation decelerate geroconversion, decreasing cellular hypertrophy and preventing loss of regenerative/proliferative potential (RP).4-9 Remarkably, rapamycin slows down aging in Rabbit polyclonal to TDT mice10-15 and prevents age-related diseases in animals,16-23 suggesting HPGDS inhibitor 2 a common basis in cellular senescence and organismal aging. Yet, organismal aging is associated not only with decreased regeneration, but also with hyper-proliferation, such as hyperplasia, fibrosis, prostate enlargement, atherosclerotic plaques, benign tumors and cancer. Inappropriate re-entry into the cell cycle is involved in many age-related pathologies. This cannot be easily explained by such a hallmark of cellular senescence as loss of regenerative/proliferative potential (RP). Furthermore, it was noticed previously that senescent cells express high levels of cyclins D1 and E.24-29 Here we investigated how these markers of proliferation (cyclins) can be associated with the loss of RP. The second question we address here is how inhibitors of mTOR affect the ability of cells to re-start proliferation after their release from p21-induced cell cycle arrest. Is it initiation of the cell cycle, or its completion affected? We also address the question of how nutlin-3, a Mdm-2 inhibitor and p53 inducer, preserves RP in HT1080-p21C9 cells (HT-p21 cells). In HT-p21 cells, nutlin-3 inhibits mTOR and, like rapamycin, suppresses geroconversion during p21-induced arrest, maintaining quiescence and preserving RP.7,8,30 Using time-lapse video microscopy, we demonstrate that preservation of RP by both rapamycin and nutlin-3 is due to preservation of mitotic competence, an ability to undergo mitosis. Results Induction of cyclins D1 and E in senescent cells First, we used a well-studied model of cellular senescence: HT-p21 cells with IPTG-inducible p21.31,32 In these cells, IPTG induces p21 and irreversible senescence, whereas nutlin-3 induces p53 and reversible arrest.7,8 Unlike nutlin-3a, IPTG strongly induced cyclin D1 and cyclin E (Fig.?1A). Cyclin E levels continued to rise from day 1 to day 3 in IPTG-treated cells (Fig.?1A). We conclude that elevated levels of cyclins D1 and E were associated with senescence but not with reversible arrest. Open in a separate window Figure?1. Immunoblot analysis of cyclins D1 and E in IPTG-induced senescence in HT-p21C9 and HT-p16 cells. (A) HT-p21C9 cells were treated with 500 nM rapamycin (R) or10 uM nutlin-3 (N) in the presence or absence of IPTG (+). Cells were lysed on day 1 and 3. (B) HT-p21C9 cells were treated with IPTG and co-treated with (+) or without (-) rapamycin (Rapa) for 3 d. On day 3, cells were either washed (W) or cultured in fresh medium with IPTG (F) plus/minus rapamycin (+ -) and lysed the next day (day 4). (C) HT-p16 cells were treated with or without IPTG (+/?) and with or without rapamycin (Rapa +/?) for 1 d before lysis. Deceleration of cyclin induction by nutlin-3a and rapamycin In IPTG-treated cells, rapamycin and nutlin-3 decreased levels of cyclin D1 and cyclin E on day 1 and also slightly decreased levels of cyclin E on day 3 (Fig.?1A). This parallels deceleration of geroconversion by inhibitors of mTOR. In fact, rapamycin delayed the appearance of hallmarks of senescence in IPTG-treated HT-p21.