MEK drives cyclin D1 hyperelevation during geroconversion

Abstract

When the cell cycle becomes arrested, MTOR (mechanistic Target of Rapamycin) converts reversible arrest into senescence (geroconversion). Hyperexpression of cyclin D1 is a universal marker of senescence along with hypertrophy, beta-Gal staining and loss of replicative/regenerative potential (RP), namely, the ability to restart proliferation when the cell cycle is released. Inhibition of MTOR decelerates geroconversion, although only partially decreases cyclin D1. Here we show that in p21-and p16-induced senescence, inhibitors of mitogen-activated/extracellular signal-regulated kinase (MEK) (U0126, PD184352 and siRNA) completely prevented cyclin D1 accumulation, making it undetectable. We also used MEL10 cells in which MEK inhibitors do not inhibit MTOR. In such cells, U0126 by itself induced senescence that was remarkably cyclin D1 negative. In contrast, inhibition of cyclin-dependent kinase (CDK) 4/6 by PD0332991 caused cyclin D1-positive senescence in MEL10 cells. Both types of senescence were suppressed by rapamycin, converting it into reversible arrest. We confirmed that the inhibitor of CDK4/6 caused cyclin D1 positive senescence in normal RPE cells, whereas U0126 prevented cyclin D1 expression. Elimination of cyclin D1 by siRNA did not prevent other markers of senescence that are consistent with the lack of its effect on MTOR. Our data confirmed that a mere inhibition of the cell cycle was sufficient to cause senescence, providing MTOR was active, and inhibition of MEK partially inhibited MTOR in a cell-type-dependent manner. Second, hallmarks of senescence may be dissociated, and hyperelevated cyclin D1, a marker of hyperactivation of senescent cells, did not necessarily determine other markers of senescence. Third, inhibition of MEK was sufficient to eliminate cyclin D1, regardless of MTOR. In proliferating cells, MTOR (mechanistic Target of Rapamycin) stimulates cellular mass growth, whereas cyclins D and E initiate S-phase transition.1-4 Withdrawal of growth factors causes reversible cell-cycle arrest (quiescence) with low levels of cyclins and MTOR activity, reversible by re-addition of growth factors. In contrast, cell-cycle arrest in the presence of growth stimuli, which activate MTOR, leads to cellular senescence. 5,6 In other words, when the cell cycle is blocked, while growth-promoting pathways such as MTOR remain active, cells continue to grow in size and undergo senescence. A process of conversion from normal reversible arrest into senescence was named as gerogenic conversion (geroconversion).7 Cellular senescence is characterized by large flat morphology (hypertrophy), beta-Gal staining, very high levels of cyclins D1 and E, senescence-associated secretory phenotype and loss of replicative/regenerative potential (RP).7-12 Loss of RP means that cells cannot proliferate even when cell-cycle block is removed and cells re-enter the cell cycle.13,14 Rapamycin decreases hypertrophy and loss of RP, in a dose-dependent manner proportional to S6 dephosphorylation (a marker of MTORC1 activity). 15Other ...

The CDK4/CDK6 inhibitor PD0332991 paradoxically stabilizes activated cyclin D3-CDK4/6 complexes

“…1B), as observed by others. 41,42,51,52 Also interestingly, PD0332991 treatment prevented the disappearance of p21 but not of p27 (Fig. 1B).…”

“…Nevertheless, other mechanisms might concur to cyclin D1 accumulation in PD0332991-arrested cells. As an early marker of conversion to senescence (geroconversion), MEK-dependent hyperinduction of cyclin D1 in response to PD0332991 52 was also observed in the absence of a large increase of p21. 42 Arrest of PD0332991 treatment induces DNA synthesis and pRb phosphorylations in serumdeprived cells…”

Nuclear accumulation of cyclin D1 following long-term fractionated exposures to low-dose ionizing radiation in normal human diploid cells

“…It has also been shown that senescent human fibroblasts express high levels of cyclin D1, [23][24][25][26] and cell cycle deregulation often induces cellular senescence. 27 Thus, cyclin D1 overexpression is considered a marker of cellular senescence. Importantly, senescent cells are negative for PCNA, because cell growth has been arrested.…”

Contact inhibition and high cell density deactivate the mammalian target of rapamycin pathway, thus suppressing the senescence program

“…Conversely, cells undergo senescence, when their cell cycle is arrested in the presence of growth stimulation (1)(2)(3)(4)(5). Recently, it was demonstrated that the difference between reversible quiescence and irreversible senescence is determined by an active mammalian target of rapamycin (mTOR) pathway in senescent cells (6)(7)(8)(9)(10)(11). When cell cycle is arrested and mTOR is stimulated by serum growth factors or oncoproteins such as Ras, the arrested cells undergo senescence (1,4,12,13).…”

“…The conversion from reversible cell cycle arrest to senescence is named gerogenic conversion (or geroconversion) (12). Conditions that inhibit mTOR (6,14,15) also inhibit geroconversion while causing or maintaining cell cycle arrest (6,7,10).…”