We tested a hypothesis that activation of growth-promoting pathways is required for cellular senescence. In the presence of serum, induction of p21 caused senescence, characterized by betaGalactosidase staining, cell hypertrophy, increased levels of cyclin D1 and active TOR (target of rapamycin, also known as mTOR). Serum starvation and rapamycin inhibited TOR and prevented the expression of some senescent markers, despite high levels of p21 and cell cycle arrest. In the presence of serum, p21-arrested cells irreversibly lost proliferative potential. In contrast, when cells were arrested by p21 in the absence of serum, they retained the capacity to resume proliferation upon termination of p21 induction. In normal human cells such as WI38 fibroblasts and retinal pigment epithelial (RPE) cells, serum starvation caused quiescence, which was associated with low levels of cyclin D1, inactive TOR and slim-cell morphology. In contrast, cellular senescence with high levels of TOR activity was induced by doxorubicin (DOX), a DNA damaging agent, in the presence of serum. Inhibition of TOR partially prevented senescent phenotype caused by DOX. Thus growth stimulation coupled with cell cycle arrest leads to senescence, whereas quiescence (a condition with inactive TOR) prevents senescence.

The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) was initially identified and studied in the regulation of glycogen synthesis. GSK-3 functions in a wide range of cellular processes. Aberrant activity of GSK-3 has been implicated in many human pathologies including: bipolar depression, Alzheimer's disease, Parkinson's disease, cancer, non-insulin-dependent diabetes mellitus (NIDDM) and others. In some cases, suppression of GSK-3 activity by phosphorylation by Akt and other kinases has been associated with cancer progression. In these cases, GSK-3 has tumor suppressor functions. In other cases, GSK-3 has been associated with tumor progression by stabilizing components of the beta-catenin complex. In these situations, GSK-3 has oncogenic properties. While many inhibitors to GSK-3 have been developed, their use remains controversial because of the ambiguous role of GSK-3 in cancer development. In this review, we will focus on the diverse roles that GSK-3 plays in various human cancers, in particular in solid tumors. Recently, GSK-3 has also been implicated in the generation of cancer stem cells in various cell types. We will also discuss how this pivotal kinase interacts with multiple signaling pathways such as: PI3K/PTEN/Akt/mTORC1, Ras/Raf/MEK/ERK, Wnt/beta-catenin, Hedgehog, Notch and others.

When the cell cycle is arrested but cellular growth is not, then cells senesce, permanently losing proliferative potential. Here we demonstrated that the duration of cell cycle arrest determines a progressive loss of proliferative capacity. In human and rodent cell lines, rapamycin (an inhibitor of mTOR) dramatically decelerated loss of proliferative potential caused by ectopic p21, p16 and sodium butyrate-induced p21. Thus, when the cell cycle was arrested by these factors in the presence of rapamycin, cells retained the capacity to resume proliferation, once p21, p16 or sodium butyrate were removed. While rapamycin prevented the permanent loss of proliferative potential in arrested cells, it did not force the arrested cells into proliferation. During cell cycle arrest, rapamycin transformed the irreversible arrest into a reversible condition. Our data demonstrate that senescence can be pharmacologically suppressed.

The tumor suppressor p53 is a canonical inducer of cellular senescence (irreversible loss of proliferative potential and senescent morphology). p53 can also cause reversible arrest without senescent morphology, which has usually been interpreted as failure of p53 to induce senescence. Here we demonstrate that p53-induced quiescence actually results from suppression of senescence by p53. In previous studies, suppression of senescence by p53 was masked by p53-induced cell cycle arrest. Here, we separated these two activities by inducing senescence through overexpression of p21 and then testing the effect of p53 on senescence. We found that in p21-arrested cells, p53 converted senescence into quiescence. Suppression of senescence by p53 required its transactivation function. Like rapamycin, which is known to suppress senescence, p53 inhibited the mTOR pathway. We suggest that, while inducing cell cycle arrest, p53 may simultaneously suppress the senescence program, thus causing quiescence and that suppression of senescence and induction of cell cycle arrest are distinct functions of p53. Thus, in spite of its ability to induce cell cycle arrest, p53 can act as a suppressor of cellular senescence.nduction of p53 can cause apoptosis, reversible cell cycle arrest, and cellular senescence (1-5). In contrast to reversible cell cycle arrest (quiescence), cellular senescence is defined by irreversible loss of proliferative potential, acquisition of characteristic morphology (large, flattened cells), and expression of specific biomarkers (e.g., senescence-associated β-galactosidase, SA-β-Gal) (6). Since p53 appeared to induce senescence in some situations, observations of p53-induced quiescence have usually been interpreted as failure of p53 to activate the senescence program, which remains poorly understood. We recently reported that in two cell lines in which ectopic expression of p21 caused senescence, activation of endogenous p21 by endogenous p53 caused quiescence (7). The simplest conventional explanation for this result is that p53 failed to activate p21 to the degree required for induction of senescence, although it was sufficient for induction of quiescence. However, an alternative possibility is that p53 acts as a suppressor of the senescence program. This model leads to the testable prediction that induction of p53 would suppress p21-mediated senescence and convert it into quiescence. Here we demonstrate that this model is indeed correct, which indicates that, despite its ability to induce cell cycle arrest, p53 is a suppressor, not an inducer, of cellular senescence. Retrospectively, this result is not completely unexpected. First, it is known that p53 inhibits the mTOR (mammalian target of rapamycin) pathway (8-12). Second, it is known that inhibition of mTOR by rapamycin converts senescence into quiescence (13-15). In turn, this predicts that p53, like rapamycin, may suppress senescence. Here we confirmed this prediction. ResultsThe p53 Activator Nutlin-3a Suppresses p21-Induced Senescence. As previously show...

Transient induction of p53 can cause reversible quiescence and irreversible senescence. Using nutlin-3a (a small molecule that activates p53 without causing DNA damage), we have previously identified cell lines in which nutlin-3a caused quiescence. Importantly, nutlin-3a caused quiescence by actively suppressing the senescence program (while still causing cell cycle arrest). Noteworthy, in these cells nutlin-3a inhibited the mTOR (mammalian Target of Rapamycin) pathway, which is known to be involved in the senescence program. Here we showed that shRNA-mediated knockdown of TSC2, a negative regulator of mTOR, partially converted quiescence into senescence in these nutlin-arrested cells. In accord, in melanoma cell lines and mouse embryo fibroblasts, which easily undergo senescence in response to p53 activation, nutlin-3a failed to inhibit mTOR. In these senescence-prone cells, the mTOR inhibitor rapamycin converted nutlin-3a-induced senescence into quiescence. We conclude that status of the mTOR pathway can determine, at least in part, the choice between senescence and quiescence in p53-arrested cells.