Wild-type p53 is normally expressed at low levels and inactive due to the action of MDM2, an E3 ubiquitin ligase that binds p53 and promotes its degradation [¹,²]. However, p53 is stabilized in response to various stresses, such as DNA damage or inappropriate oncogene signaling, that might otherwise predispose a normal cell toward tumorigenesis [³]. The stress-induced stabilization of p53 results from disruption of p53-MDM2 binding. The majority of stabilized p53 accumulates in the nucleus where it functions as a transcription factor, activating expression of genes that induce either apoptosis or cell cycle arrests that can be either transient (quiescence) or permanent (senescence). Thus, p53 eliminates cells with potentially cancer-promoting lesions by inhibiting their growth or causing them to die. mTOR is a cytoplasmic kinase whose activity is often elevated in cancer [⁴]. mTOR converts signals from activated growth factor receptors into downstream events that promote cell proliferation and survival. Previous studies have demonstrated cross-talk between the p53 and mTOR signaling pathways. For example, p53 can activate expression of several genes, including TSC2, PTEN, IGF-BP3, and others, whose protein products can directly or indirectly inhibit mTOR activity [⁵]. These observations make sense given that p53 is a tumor suppressor and mTOR has more an oncogenic role in promoting cancer cell survival and proliferation. However, more recent studies indicate that the outcome of mTOR signaling can be context-dependent. Thus, while mTOR signaling promotes proliferation and survival under normal conditions, mTOR signaling can promote senescence under conditions in which the cell cycle is blocked [⁶,⁷]. These observations support mTOR as a hub for receipt of multiple inputs that ultimately determine cell fate. When conditions are favorable mTOR activation promotes proliferation and survival, however, in the context of conflicting signals (e.g. growth factor signaling vs. cell cycle arrest), the effect of mTOR activation is permanent cell cycle exit (senescence). P21 is a cyclin-cdk inhibitor, transcriptional target of p53, and potent inducer of senescence [⁸,⁹]. Blagosklonny and colleagues noted that in some cases p53 induction did not induce senescence while ectopic expression of p21 did [¹⁰]. This led to them to question the role of p53 in the senescence program, and whether p53 may actively suppress senescence. To address this, they used a cell line in which p21 was expressed from an inducible (IPTG-driven) promoter [¹¹]. In this cell line, transient p21 expression induced by IPTG caused the cells to undergo a senescent arrest characterized by flat-cell phenotype, expression of senescence-associated beta galactosidase, and a complete loss of proliferative potential after IPTG removal [¹²]. To test the effect of p53 on this senescent arrest, the authors first induced p21 by IPTG, and then induced p53 expression in the same cells by addition of Nutlin-3a, a small molecule MDM2 antagonist and potent p53 stabilizer. Remarkably, cells in which p53 was induced by Nutlin-3a were able to resume cycling and fully recover after IPTG removal. These results indicated that p53 expression converted the senescence response in these cells to quiescence. The suppression of senescence they observed was associated with p53-dependent inhibition of mTOR activity [¹²]. In the current issue of Aging, Korotchkina et al. [¹³] demonstrate that shRNA-mediated knockdown of TSC2, a negative regulator of mTOR and p53 target gene [⁵], imposed senescence in these Nutlin-3a treated cells. The results support a model in which p53 can suppress senescence through upregulation of TSC2 and inhibition of mTOR.

Our lab has also examined responses to p53 activation by Nutlin-3a. Our original report demonstrated that Nutlin-3a promoted a non-permanent, tetraploid G1-arrest in two different p53 wild-type cancer cell lines (HCT116 and U2OS). Both cell lines underwent endoreduplication after Nutlin-3a removal, giving rise to tetraploid clones resistant to therapy-induced apoptosis [¹⁴]. More recently, we demonstrated that Nutlin-3a could promote a tetraploid G1-arrest in multiple p53 wild-type cell lines [¹⁵]. However, some cell lines underwent endoreduplication to relatively high extents after Nutlin-3a removal while other cell lines did not. The resistance to endoreduplication observed in some cell lines was associated with a prolonged 4N arrest after Nutlin-3a removal. Knockdown of either p53 or p21 immediately after Nutlin-3a removal could drive endoreduplication in otherwise resistant 4N cells. Finally, 4N arrested cells had diminished p53 expression, but retained high levels of p21. Moreover, these cells expressed senescence-associated beta galactosidase, had a flattened cell phenotype, and underwent a permanent proliferation block (senescence) after Nutlin-3a removal. These findings demonstrated that transient Nultin-3a treatment can promote senescence in 4N cells of certain cell lines associated with persistent p21 expression and resistance to endoreduplication. In terms of a model, the results suggest p53 is required to initiate Nutlin-3a-induced senescence by increasing p21 expression, but is not required to maintain senescence. In light of the findings by Blagosklonny and colleagues, we would speculate that the diminished level of p53 restores mTOR activity in these 4N cells, and that mTOR activity, as well as elevated p21, are required for their senescent arrest.