In this study we tested the idea that while causing senescence in proliferating cells, DNA damaging drugs and induced p53 will not cause senescence in quiescent cells. To this point we 1) induced quiescence prior to p53 induction and 2) used p53-inducing agents that could be washed out to observe whether treated cells would retain proliferative potential. Etoposide, which causes DNA damage, was used for normal cells and nutlin-3a, which induces p53 without DNA damage, was used for cancer cells. Both agents could be washed out to check the reversibility of arrest. We used these drugs at concentrations that caused senescence in proliferating cells. When applied to serum-starved and rapamycin-treated cells, p53-inducing drugs did not completely convert quiescence into senescence. Cells retained mostly quiescent morphology and some degree of proliferative potential, resuming proliferation in fresh (drug-free, serum-containing) medium.
Although not causing senescence, induction of p53 in quiescent cells ‘locked’ the cell cycle. In quiescence caused by serum starvation, the cell cycle is inactive (due to low levels of cyclins) but not blocked. In quiescent cells, induction of p53 blocks the cell cycle (in addition to its deactivation). Re-addition of serum to such blocked (locked) quiescent cells did not cause proliferation. Instead it caused senescence. This is in agreement with the notion that senescence is a form of growth, a continuation of growth, when proliferation is impossible [18
Thus, induction of p53 by different agents (including DNA damaging drugs) did not cause senescence in serum-starved and rapamycin-treated cells. It is less clear whether DNA damaging agents induced identical DNA damage in all conditions. One potential problem is that DNA damaging drugs may induce a lesser DNA damage in quiescent cells. However, first, we utilized etoposide, which was reported to induce damage in all phases of the cell cycle [40
]. Second, etoposide induced the same levels of p53 in proliferating, serum-starved and rapamycin-treated cells. (Note: Although p53 could be induced independently from DNA damage, this has not been described for etoposide. DNA damage response such as gamma-H2AX is also not absolutely reliable marker because it may occur in the absence of DNA damage in senescent cells [46
]). Therefore, direct measurement of DNA damage by comet assay is warranted. 0.5 μg/ml etoposide did not induce obvious comets both in control and serum-free conditions. 10 μg/ml etoposide induced comets in serum-starved cells (Supplemental Figure 1
). We conclude that, in agreement with literature data, etoposide induces DNA damage in serum-starved cells (some of which may still cycling at the moment of etoposide treatment) but more detailed measurements are needed for quantitative results. Third, simultaneous serum-withdrawal and addition of doxorubicin also suppressed senescence and this effect cannot be explained by cell cycle arrest caused by serum withdrawal. Fourth, DNA damaging drugs were even more cytotoxic in serum free-medium, indicating damage. Thus, it is not that etoposide is less cytotoxic in serum-free medium but rather that cells retain lean morphology and prolifertative potential (the ability to proliferate in fresh medium). At high concentrations, damaging agents and p53 can induce cell death rather than senescence in serum-starved cells. Yet, according to our preliminary data, if drugs are removed before death occurs, serum-restimulated survived cells can become senescent. TOR-independent latent senescence caused by high levels of DNA damage is an intriguing topic for further investigations.
In conclusion, quiescence is characterized by inactive mTOR both in cell culture [18
] and in the organism [48
]. The inability of p53 to cause senescence in quiescent cells has important physiological applications. Most cells of an adult organism are resting and there-fore induction of p53 and DNA damage cannot cause senescence. In contrast, stimulation of GF-sensing mTOR-centric pathways can. ‘Locked’ quiescent cells represent post-mitotic cells in the organism, including muscle cells, adipocytes and neurons. While not triggering proliferation of such ‘locked’ cells, stimulation with growth factors, hormones and nutrients may cause their senescence. Conversion of quiescence to senescence is a model of physiological senescence. Locked (non-senes-cent) cells undergo chronic over-stimulation and event-ually senesce. At least in some in vitro cellular models, conversion of quiescence to senescence (physiological senescence) can be suppressed by rapamycin.