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

During cell cycle arrest caused by contact inhibition (CI), cells do not undergo senescence, thus resuming proliferation after replating. The mechanism of senescence avoidance during CI is unknown. Recently, it was demonstrated that the senescence program, namely conversion from cell cycle arrest to senescence (i.e., geroconversion), requires mammalian target of rapamycin (mTOR). Geroconversion can be suppressed by serum starvation, rapamycin, and hypoxia, which all inhibit mTOR. Here we demonstrate that CI, as evidenced by p27 induction in normal cells, was associated with inhibition of the mTOR pathway. Furthermore, CI antagonized senescence caused by CDK inhibitors. Stimulation of mTOR in contact-inhibited cells favored senescence. In cancer cells lacking p27 induction and CI, mTOR was still inhibited in confluent culture as a result of conditioning of the medium. This inhibition of mTOR suppressed p21-induced senescence. Also, trapping of malignant cells among contact-inhibited normal cells antagonized p21-induced senescence. Thus, we identified two nonmutually exclusive mechanisms of mTOR inhibition in high cell density: (i) CI associated with p27 induction in normal cells and (ii) conditioning of the medium, especially in cancer cells. Both mechanisms can coincide in various proportions in various cells. Our work explains why CI is reversible and, most importantly, why cells avoid senescence in vivo, given that cells are contact-inhibited in the organism.When cells are deprived of serum growth factors, they cease proliferation and rest in a reversible state known as quiescence. Conversely, cells undergo senescence, when their cell cycle is arrested in the presence of growth stimulation (1–5). 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 (6–11). 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).Of note, in cell culture, quiescence and senescence are observed at low or regular cell density. There is a third type of cell cycle arrest. When normal cells reach high density (HD), they stop proliferation i.e., contact inhibition (CI)] and can stay arrested for weeks. However, when the culture is split and replated, the cells restart proliferation. This condition resembles quiescence, even though it occurs in the presence of growth stimulation by serum. Perhaps CI is the most important and physiological type of cell cycle arrest. First, most cells in the organism are contact inhibited. Like in confluent cell culture, wounding causes cells to restart proliferation and to fill the wound. Second, the most noticeable difference between normal and cancer cells in culture is the lack of CI in cancer cells (16, 17). Cancer cells continue to proliferate, acidifying culture medium and damaging themselves (18).Whether CI is reversible and why it is reversible remain unknown. Irreversible senescence is characterized by active mTOR pathway, high metabolism, and large flat cell morphology (8, 19–21). In contrast, contact-inhibited cells are characterized by a small vertical morphology and low protein synthesis and metabolism. We speculated that mTOR might be inhibited in high cell density. This would explain the peculiarity of CI. This further would predict that CDK inhibitors, which cause cell senescence at low and regular cell density, would not cause it at HD. Here we tested this hypothesis.