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

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

Proceedings of the National Academy of Sciences of the United States of America (2014-06-04)
Olga V Leontieva, Zoya N Demidenko, Mikhail V Blagosklonny
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Etoposide for system suitability, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Rapamycin, Ready Made Solution, 2.5 mg/mL in DMSO (2.74 mM), from Streptomyces hygroscopicus
Supelco
Rapamycin, VETRANAL®, analytical standard
Etoposide, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Etoposide, synthetic, 95.0-105.0%, powder