Reversal of human cellular senescence: roles of the p53 and p16 pathways

Abstract

Telomere erosion and subsequent dysfunction limits the proliferation of normal human cells by a process termed replicative senescence. Replicative senescence is thought to suppress tumorigenesis by establishing an essentially irreversible growth arrest that requires activities of the p53 and pRB tumor suppressor proteins. We show that, depending on expression of the pRB regulator p16, replicative senescence is not necessarily irreversible. We used lentiviruses to express specific viral and cellular proteins in senescent human fibroblasts and mammary epithelial cells. Expression of telomerase did not reverse the senescence arrest. However, cells with low levels of p16 at senescence resumed robust growth upon p53 inactivation, and limited growth upon expression of oncogenic RAS. In contrast, cells with high levels of p16 at senescence failed to proliferate upon p53 inactivation or RAS expression, although they re-entered the cell cycle without growth after pRB inactivation. Our results indicate that the senescence response to telomere dysfunction is reversible and is maintained primarily by p53. However, p16 provides a dominant second barrier to the unlimited growth of human cells.

Fig. 1. hTERT does not reverse the senescent phenotype. (A) Lenti-hTERT confers telomerase activity. Senescent (S) or pre-senescent (P) WI-38 (WI) cells were infected with lenti-hTERT, lenti–GFP or pBABE-hTERT, and telomerase activity was measured using the TRAP assay, as described in Materials and methods. ‘+’ is a positive TRAP control, and ‘–’ and ‘Heat inactivated’ are negative controls. (B) Lenti-hTERT alters telomere length in pre-senescent, but not senescent cells. Terminal restriction fragment (TRF) lengths in P- or S-WI cells infected with lenti–GFP (GFP) or lenti-hTERT (hTERT) were determined as described in Materials and methods. (C) Lenti-hTERT does not alter senescent morphology. S-WI cells were infected with lenti–GFP (+GFP) or lenti-hTERT (+hTERT), and viewed and photographed 7 days later.

Fig. 2. p53 inactivation reverses senescence of BJ, but not WI-38 fibroblasts. (A) S-WI cells synthesize DNA, but do not proliferate. S-WI cells were infected with lentiviruses expressing GFP, hTERT, GSE, LgT, LgTK1 and CDK4m as indicated; 72 h later, DNA synthesis was determined by % LN, and percentage growth monitored, as described in the text. (B) S-BJ cells synthesize DNA and proliferate. S-BJ cells were infected and monitored, as described in (A). (C) Morphology of control and rescued S-BJ cells. S-BJ cells infected with GFP or GSE-expressing lentivirus were photographed 6 days later. (D) Lifespan assays. S-BJ cells were infected with lentiviruses expressing the indicated proteins, serially passaged, and cell number determined at each passage, as described in Materials and methods.

Fig. 3. Senescence reversal correlates inversely with p16 expression. (A) p16 expression. p16 and actin (control) protein levels were assessed in pre-senescent (P) and senescent (S) BJ and WI-38 (WI) cells by western blotting (WB). The labeling index of the cultures is shown below the blot (% LN). (B) p16 immunostaining. Pre-senescent (P) and senescent (S) BJ and WI-38 cells were immunostained for p16, and nuclei stained with DAPI, as described in Materials and methods. HeLa cells served as a positive control. (C) p16 and p21 levels after rescue from senescence by GSE-22. S-BJ cells were infected with control (GFP) or GSE-22-expressing (rescued GSE) lentivirus. Rescued cells were harvested while proliferating (16 PDs) or after proliferation ceased (23 PDs), and analyzed for p21, p16 and actin (control) by western blotting. (D) Senescence reversal in HMECs. Post-selection HMECs were infected with lentiviruses expressing the indicated proteins, and monitored for growth, as described in Materials and methods. Shown are cells 72 h following infection.

Fig. 4. p16 suppression allows senescence reversal in WI-38 cells. (A) shRNA-p16 suppresses p16 expression in WI-38 cells. P-WI cells were mock infected (–) or infected with shRNA-p16 expressing pMSCV retroviruses, and passaged until replicatively senescent (S-WI; % LN <1%). p16, p21 and actin (control) levels were assessed by western blotting (WB). (B) % LN. P-WI were mock infected (–) or infected with shRNA-p16 expressing retrovirus (+) and cultured until senescent (S-WI). Cells were then monitored for ability to synthesize DNA (% LN) upon p53 inactivation by subsequent infection with lenti-GSE-22 (GSE). Parallel cultures were monitored for proliferation (% growth), as described in Materials and methods. (C) Ectopic p16 expression. P-WI cells were mock infected (–) or infected (+) with lenti-p16; p16 and actin (control) were assessed by western blotting (WB). At the exposure shown, endogenous p16 in mock-infected P-WI cells is undetectable.

Fig. 5. Sequential inactivation of p16/Rb determines senescence reversibility. (A) p16 followed by LgT. P-WI cells were infected with lenti-p16, and then mock infected (–) or infected with lenti LgT (+). Cells were monitored for ability to synthesize DNA (% LN) and proliferate (% growth), as described in Materials and methods. (B) LgT followed by p16. P-WI cells were infected with lenti-LgT, followed by mock infection (–) or lenti-p16 infection (+). The infected cells were monitored for DNA synthesis (% LN) and proliferation (% growth). (C) Silencing p16 after senescence. S-WI cells were infected with lenti-shRNA-p16, and then mock infected (–) or infected with lenti-GSE-22 (+). The infected cells were monitored for DNA synthesis (% LN) and proliferation (% growth).

Fig. 6. Oncogenic Ras partially reverses p16-independent senescence. (A) S-WI cells do not synthesize DNA in response to oncogenic Ras. S-WI cells were infected with the indicated lentiviruses, and 72 h later assessed for ability to synthesize DNA (% LN) and proliferate (% growth), as described in Materials and methods. (B) S-BJ cells synthesize DNA and undergo limited proliferation in response to oncogenic Ras. S-BJ cells were infected with the indicated lentiviruses, and assessed for % LN and % growth. The asterisk indicates that the cells underwent limited proliferation, amounting to 3 PDs or less. (C) Mitogenic effects of Ras are concentration- and p16-dependent. S-BJ cells were infected with 1× (low Ras) or 3× (high Ras) lenti-Ras virus concentrations (determined by p24 levels, as described in Materials and methods) and % LN was measured. Where indicated, S-BJ cells were infected with lenti-p16 5 days prior to subsequent infection with high lenti-Ras. (D) Ras immunostaining. S-BJ cells were mock infected or infected with lenti-Ras at 1× or 3× virus concentrations, and immunostained for Ras. Nuclei were identified by DAPI staining.

Fig. 7. Pathways leading to reversible and essentially irreversible senescence growth arrests in human cells. Proliferating cells (Presenescent) arrest growth with a senescent phenotype in response to telomere erosion, which is p53 dependent, or a combination of telomere erosion and an as yet unidentified stimulus that induces p16. The p53-dependent arrest increases p21 expression, and is reversed by p53 inactivation or oncogenic Ras. p53 inactivation results in extensive proliferation (growth) culminating in crisis, whereas Ras causes limited proliferation. Cells that senesce with high p16 can be stimulated to synthesize DNA (S-phase) upon inactivation of p53 and pRb, or pRb inactivation plus oncogenic Ras, but do not proliferate (no growth).