Senescence from G2 arrest, revisited

Abstract

Senescence was classically defined as an irreversible cell cycle arrest in G1 phase (G1 exit) triggered by eroded telomeres in aged primary cells. The molecular basis of this G1 arrest is thought to be due to a DNA damage response, resulting in accumulation of the cyclin dependent kinase (Cdk) inhibitors p21 and p16 that block the inactivating phosphorylation of the retinoblastoma tumor suppressor pRb, thereby preventing DNA replication. More than a decade ago, several studies showed that p21 also mediates permanent DNA damage-induced cell cycle arrest in G2 (G2 exit) by inhibiting mitotic Cdk complexes and pRb phosphorylation. The idea that the senescence program can also be launched after G2 arrest has gained support from several recent publications, including evidence for its existence in vivo.

Keywords: Cdk inhibitors; DNA damage; G2/M checkpoint; p53; pRb; senescence; telomeres.

Figure 1.

Molecular pathways involved in irreversible G1 arrest proceeding senescence. Dysfunctional telomeres, hyperactive oncogenes and various genotoxic agents stimulate the ATM/ATR-mediated DNA damage response (DDR) pathway. The ATM/ATR kinases induce the early response by activating Chk1/2 checkpoint kinases that, in turn, by inhibiting Cdc25 phosphatases, prevent activation of cyclin E1-Cdk2, which is a key regulator of G1/S transition. ATM also phosphorylates the p53 tumor suppressor that, by inducing the Cdk inhibitor p21^Waf1/Cip1 (p21), plays a central role in the G1 exit program (late response). In addition to cyclin E1-Cdk2 (cE1-K2) complexes, p21 also inhibits cyclin D1-Cdk4/6 (cD1-K4/6) complexes that phosphorylate and inactivate pRb family pocket proteins. In turn, active pRb inhibits the E2F1-dependent expression of genes controlling G1/S progression, thus irreversibly blocking the cell cycle entry. Senescence is also associated with p16^Ink4A (p16) upregulation, but the pathways leading to its induction are not entirely elucidated. This Cdk inhibitor specifically targets Cdk4/6 and prevents their association with D-type cyclins. While p16 does not intervene in G1 arrest (p16 is upregulated after induction of p21 and G1 arrest), it plays a key role in senescence maintenance.

Figure 2.

DNA damage response pathways involved in the G2 exit program leading to senescence. Pro-senescence stimuli activate the ATM/ATR DNA damage-signaling pathway, leading to stable G2 arrest that entails mitotic bypass (see also Fig. 3) and permanent cell cycle arrest in a tetraploid G1 phase. The early response involves Chk1/2-mediated inhibition of Cdc25 phosphatases, which promote mitosis by activating cyclin B1-Cdk1 complexes (cB1/K1). In p53-deficient cells, this pathway transiently blocks G2/M progression, but the p53/p21 pathway is required to stabilize G2 arrest. In addition to inhibiting cyclin B1-Cdk1 complexes, p21 mediates premature activation of APC/C-Cdh1, leading to degradation of cyclin B1 and other mitotic regulators. Like in G1 arrest, p21 also activates pRb by blocking Cdk-mediated pRb phosphorylation. Active pRb inhibits the expression of genes that control G2/M progression, leading to irreversible G2 arrest of the cell cycle. It is proposed that p53 might induce senescence independently of p21 through mechanisms that are not fully clear. Like in G1 arrest, p16 stabilizes senescence in G2 presumably by targeting pRb kinases. However, the pathways involved have not been elucidated.

Figure 3.

Mitotic bypass associated with p21 induction triggers senescence in tetraploid G1 cells. In p53-proficient cells, DNA damage-induced G2 arrest leads to p21-dependent nuclear sequestration of cyclin B1-Cdk1 (cB1) complexes, thus locking them in the inactive state (cf. Fig. 2). Subsequent APC/C-Cdh1-mediated degradation of cyclin B1 and other mitotic regulators precedes mitotic bypass (skip), leading to irreversible arrest (senescence) in a tetraploid (4N) G1 phase, characterized by accumulation of G1 cyclins (cyclin D1, cD1). In p53-deficient cells, in the case of persistent telomere dysfunction, DNA damage induces strong and sustained Chk1/2 activation leading to stable G2 arrest that, via mitotic bypass, leads to endoreplication, resulting in tetraploidy. In the absence of sustained Chk1/2 activation, p53/p21-deficient cells exposed to genotoxic agents arrest only transiently in G2 and then progress into mitosis and eventually die. Similarly, in aging p53/pRb-deficient cells, extensive telomere erosion induces crisis with massive cell death and tetraploidization through endoreplication or mitotic failure.