Differential Roles for Cyclin-Dependent Kinase Inhibitors p21 and p16 in the Mechanisms of Senescence and Differentiation in Human Fibroblasts

The irreversible G(1) arrest in senescent human diploid fibroblasts is probably caused by inactivation of the G(1) cyclin–cyclin-dependent kinase (Cdk) complexes responsible for phosphorylation of the retinoblastoma protein (pRb). We show that the Cdk inhibitor p21(Sdi1,Cip1,Waf1), which accumulates progressively in aging cells, binds to and inactivates all cyclin E-Cdk2 complexes in senescent cells, whereas in young cells only p21-free Cdk2 complexes are active. Furthermore, the senescent-cell-cycle arrest occurs prior to the accumulation of the Cdk4-Cdk6 inhibitor p16(Ink4a), suggesting that p21 may be sufficient for this event. Accordingly, cyclin D1-associated phosphorylation of pRb at Ser-780 is lacking even in newly senescent fibroblasts that have a low amount of p16. Instead, the cyclin D1-Cdk4 and cyclin D1-Cdk6 complexes in these cells are associated with an increased amount of p21, suggesting that p21 may be responsible for inactivation of both cyclin E- and cyclin D1-associated kinase activity at the early stage of senescence. Moreover, even in the late stage of senescence when p16 is high, cyclin D1-Cdk4 complexes are persistent, albeit reduced by ≤50% compared to young cells. We also provide new evidence that p21 may play a role in inactivation of the DNA replication factor proliferating cell nuclear antigen during early senescence. Finally, because p16 accumulates in parallel with the increases in senescence-associated β-Gal activity and cell volume that characterize the senescent phenotype, we suggest that p16 upregulation may be part of a differentiation program that is turned on in senescent cells. Since p21 decreases after senescence is achieved, this upregulation of p16 may be essential for maintenance of the senescent-cell-cycle arrest

Nuclear Accumulation of p21(Cip1) at the Onset of Mitosis: a Role at the G(2)/M-Phase Transition

Cell cycle arrest in G(1) in response to ionizing radiation or senescence is believed to be provoked by inactivation of G(1) cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21(Cip1/Waf1/Sdi1). We provide evidence that in addition to exerting negative control of the G(1)/S phase transition, p21 may play a role at the onset of mitosis. In nontransformed fibroblasts, p21 transiently reaccumulates in the nucleus near the G(2)/M-phase boundary, concomitant with cyclin B1 nuclear translocation, and associates with a fraction of cyclin A-Cdk and cyclin B1-Cdk complexes. Premitotic nuclear accumulation of cyclin B1 is not detectable in cells with low p21 levels, such as fibroblasts expressing the viral human papillomavirus type 16 E6 oncoprotein, which functionally inactivates p53, or in tumor-derived cells. Moreover, synchronized E6-expressing fibroblasts show accelerated entry into mitosis compared to wild-type cells and exhibit higher cyclin A- and cyclin B1-associated kinase activities. Finally, primary embryonic fibroblasts derived from p21(−/−) mice have significantly reduced numbers of premitotic cells with nuclear cyclin B1. These data suggest that p21 promotes a transient pause late in G(2) that may contribute to the implementation of late cell cycle checkpoint controls

La régulation réciproque de p21 et de Chk1 contrôle la voie de la cycline D1-RB pour induire le début de la sénescence après l'arrêt de G2

International audienceABSTRACT Senescence is an irreversible withdrawal from cell proliferation that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 requires p53 (also known as TP53) and retinoblastoma protein (RB, also known as RB1) family tumour suppressors, but how they are regulated to convert a temporary cell cycle arrest into a permanent one remains unknown. Here, we show that a previously unrecognised balance between the cyclin-dependent kinase (CDK) inhibitor p21 and the checkpoint kinase Chk1 controls cyclin D–CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting cyclin D1 complexed with CDK2 or CDK4. The resulting G2 exit, which precedes the appearance of senescence markers, is associated with a mitotic bypass, Chk1 downregulation and reduction in the number of DNA damage foci. In p53/RB-proficient cancer cells, a compromised G2 exit correlates with sustained Chk1 activity, delayed p21 induction, untimely cyclin E1 re-expression and genome reduplication. Conversely, Chk1 depletion promotes senescence by inducing p21 binding to cyclin D1– and cyclin E1–CDK complexes and downregulating CDK6, whereas knockdown of the checkpoint kinase Chk2 enables RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus promoting the onset of senescence induced by DNA damage in G2.La sénescence est un retrait irréversible de la prolifération cellulaire qui peut être initié après l'arrêt du cycle cellulaire induit par des dommages à l'ADN en phase G2 pour prévenir l'instabilité génomique. L'apparition de la sénescence dans G2 nécessite des suppresseurs de tumeurs de la famille p53 (également connu sous le nom de TP53) et de la protéine du rétinoblastome (RB, également connu sous le nom de RB1), mais la façon dont ils sont régulés pour convertir un arrêt temporaire du cycle cellulaire en un arrêt permanent reste inconnue. Ici, nous montrons qu'un équilibre précédemment non reconnu entre l'inhibiteur p21 de la kinase dépendante de la cycline (CDK) et la kinase de point de contrôle Chk1 contrôle l'activité de la cycline D – CDK lors de l'arrêt de G2. Dans les cellules non transformées, p21 active RB dans G2 en inhibant la cycline D1 complexée avec CDK2 ou CDK4. La sortie G2 qui en résulte, qui précède l'apparition des marqueurs de sénescence, est associée à un pontage mitotique, à une régulation négative de Chk1 et à une réduction du nombre de foyers de dommages à l'ADN. Dans les cellules cancéreuses compétentes en p53 / RB, une sortie G2 compromise est corrélée à une activité soutenue de Chk1, à une induction retardée de p21, à une réexpression intempestive de la cycline E1 et à une reduplication du génome. Inversement, la déplétion de Chk1 favorise la sénescence en induisant la liaison de p21 aux complexes cycline D1 et cycline E1-CDK et en régulant à la baisse CDK6, tandis que l'inactivation de la kinase de point de contrôle Chk2 permet la phosphorylation de RB et retarde la sortie de G2. En conclusion, p21 et Chk2 s'opposent à Chk1 pour maintenir l'activité RB, favorisant ainsi l'apparition de la sénescence induite par les dommages à l'ADN dans la phase G2