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

Abstract

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 p16Ink4a , 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 ␤-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.Human diploid fibroblasts (HDF) have a finite proliferative lifespan, at the end of which they are unable to enter S phase in response to mitogenic stimuli. Senescent HDF are also enlarged and flattened and synthesize an altered repertoire of cell-type-specific proteins, suggesting that they have differentiated as well as aged (5). Serum-stimulated senescent HDF fail to phosphorylate the retinoblastoma protein (pRb) (51), an event that is necessary for the release of E2F transcription factors that promote the expression of late G 1 genes whose products are required for S-phase initiation and progression (39, 55). The inhibition of DNA synthesis in senescent nuclei can be overcome by factors or treatments that block or inactivate the inhibitory activity of pRb and its family of related proteins. For example, transfection or microinjection of simian virus 40 (SV40) T antigen into senescent HDF induces DNA synthesis, but this effect is lost when SV40 T antigen deficient in pRb binding is used (22). These data suggest that failure to phosphorylate pRb is a key mechanism for the cell cycle arrest of senescent cells.Phosphorylation of pRb during G 1 phase is carried out by cyclin D-Cdk4 and cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes (44,50,55). In quiesce...

Survivin initiates cell cycle entry by the competitive interaction with Cdk4/p16INK4a and Cdk2/Cyclin E complex activation

“…In contrast, INK4 family members act only as suppressors for cell cycle progression Roberts, 1995, 1999;Reynisdottir and Massague, 1997). p16 INK4a is known as a speci®c suppressor for Cdk4 and its homolog Cdk6 (Byeon et al, 1998;Parry et al, 1999;Stein et al, 1999). Since we demonstrated that Survivin interacts with Cdk4 during the cell cycle, we investigated whether Survivin in¯uences the Cdk4/p16 INK4a complex.…”

Mn‐SOD antisense upregulates in vivo apoptosis of squamous cell carcinoma cells by anticancer drugs and γ‐rays regulating expression of the BCL‐2 family proteins, COX‐2 and p21

“…54 The roles of both families in G 1 phase arrest and induction of differentiation and apoptosis have been thoroughly studied. [55][56][57][58] In SCC cells, the CIP1/KIP1 family appears to play a more important role than the INK4 family because p21 waf1/cip1 is strongly expressed but p16 INK4a is weakly expressed. 59,60 In the present study, p16 INK4a expression was not increased in the Mn-SOD antisense-transfected SCC cells, while p21 waf1/cip1 expression was increased and coordinated with the upregulation of Bax and downregulation of Bcl-2 expression.…”

p21Waf1/Cip1/Sdi1-induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells

“…We suggest that such changes may be due at least in part to genetic destabilization that results from accidental re-entry into the cell cycle of cells that underwent prolonged p21 induction in the course of senescence. Transient p21 induction triggers growth arrest of senescent cells, whereas another CDK inhibitor, p16, appears to be responsible for maintaining this growth arrest after the decay of p21 (Alcorta et al, 1996;Stein et al, 1999). p16 (in striking contrast to p21) is frequently mutated in human tumors (Hall and Peters, 1996), including HT1080 ®brosarcoma used in the present study.…”

“…p21 is also induced at the onset of terminal growth arrest during replicative senescence of aging ®broblasts (Noda et al, 1994). A transient surge of p21 expression in both replicative and accelerated senescence is followed by the induction of another CDK inhibitor, p16 INK4A , which remains at a high level and may be responsible for the maintenance of growth arrest in senescent cells after the decay of p21 (Alcorta et al, 1996;Robles and Adami, 1998;Stein et al, 1999).…”