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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...

G1 cyclins control the G1 to S phase transition in the budding yeast, Saccharomyces cerevisiae. Cyclin E was discovered in the course of a screen for human complementary DNAs that rescue a deficiency of G1 cyclin function in budding yeast. The amounts of both the cyclin E protein and an associated protein kinase activity fluctuated periodically through the human cell cycle; both were maximal in late G1 and early S phases. Cyclin E-associated kinase activity was correlated with the appearance of complexes containing cyclin E and the cyclin-dependent kinase Cdk2. Thus, the cyclin E-Cdk2 complex may constitute a human G1-S phase-specific regulatory protein kinase.