Uncoupling between Phenotypic Senescence and Cell Cycle Arrest in Aging p21-Deficient Fibroblasts

Abstract

Irreversible G 1 arrest in senescent human fibroblasts is mediated by two inhibitors of cyclin-dependent kinases (Cdks), p21Cip1/SDI1/WAF1 and p16 Ink4A . To determine the physiological and molecular events that specifically require p21, we studied senescence in human diploid fibroblasts expressing the human papillomavirus type 16 E6 oncogene, which confers low p21 levels via enhanced p53 degradation. We show that in late-passage E6 cells, high Cdk activity drives the cell cycle, but population expansion is slowed down by crisis-like events, probably owing to defective cell cycle checkpoints. At the end of lifespan, terminal-passage E6 cells exhibited several aspects of the senescent phenotype and accumulated unphosphorylated pRb and p16. However, both replication and cyclin-Cdk2 kinase activity were still not blocked, demonstrating that phenotypic and replicative senescence are uncoupled in the absence of normal p21 levels. At this stage, E6 cells also failed to upregulate p27 and inactivate cyclin-Cdk complexes in response to serum deprivation. Eventually, irreversible G 1 arrest occurred coincident with inactivation of cyclin E-Cdk2 owing to association with p21. Similarly, when p21 ؊/؊ mouse embryo fibroblasts reached the end of their lifespan, they had the appearance of senescent cells yet, in contrast to their wild-type counterparts, they were deficient in downregulating bromodeoxyuridine incorporation, cyclin E-and cyclin A-Cdk2 activity, and inhibiting pRb hyperphosphorylation. These data support the model that the critical event ensuring G 1 arrest in senescence is p21-dependent Cdk inactivation, while other aspects of senescent phenotype appear to occur independently of p21.Human diploid fibroblasts (HDFs) have a finite proliferative lifespan, at the end of which they cease irreversibly to divide and they undergo a series of phenotypic changes that distinguish senescence from quiescence (26). These phenotypic changes include altered morphology, increased cell volume, expression of a neutral senescence-associated ␤-galactosidase activity (SA-␤-Gal), and increased production of extracellular matrix degradative enzymes such as collagenase and stromelysin (26,40,61). It is now generally accepted that two inhibitors of cyclin-dependent kinases (Cdks), p16Ink4a (p16) and p21Cip1/ Waf1/Sdi1 (p21), whose amounts increase with age, have an essential role in inactivating Cdks in senescent fibroblasts (1,24,42,44,60). Cdk inactivation, in turn, allows the accumulation of unphosphorylated retinoblastoma protein (pRb) (59), a growth suppressor whose function is modulated by Cdks. Unphosphorylated pRb exerts negative regulation of cell cycle progression by forming complexes with members of the E2F transcription factor family (23, 28). In spite of their undisputed role in mediating senescence, the precise contribution of each Cdk inhibitor (CKI) is not fully established. The CKI p21 binds to and inactivates most cyclin-Cdk complexes, whereas p16 blocks cyclin D-Cdk activation by binding specifically to Cdk4 and Cdk6,...

Manganese Superoxide Dismutase Protects the Proliferative Capacity of Confluent Normal Human Fibroblasts

“…It is possible that MnSOD-induced accumulation of p21 protein levels would protect cyclin E(A)-Cdk 2 kinase complexes, which upon subculturing would facilitate a Rb phosphorylation-independent entry into S-phase. Such an assumption is supported by previous studies in the literature demonstrating cyclin E-dependent and Rb phosphorylationindependent entry into S-phase (24,52). Nonetheless, the observation that MnSOD induces p21 protein levels might be of broad interest because of the possibility that antioxidants, in particular MnSOD, could have a protective role in maintaining the reproductive integrity of a variety of proliferation-competent quiescent cells, including stem cells.…”

“…Increases in both p16 and p21 Cdk inhibitors during an irreversible growth arrest are generally thought to be associated with accumulation of hypophosphorylated Rb (24). Hypophosphorylated Rb is known to sequester members of the E2F transcription factor family, and the unavailability of E2F inhibits progression through the cell cycle.…”

CDK2 Transcriptional Repression Is an Essential Effector in p53-Dependent Cellular Senescence—Implications for Therapeutic Intervention

“…As mentioned above, previous work has established that CDK2 kinase activity (but not CDK2 protein levels) is decreased during replicative senescence (13)(14)(15)36). Expression of recombinant CYCLIN E-CDK2 in replicatively senescent human fibroblasts resulted in DNA synthesis in approximately 10% of cells (17).…”

“…Cip1 , whereas CDK2 protein levels remain unchanged (13)(14)(15); whereas in replicative senescence of cultured human umbilical vein endothelial cells (HUVEC), reduction of both CDK2 activity and protein levels were noted (16). Transduction of replicatively senescent cells with Cyclin E-CDK2 complexes results in cellcycle reentry in a subset of cells, indicating that reduction of CDK2 activity is indeed essential for replicative senescence (17).…”

Reactive Oxygen Species and Mitochondrial Sensitivity to Oxidative Stress Determine Induction of Cancer Cell Death by p21

“…Senescence is also a tumor suppressor mechanism that prevents emergence of transformed cells (9). It has been shown that lack of p21 delays or abolishes the onset of senescence (12) and that continuous p21 expression induces a senescent arrest in normal and cancer cells in a p53-independent manner (13)(14)(15).…”