Functions of p21Waf1 in Norm and in Stress

Cyclin-dependent kinase inhibitor p21^Waf1/Cip1 plays the key part in cell cycle arrest at the G1/S checkpoint in response to DNA damage, and is involved in the assembly of active cyclin–kinase complexes, in particular, cyclin D–Cdk4/6. Recent studies extended the range of known p21^Waf1/Cip1 functions. In addition to the cell-cycle control, p21^Waf1/Cip1 participates in important cell processes such as differentiation, senescence, and apoptosis. The balance of p21^Waf1/Cip1 functional activity appears to shift depending on the cell state (senescence, exposure to stress, expression of viral oncogenes). This is due to direct or indirect interaction with various modulators or to modification (phosphorylation, partial proteolysis) of p21^Waf1/Cip1. The review considers the structure of p21^Waf1/Cip1, its posttranslational modification, interactions with various cell or viral proteins, and their effects on the p21^Waf1/Cip1 function and on the cell.     

Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

Senescence of alveolar type 2 (ATII) cells, progenitors of the alveolar epithelium, is implicated in the pathogeneses of idiopathic pulmonary fibrosis (IPF), an aging‐related progressive fatal lung disorder with unknown etiology. The mechanism underlying ATII cell senescence in fibrotic lung diseases, however, remains poorly understood. In this study, we report that ATII cells in IPF lungs express higher levels of serpine 1, also known as plasminogen activator inhibitor 1 (PAI‐1), and cell senescence markers p21 and p16, compared to ATII cells in control lungs. Silencing PAI‐1 or inhibition of PAI‐1 activity in cultured rat ATII (L2) cells leads to decreases in p53 serine 18 phosphorylation (p53^S18P), p53 and p21 protein expressions; an increase in retinoblastoma protein phosphorylation (ppRb); and a reduction in the sensitivity to bleomycin‐ and doxorubicin‐induced senescence. Silencing p53, on the other hand, abrogates PAI‐1 protein‐stimulated p21 expression and cell senescence. In vivo studies, using ATII cell‐specific PAI‐1 conditional knockout mouse model generated recently in this laboratory, further support the role of PAI‐1 in the activation of p53‐p21‐Rb cell cycle repression pathway, ATII cell senescence, and lung fibrosis induced by bleomycin. This study reveals a novel function of PAI‐1 in regulation of cell cycle and suggests that elevation of PAI‐1 contributes importantly to ATII cell senescence in fibrotic lung diseases.     

ECRG1, a novel candidate of tumor suppressor gene in the esophageal carcinoma, triggers a senescent program in NIH3T3 cells

Esophageal cancer-related gene 1 (ECRG1) is a novel tumor-suppressor gene candidate identified from the human esophagus. Previous studies showed that ECRG1 overexpression could inhibit cell growth and induce G1 cell cycle arrest and p15(INK4b) expression by interacting with Miz-1 (Myc-interacting zinc finger protein). Such evidence suggests the alterations in ECRG1 may play an important role in tumorigenesis. To further study the biological function of the ECRG1 gene, we transfected ECRG1 into NIH3T3 cells. Expression of ECRG1 in these cells caused senescence-like changes characterized in terms of altered cell morphology, cell cycle arrest at the G1/S phase, and significantly impaired cell proliferation (P < 0.01). Moreover, NIH3T3 cells transfected with ECRG1 stained positive for SA-beta-gal staining (pH 6.0), which is a specific marker of cellular senescence. We also studied changes in telomerase activity and the related senescence genes, such as p21 and p16. The results indicated that when ECRG1 induced a senescence-like state, telomerase activity was markedly decreased (P < 0.05), and expression of p21 was distinctly increased, whereas no changes were detected in p16 and telomerase-component RNA levels. These findings suggest that ECRG1 may be of importance in murine cell senescence, promoting senescence by regulating expression of p21.     

ZNF313 is a novel cell cycle activator with an E3 ligase activity inhibiting cellular senescence by destabilizing p21WAF1

ZNF313 encoding a zinc-binding protein is located at chromosome 20q13.13, which exhibits a frequent genomic amplification in multiple human cancers. However, the biological function of ZNF313 remains largely undefined. Here we report that ZNF313 is an ubiquitin E3 ligase that has a critical role in the regulation of cell cycle progression, differentiation and senescence. In this study, ZNF313 is initially identified as a XIAP-associated factor 1 (XAF1)-interacting protein, which upregulates the stability and proapoptotic effect of XAF1. Intriguingly, we found that ZNF313 activates cell cycle progression and suppresses cellular senescence through the RING domain-mediated degradation of p21^WAF1. ZNF313 ubiquitinates p21^WAF1 and also destabilizes p27^KIP1 and p57^KIP2, three members of the CDK-interacting protein (CIP)/kinase inhibitor protein (KIP) family of cyclin-dependent kinase inhibitors, whereas it does not affect the stability of the inhibitor of CDK (INK4) family members, such as p16^INK4A and p15^INK4B. ZNF313 expression is tightly controlled during the cell cycle and its elevation at the late G1 phase is crucial for the G1-to-S phase transition. ZNF313 is induced by mitogenic growth factors and its blockade profoundly delays cell cycle progression and accelerates p21^WAF1-mediated senescence. Both replicative and stress-induced senescence are accompanied with ZNF313 reduction. ZNF313 is downregulated during cellular differentiation process in vitro and in vivo, while it is commonly upregulated in many types of cancer cells. ZNF313 shows both the nuclear and cytoplasmic localization in epithelial cells of normal tissues, but exhibits an intense cytoplasmic distribution in carcinoma cells of tumor tissues. Collectively, ZNF313 is a novel E3 ligase for p21^WAF1, whose alteration might be implicated in the pathogenesis of several human diseases, including cancers.     

Downregulation of caveolin-1 affects bleomycin-induced growth arrest and cellular senescence in A549 cells

Bleomycin is an anti-cancer drug that induces both apoptosis and senescence, two processes thought to involve caveolin-1. Here we investigate the role of caveolin-1 in bleomycin-induced senescence. We show that bleomycin-treated A549 cells exhibit: senescence-like cell morphology; a senescence-associated increase in SA-beta-galactosidase activity; cell cycle arrest; and upregulation of p53 and p21. As predicted, we find that caveolin-1 amount increases in response to bleomycin-treatment and that modulation of caveolin-1 affects p21 and p53 levels, cell cycling, and senescence (SA-beta-galactosidase activity). Interestingly, senescence-associated cell cycle arrest via p53 and p21 and SA-beta-galactosidase activity is reduced in young A549 cells when short hairpin RNA specific for caveolin-1 was applied before bleomycin-treatment. Our results support the hypothesis that downregulation of caveolin-1 expression affects bleomycin-induced cell cycle arrest and subsequent cellular senescence that is driven by p53 and p21.     

Loss of retinoblastoma but not p16 function allows bypass of replicative senescence in human fibroblasts

Current models envision replicative senescence to be under dual control by the p53 and retinoblastoma (RB) tumour-suppressor pathways. The role of the p16^INK4a–RB pathway is controversial, and the function of RB in human cells has not been tested directly. We used targeted homologous recombination to knock out one copy of RB in presenescent human fibroblasts. During entry into senescence, RB^+/− cells underwent spontaneous loss of heterozygosity and the resultant RB^−/− clones bypassed senescence. The extended lifespan phase was eventually terminated by a crisis-like state. The same phenotype was documented for p21 ^CIP1/WAF1 and p53 heterozygous cells, indicating that loss of function of all three genes results in failure to establish senescence. By contrast, the abolition of p16 function by the expression of a p16-insensitive cyclin-dependent kinase 4 protein or siRNA-mediated knockdown provided only minimal lifespan extension that was terminated by senescence. We propose that p53, p21 and RB act in a linear genetic pathway to regulate cell entry into replicative senescence.     

p21Waf1 is required for cellular senescence but not for cell cycle arrest induced by the HDAC inhibitor sodium butyrate

Cell senescence is characterized by senescent morphology and permanent loss of proliferative potential. HDAC inhibitors (HDACI) induce senescence and/or apoptosis in many types of tumor cells. Here, we studied the role of cyclin-kinase inhibitor p21^waf1(Cdkn1n gene) in cell cycle arrest, senescence markers (cell hypertrophy, SA-bGal staining and accumulation of gH2AX foci) in p21^Waf1+/+ versus p21^Waf1-/- mouse embryonic fibroblast cells transformed with E1A and cHa-Ras oncogenes (mERas). While short treatment with the HDACI sodium butyrate (NaB) induced a reversible G1 cell cycle arrest in both parental and p21^Waf1-/- cells, long-term treatment led to dramatic changes in p21^Waf1+/+ cells only: cell cycle arrest became irreversible and cells become hypertrophic, SA-bGal-positive and accumulated gH2AX foci associated with mTORC1 activation. The p21^Waf1+/+ cells lost their ability to migrate into the wound and through a porous membrane. Suppression of migration was accompanied by accumulation of vinculin-staining focal adhesions and Ser3-phosphorylation of cofilin, incapable for F-actin depolymerization. In contrast, the knockout of the p21^Waf1 abolished most of the features of NaB-induced senescence, including irreversibility of cell cycle arrest, hypertrophy, additional focal adhesions and block of migration, gH2AX foci accumulation and SA-bGal staining. Rapamycin, a specific inhibitor of mTORC1 kinase, decreased cellular hypertrophy, canceled coffilin phosphorylation and partially restored cell migration in p21^Waf1+/+ cells. Taken together, our data indicate a new role of p21^Waf1 in cell senescence, which may be connected not with execution of cell cycle arrest, but also with the development of mTOR-dependent markers of cellular senescence.     

Dissecting the influence of cellular senescence on cell mechanics and extracellular matrix formation in vitro

Tissue formation and healing both require cell proliferation and migration, but also extracellular matrix production and tensioning. In addition to restricting proliferation of damaged cells, increasing evidence suggests that cellular senescence also has distinct modulatory effects during wound healing and fibrosis. Yet, a direct role of senescent cells during tissue formation beyond paracrine signaling remains unknown. We here report how individual modules of the senescence program differentially influence cell mechanics and ECM expression with relevance for tissue formation. We compared DNA damage-mediated and DNA damage-independent senescence which was achieved through over-expression of either p16^Ink4a or p21^Cip1 cyclin-dependent kinase inhibitors in primary human skin fibroblasts. Cellular senescence modulated focal adhesion size and composition. All senescent cells exhibited increased single cell forces which led to an increase in tissue stiffness and contraction in an in vitro 3D tissue formation model selectively for p16 and p21-overexpressing cells. The mechanical component was complemented by an altered expression profile of ECM-related genes including collagens, lysyl oxidases, and MMPs. We found that particularly the lack of collagen and lysyl oxidase expression in the case of DNA damage-mediated senescence foiled their intrinsic mechanical potential. These observations highlight the active mechanical role of cellular senescence during tissue formation as well as the need to synthesize a functional ECM network capable of transferring and storing cellular forces.     

Cyclin-dependent kinase inhibitor p21<Superscript>Waf1</Superscript>: Contemporary view on its role in senescence and oncogenesis

p21^Waf1 was identified as a protein suppressing cyclin E/A-CDK2 activity and was originally considered as a negative regulator of the cell cycle and a tumor suppressor. It is now considered that p21^Waf1 has alternative functions, and the view of its role in cellular processes has begun to change. At present, p21^Waf1 is known to be involved in regulation of fundamental cellular programs: cell proliferation, differentiation, migration, senescence, and apoptosis. In fact, it not only exhibits antioncogenic, but also oncogenic properties. This review provides a contemporary understanding of the functions of p21^Waf1 depending on its intracellular localization. On one hand, when in the nucleus, it serves as a negative cell cycle regulator and tumor suppressor, in particular by participating in the launch of a senescence program. On the other hand, when p21^Waf1 is localized in the cytoplasm, it acts as an oncogene by regulating migration, apoptosis, and proliferation.     

Loss of p21CDKN1A impairs entry to quiescence and activates a DNA damage response in normal fibroblasts induced to quiescence

The cell cycle inhibitor p21^CDKN1A induces cell cycle arrest under different conditions, including senescence and terminal differentiation. Still debated is its involvement in the reversible transition from proliferation to a non-dividing quiescent state (G0), in which a significant role has been attributed to cell cycle inhibitor p27^CDKN1B. Here we provide evidence showing that high p21 protein levels are necessary to enter and maintain the quiescence state following contact inhibition and growth factor withdrawal. In fact, entry into quiescence was impaired, both in human fibroblasts in which p21 gene has been deleted, or protein expression knocked-down by RNA interference. Importantly, in the absence of p21, human fibroblasts activate a DNA damage-like signalling pathway, as shown by phosphorylation of histone H2AX and Chk1 proteins. In addition, we show that in the absence of p21, checkpoint is activated by an unscheduled entry into S phase, with a reduced efficiency in DNA maturation, in the presence of high c-myc protein levels. These results highlight the role of p21 in counteracting inappropriate proliferation stimuli for genome stability maintenance.     

CSIG inhibits PTEN translation in replicative senescence

Using a suppressive subtractive hybridization system, we identified CSIG (cellular senescence-inhibited gene protein; RSL1D1) that was abundant in young human diploid fibroblast cells but declined upon replicative senescence. Overexpression or knockdown of CSIG did not influence p21^Cip1 and p16^INK4a expressions. Instead, CSIG negatively regulated PTEN and p27^Kip1 expressions, in turn promoting cell proliferation. In PTEN-silenced HEK 293 cells and PTEN-deficient human glioblastoma U87MG cells, the effect of CSIG on p27^Kip1 expression and cell division was abolished, suggesting that PTEN was required for the role of CSIG on p27^Kip1 regulation and cell cycle progression. Investigation into the underlying mechanism revealed that the regulation of PTEN by CSIG was achieved through a translational suppression mechanism. Further study showed that CSIG interacted with PTEN mRNA in the 5′ untranslated region (UTR) and that knockdown of CSIG led to increased luciferase activity of a PTEN 5′ UTR-luciferase reporter. Moreover, overexpression of CSIG significantly delayed the progression of replicative senescence, while knockdown of CSIG expression accelerated replicative senescence. Knockdown of PTEN diminished the effect of CSIG on cellular senescence. Our findings indicate that CSIG acts as a novel regulatory component of replicative senescence, which requires PTEN as a mediator and involves in a translational regulatory mechanism.     

Myc,Cdk2 and cellular senescence: Old players,new game

The aberrant activation of oncogenic pathways promotes tumor progression, but concomitantly elicits compensatory tumor-suppressive responses, such as apoptosis or senescence. For example, Ras induces senescence, while Myc generally triggers apoptosis. Myc is in fact viewed as an anti-senescence oncogene, as it is a potent inducer of cell proliferation and immortalization, bypasses growth-inhibitory signals, and cooperates with Ras in cellular transformation. Recent reports prompt re-evaluation of Myc-induced senescence, and of its role in tumor progression and therapy. We have shown that the cyclin-dependent kinase Cdk2, although redundant for cell cycle progression, has a unique role in suppressing a Myc-induced senescence program: Myc activation elicited expression of p16^INK4a and p21^Cip1, and caused senescence in cell lacking Cdk2, but not in Cdk2-proficient cells. Additional cellular activities have been identified that suppress Myc-induced senescence, including the Wrn helicase, Telomerase and Miz1. These senescence-suppressing activities were critical for tumor progression, as deficiency in Cdk2, telomerase or Miz1 reduced the onset of Myc-induced lymphoma in transgenic mice. Other gene products like p53, SUV39H1 or TGFß promoted senescence, which together with apoptosis contributed to tumor suppression. Paradoxically, Myc directly counteracted the very same senescence program that it potentially elicits, since it positively regulated Wrn, Telomerase and Cdk2 activity, and Cdk2 inhibition re-activated the latent senescence program in Myc expressing cells. Hence, while these molecules are instrumental to the oncogenic action of Myc, they may simultaneously constitute its Achille's heel for therapeutic development.     

Single‐cell analysis of p16INK4a and p21WAF1 expression suggests distinct mechanisms of senescence in normal human and Li‐Fraumeni Syndrome fibroblasts

Herein we used single‐cell observation methods to gain insight into the roles of p16^INK4A and p21^WAF1 (hereafter p16 and p21) in replicative senescence and ionizing radiation‐induced accelerated senescence in human [normal, ataxia telangiectasia (AT) and Li‐Fraumeni syndrome (LFS)] fibroblast strains. Cultures of all strains entered a state of replicative senescence at late passages, as evident from inhibition of growth, acquisition of flattened and enlarged cell morphology, and positive staining for senescence‐associated β‐galactosidase. In addition, proliferating early‐passage cultures of these strains exhibited accelerated senescence in response to ionizing radiation. Immunofluorescence microscopy revealed the heterogeneous expression of p16 in normal and AT fibroblast strains, with the majority of the cells exhibiting undetectable levels of p16 irrespective of in vitro culture age. Importantly, replicative senescence as well as accelerated senescence triggered by ionizing radiation were accompanied by sustained nuclear accumulation of p21, but did not correlate with p16 expression in p53‐proficient (normal and AT) fibroblasts. In p53‐deficient (LFS) fibroblasts, on the other hand, replicative senescence and ionizing radiation‐triggered accelerated senescence strongly correlated with expression of p16 but not of p21. Furthermore, senescence in LFS fibroblasts was associated with genomic instability encompassing polyploidy. Our findings are compatible with a model in which p16 serves as a backup regulator of senescence, triggering this response preferentially in the absence of wild‐type p53 activity. The possibility that one of the tumor‐suppressor functions of p16 may be associated with genomic instability, preventing the emergence of malignant progeny from polyploid giant cells, is also supported by these results. J. Cell. Physiol. 223: 57–67, 2010. © 2009 Wiley‐Liss, Inc.     

MYC accelerates p21CIP‐induced megakaryocytic differentiation involving early mitosis arrest in leukemia cells

p21^CIP is a potent cell cycle inhibitor often up‐regulated in differentiation. Protooncogene MYC induces cell growth and proliferation, inhibits differentiation and represses p21^CIP. However, both molecules are involved in processes of polyploidisation, cell size increase, differentiation and senescence. It is unclear why MYC has a dual role in differentiation. We have previously shown that overexpression of p21^CIP in K562 myeloid cells induces megakaryocytic differentiation with polyploidy. We have now investigated the requirements for p21^CIP to block mitosis and induce differentiation in the presence of overactivated MYC. Silencing and over‐expression studies showed that p21^CIP is required to induce differentiation. However, the expression of p21^CIP needs to be transient to irreversibly inhibit mitosis but not DNA replication, what leads to polyploidy. Transient overexpression of p21^CIP caused early down‐regulation of mitotic Cyclins and up‐regulation of G1/S Cyclins D and E, changes typical of endoreplication. Interestingly, over‐activation of MYC did not release the proliferative block imposed by p21^CIP and instead, accelerated cell size increase, megakaryocytic differentiation and polyploidisation. Our data suggests that in some systems p21^CIP takes part in a mitosis control driving MYC‐induced cellular growth into differentiation. J. Cell. Physiol. 227: 2069–2078, 2012. © 2011 Wiley Periodicals, Inc.