Weak p53 permits senescence during cell cycle arrest

Cell cycle arrest coupled with hyper-active mTOR leads to cellular senescence. While arresting cell cycle, high levels of p53 can inhibit mTOR (in some cell lines), thus causing reversible quiescence instead of senescence. Nutlin-3a-induced p53 inhibited mTOR and thus caused quiescence in WI-38 cells. In contrast, while arresting cell cycle, the DNA-damaging drug doxorubicin (DOX) did not inhibit mTOR and caused senescence. Super-induction of p53 by either nutlin-3a or high concentrations of DOX (high-DOX) prevented low-DOX-induced senescence, converting it into quiescence. This explains why in order to cause senescence, DNA damaging drugs must be used at low concentrations, which arrest cell cycle but do not induce p53 at levels sufficient to suppress mTOR. Noteworthy, very prolonged treatment with nutlin-3a also caused senescence preventable by rapamycin. In RPE cells, low concentrations of nutlin-3a caused a semi-senescent morphology. Higher concentrations of nutlin-3a inhibited mTOR and caused quiescent morphology. We conclude that low p53 levels during prolonged cell cycle arrest tend to cause senescence, whereas high levels of p53 tend to cause either quiescence or cell death.     

Cooperation between p21 and Akt is required for p53‐dependent cellular senescence

Cellular senescence has been implicated in normal aging, tissue homeostasis, and tumor suppression. Although p53 has been shown to be a central mediator of cellular senescence, the signaling pathway by which it induces senescence remains incompletely understood. In this study, we have shown that both Akt and p21 are required to induce cellular senescence in response to p53 expression. In a p53‐induced senescence model, we found that Akt activation was essential for inducing a cellular senescence phenotype. Surprisingly, Akt inhibition did not abolish p53‐induced cell cycle arrest, but it suppressed the increase in intracellular reactive oxygen species (ROS) levels. The results of the cell cycle and morphological analysis suggest that p53 induced quiescence, not senescence, following Akt inhibition. Conversely, the inhibition of p21 induction abolished cell cycle arrest but did not affect the p53‐induced increase in ROS levels. Additionally, p21 and Akt separately controlled cell cycle arrest and ROS levels, respectively, during H‐Ras‐induced senescence in human normal fibroblasts. The mechanistic analysis revealed that Akt increased ROS levels through NOX4 induction, and increased Akt‐dependent NF‐κB binding to the NOX4 promoter is responsible for NOX4 induction upon p53 expression. We further showed that Akt activation upon p53 expression is mediated by mammalian target of rapamycin complex 2. In addition, p53‐mediated IL6 and IL8 induction was abrogated by Akt inhibition, suggesting that Akt activation is also required for the senescence‐associated secretory phenotype. Collectively, these results suggest that p53 simultaneously controls multiple pathways to induce cellular senescence through p21 and Akt.     

Program of premature senescence is not induced by sodium butyrate in transformants with JNK1,2 knockout

The role of JNK1,2 stress kinases in the regulation of premature senescence stimulated by sodium butyrate (NaB), a histone deacetylase inhibitor, has been studied. It was found that NaB did not block the cell cycle in E1A+cHa-ras transformants selected from embryonic mouse fibroblasts with jnk1,2 stress-kinase gene knockout (mERasJNK^?/? cells). Even long-term (five days) NaB treatment did not block cell cycle distribution or cell proliferation, nor did it induce cellular hypertrophy or activate SA-β-galactosidase activity, a senescence marker. The data show that JNK stress kinases are involved in senescence induced in E1A+cHa-ras mouse transformants by NaB. It is possible to suggest that JNK1,2 have tumor suppressor properties because the process of senescence, which prevents tumor cell proliferation, does not occur if they are absent.     

p21cip1 is required for the differentiation of oligodendrocytes independently of cell cycle withdrawal

Differentiation of most cell types requires both establishment of G₁ arrest and the induction of a program related to achieving quiescence. We have chosen to study the differentiation of oligodendrocyte cells to determine the role of p27 and p21 in this process. Here we report that both p27 and p21 are required for the appropriate differentiation of these cells. p27 is required for proper withdrawal from the cell cycle, p21 is not. Instead, p21 is required for the establishment of the differentiation program following growth arrest. Similar observations were made in vivo. We show that p21^–/– cells withdraw from the cell cycle similar to wild-type cells; however, early in animal life, the brain is hypomyelinated, inferring that the loss of p21 delayed myelination in the cerebellum. We found that we could complement or bypass the differentiation failure in p21^–/– cells with either PD98059, an inhibitor of Mek1, or by transducing them with a tat–p16^Ink4a protein. We concluded that the two cdk inhibitors serve non-redundant roles in this program of differentiation, with p27 being responsible for arrest and p21 having a function in differentiation independent of its ability to control exit from the cell cycle.     

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.     

Regulation of Cdc2p and Cdc13p is required for cell cycle arrest induced by defective RNA splicing in fission yeast

Screening of cdc mutants of fission yeast for those whose cell cycle arrest is independent of the DNA damage checkpoint identified the RNA splicing-deficient cdc28 mutant. A search for mutants of cdc28 cells that enter mitosis with unspliced RNA resulted in the identification of an orb5 point mutant. The orb5+ gene, which encodes a catalytic subunit of casein kinase II, was found to be required for cell cycle arrest in other mutants with defective RNA metabolism but not for operation of the DNA replication or DNA damage checkpoints. Loss of function of wee1+ or rad24+ also suppressed the arrest of several splicing mutants. Overexpression of the major B-type cyclin Cdc13p induced cdc28 cells to enter mitosis. The abundance of Cdc13p was reduced, and the phosphorylation of Cdc2p on tyrosine 15 was maintained in splicing-defective cells. These results suggest that regulation of Cdc13p and Cdc2p is required for G2 arrest in splicing mutants.     

p21(Cip1) and p27(Kip1) regulate cell cycle reentry after hypoxic stress but are not necessary for hypoxia-induced arrest

We investigated the role of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) in cell cycle regulation during hypoxia and reoxygenation. While moderate hypoxia (1 or 0.1% oxygen) does not significantly impair bromodeoxyuridine incorporation, at very low oxygen tensions (0.01% oxygen) DNA replication is rapidly shut down in immortalized mouse embryo fibroblasts. This S-phase arrest is intact in fibroblasts lacking the cyclin kinase inhibitors p21(Cip1) and p27(Kip1), indicating that these molecules are not essential elements of the arrest pathway. Hypoxia-induced arrest is accompanied by dephosphorylation of pRb and inhibition of cyclin-dependent kinase 2, which results in part from inhibitory phosphorylation. Interestingly, cells lacking the retinoblastoma tumor suppressor protein also display arrest under hypoxia, suggesting that pRb is not an essential mediator of this response. Upon reoxygenation, DNA synthesis resumes by 3.5 h and reaches aerobic levels by 6 h. Cells lacking p21, however, resume DNA synthesis more rapidly upon reoxygenation than wild-type cells, suggesting that this inhibitor may play a role in preventing premature reentry into the cell cycle upon cessation of the hypoxic stress. While p27 null cells did not exhibit rapid reentry into the cell cycle, cells lacking both p21 and p27 entered S phase even more aggressively than those lacking p21 alone, revealing a possible secondary role for p27 in this response. Cdk2 activity is also restored more rapidly in the double-knockout cells when returned to normoxia. These studies reveal that restoration of DNA synthesis after hypoxic stress, but not the S phase arrest itself, is regulated by p21 and p27.     

Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1.

The Raf family of protein kinases display differences in their abilities to promote the entry of quiescent NIH 3T3 cells into the S phase of the cell cycle. Although conditional activation of deltaA-Raf:ER promoted cell cycle progression, activation of deltaRaf-1:ER and deltaB-Raf:ER elicited a G1 arrest that was not overcome by exogenously added growth factors. Activation of all three deltaRaf:ER kinases led to elevated expression of cyclin D1 and cyclin E and reduced expression of p27Kip1. However, activation of deltaB-Raf:ER and deltaRaf-1:ER induced the expression of p21Cip1, whereas activation of deltaA-Raf:ER did not. A catalytically potentiated form of deltaA-Raf:ER, generated by point mutation, strongly induced p21Cip1 expression and elicited cell cycle arrest similarly to deltaB-Raf:ER and deltaRaf-1:ER. These data suggested that the strength and duration of signaling by Raf kinases might influence the biological outcome of activation of this pathway. By titration of deltaB-Raf:ER activity we demonstrated that low levels of Raf activity led to activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes and to cell cycle progression whereas higher Raf activity elicited cell cycle arrest correlating with p21Cip1 induction and inhibition of cyclin-cdk activity. Using green fluorescent protein-tagged forms of deltaRaf-1:ER in primary mouse embryo fibroblasts (MEFs) we demonstrated that p21Cip1 was induced by Raf in a p53-independent manner, leading to cell cycle arrest. By contrast, activation of Raf in p21Cip1(-/-) MEFs led to a robust mitogenic response that was similar to that observed in response to platelet-derived growth factor. These data indicate that, depending on the level of kinase activity, Raf can elicit either cell cycle progression or cell cycle arrest in mouse fibroblasts. The ability of Raf to elicit cell cycle arrest is strongly associated with its ability to induce the expression of the cyclin-dependent kinase inhibitor p21Cip1 in a manner that bears analogy to alpha-factor arrest in Saccharomyces cerevisiae. These data are consistent with a role for Raf kinases in both proliferation and differentiation of mammalian cells.     

p21(Waf1/Cip1) is an assembly factor required for platelet-derived growth factor-induced vascular smooth muscle cell proliferation

The cyclin-dependent kinase inhibitors interact with cyclin-cdk complexes to arrest mitogen-stimulated transit through the cell cycle, but these proteins have recently been shown to have positive regulatory effects on cyclin-cdk complex activity as well. Most of the previous work in this area has focussed on the finding that overexpressed p21(Waf1/Cip1) causes growth arrest. However, mice lacking p21(Waf1/Cip1) showed normal development with no aberrancy in their cell cycles, and antisense p21(Waf1/Cip1) has only been shown to prevent cell cycle arrest, leading to the conclusion that the cyclin kinase inhibitors may not be required for cell cycle progression. We found that transfection of several lines of vascular smooth muscle cells with antisense oligodeoxynucleotide specific to p21(Waf1/Cip1) correlates with decreased cyclin D1/cdk 4, but not cyclin E/cdk 2, association, yet, unexpectedly, results in dose-dependent inhibition of platelet-derived growth factor-BB-stimulated DNA synthesis and cell proliferation. Our finding that p21(Waf1/Cip1) exhibits permissive effects on growth factor-induced vascular smooth muscle cell cycle progression, such that its presence is required for growth factor-induced proliferation, is the first such report and opens up a fertile area of research relevant to diseases involving vascular cell proliferation.     

2‐methoxyestradiol‐induced cell death in osteosarcoma cells is preceded by cell cycle arrest

The article to which this erratum refers was published in J. Cell. Biochem. 104: 1937–1945, 2008. © 2008 Wiley‐Liss, Inc.     

High-intensity Raf signal causes cell cycle arrest mediated by p21Cip1.

Activated Raf has been linked to such opposing cellular responses as the induction of DNA synthesis and the inhibition of proliferation. However, it remains unclear how such a switch in signal specificity is regulated. We have addressed this question with a regulatable Raf-androgen receptor fusion protein in murine fibroblasts. We show that Raf can cause a G1-specific cell cycle arrest through induction of p21Cip1. This in turn leads to inhibition of cyclin D- and cyclin E-dependent kinases and an accumulation of hypophosphorylated Rb. Importantly, this behavior can be observed only in response to a strong Raf signal. In contrast, moderate Raf activity induces DNA synthesis and is sufficient to induce cyclin D expression. Therefore, Raf signal specificity can be determined by modulation of signal strength presumably through the induction of distinct protein expression patterns. Similar to induction of Raf, a strong induction of activated Ras via a tetracycline-dependent promoter also causes inhibition of proliferation and p21Cip1 induction at high expression levels. Thus, p21Cip1 plays a key role in determining cellular responses to Ras and Raf signalling. As predicted by this finding we show that Ras and loss of p21 cooperate to confer a proliferative advantage to mouse embryo fibroblasts.