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.     

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.     

Senescence-like changes induced by expression of p21(waf1/Cip1) in NIH3T3 cell line

P21(Waf1/Cip1) is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21(Waf1/Cip1) involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidence for a link between p21(Waf1/Cip1) and cellular senescence. While in murine cells, the role of p21(Waf1/Cip1) is indefinite. We explored this issue using NIH3T3 cells with inducible p21(Waf1/Cip1) expression. Induction of p21(Waf1/Cip1) triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features, such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed that p21(Waf1/Cip1)-transduced NIH3T3 cells expressed beta-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Our results suggest that p2l(Waf1/Cip1) can also induce senescence-like changes in murine cells.     

p21Waf1 is required for complete oncogenic transformation of mouse embryo fibroblasts by E1Aad5 and c-Ha-ras oncogenes

Cyclin-dependent kinase inhibitor p21^Waf1 is known to have alternative functions associated with positive regulation of proliferation, actin cytoskeleton remodeling and suppression of apoptosis. The goal of the present study was to assess the role of p21^Waf1 in the establishment of the transformed phenotype of mouse embryo fibroblasts with stable expression of E1Aad5 and c-Ha-ras complementary oncogenes. Herein, we demonstrate that E1A/c-Ha-Ras-transformed p21^Waf1-null fibroblasts possess some characteristic features of transformed cells, such as loss of contact inhibition, high saturation density, shortened cell cycle, inability to undergo cell-cycle arrest after DNA damage and serum deprivation, but, at the same time, they are not completely transformed in that they are unable to proliferate at clonal density, are anchorage-dependent, retain a fibroblast-like morphology with pronounced actin cytoskeleton and show reduced migration and invasion. Our data support the concept of p21^Waf1 “tumor suppressor” having alternative oncogenic functions in the cytoplasm and for the first time indicate that p21^Waf1 can be indispensable for complete oncogenic transformation.     

HDAC inhibitors induce apoptosis but not cellular senescence in Gadd45α-deficient E1A+Ras cells

HDAC inhibitors (HDIs) induce irreversible cell cycle arrest and senescence in E1A+Ras expressing cells. Furthermore, HDIs activate Gadd45α/NF-κB signaling pathway to suppress apoptosis thereby promoting the cell survival. Here, to clarify the role of Gadd45α in realization of the antiapoptotic program, we compared wild-type E1A+Ras cells and the cells with knockout of gadd45α gene (Gadd45α−/− cells). As in Gadd45α-expressing E1A+Ras cells, HDIs induce irreversible cell cycle arrest in Gadd45α−/− cells, but the arrested cells do not senesce and eventually die due to activation of the apoptotic death program. These data suggest that the expression of Gadd45α is involved in maintaining the balance of pro- and anti-apoptotic stimuli, while lack or loss of Gadd45 directs the cells to apoptosis after HDIs treatment. Appropriately Gadd45α-deficient cells demonstrate a higher level of pro-apoptotic signals, whereas the anti-apoptotic program is suppressed. The elevated apoptotic background of Gadd45α−/− cells is accompanied by higher levels of Ser15-phosphorylated p53 and p21/Waf1 proteins that additionally commit the cells to HDIs-induced apoptosis. Additionally, loss of Gadd45α protein activates the DDR signaling pathway as demonstrated by nuclear pATM staining, accumulation of γH2AX foci and an increase of single-strand DNA breaks. Thus, in wild-type E1A+Ras cells the p53-dependent expression of Gadd45α is necessary not only for DNA repair and HDI-induced cellular senescence, but also to withstand to apoptosis after DNA damage and stress. Therefore the use of HDIs in combination with agents that block Gadd45α function may have promise for cancer therapy.     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21(Cip1/Waf1) axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H(2)O(2). The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H(2)O(2)-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21(Cip1/Waf1) axis; but inhibiting the canonical NF-κB pathway exacerbated H(2)O(2)-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H(2)O(2)-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.     

Activation of ATM/ATR signaling in human embryonic stem cells after DNA damage

Embryonic stem cells (ESCs) are the progenitors of all adult cells; consequently, genomic abnormalities in them may be catastrophic for the developing organism. ESCs are characterized by high proliferation activity and do not stop in checkpoints upon DNA-damage executing only G₂/M delay after DNA damage. ATM and ATR kinases are key sensors of double-strand DNA breaks and activate downstream signaling pathways involving checkpoints, DNA repair, and apoptosis. We examined activation of ATM/ATR signaling in human ESCs and revealed that irradiation induced ATM, ATR, and Chk2 phosphorylation, and γH2AX foci formation and their colocalization with 53BP1 and Rad51 proteins. Interestingly, human ESCs exhibit noninduced γH2AX foci colocalized with Rad51 and marking single-strand DNA breaks. Next, we revealed the significant contribution of ATM, Chk1, and Chk2 kinases to G₂/M block after irradiation and ATM-dependent activation (phosphorylation) of p53 in human ESCs. However, p53 activation and subsequent induction of p21 ^Waf1 gene expression after DNA damage do not result in p21^Waf1 protein accumulation due to its proteasomal degradation.     

Functions of p21Waf1 in norm and in stress

Cyclin-dependent kinase inhibitor p2(Waf1/Cip1/Sdi1/CAP20) 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 p21Waf1 functions. In addition to the cell-cycle control, p21Waf1 participates in important cell processes such as differentiation, senescence, and apoptosis. A balance of p21Waf1 functional activity seems 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 p21Waf1. The review considers the structure of p21Waf1, its posttranslational modification, interactions with various cell or viral proteins, and their effects on the p21Waf1 function and the cell.     

Involvement of MAP-kinase cascades in regulation of sodium-butyrate-induced premature senescence

We have studied the role of the stress kinases p38 and JNK1,2 in premature senescence induced by sodium butyrate (NaBut), a histone deacetylase inhibitor, in mouse embryonic fibroblasts transformed by E1A+cHa-Ras oncogenes. It was found that transformants from p38 knockout cells are able to implement NaBut-induced senescence exhibited by cell cycle arrest, inhibition of proliferation, hypertrophic changes associated with mTORC1 activation and SA-β-galactosidase activity. In jnk1,2 knockouts, the NaButinduced senescence program was inhibited. NaBut-induced senescence in p38 knockouts closely correlates with mTORC1 activation shown by inhibiting mTORC1 with rapamycin. In jnk1,2 knockouts, mTORC1 complex is not activated. We believe that JNK1,2 kinases are required for mTORC1 activation and exhibition of premature senescence markers induced by NaBut in E1A+cHa-Ras transformants.     

DNA damage and senescence in osteoprogenitors expressing Osx1 may cause their decrease with age

Age‐related bone loss in mice results from a decrease in bone formation and an increase in cortical bone resorption. The former is accounted by a decrease in the number of postmitotic osteoblasts which synthesize the bone matrix and is thought to be the consequence of age‐dependent changes in mesenchymal osteoblast progenitors. However, there are no specific markers for these progenitors, and conclusions rely on results from in vitro cultures of mixed cell populations. Moreover, the culprits of such changes remain unknown. Here, we have used Osx1‐Cre;TdRFP mice in which osteoprogenitors express the TdRFP fluorescent protein. We report that the number of TdRFP‐Osx1 cells, freshly isolated from the bone marrow, declines by more than 50% between 6 and 24 months of age in both female and male mice. Moreover, TdRFP‐Osx1 cells from old mice exhibited markers of DNA damage and senescence, such as γH2AX foci, G1 cell cycle arrest, phosphorylation of p53, increased p21^CIP1 levels, as well as increased levels of GATA4 and activation of NF‐κB – two major stimulators of the senescence‐associated secretory phenotype (SASP). Bone marrow stromal cells from old mice also exhibited elevated expression of SASP genes, including several pro‐osteoclastogenic cytokines, and increased capacity to support osteoclast formation. These changes were greatly attenuated by the senolytic drug ABT263. Together, these findings suggest that the decline in bone mass with age is the result of intrinsic defects in osteoprogenitor cells, leading to decreased osteoblast numbers and increased support of osteoclast formation.     

Nuclear Accumulation of p21Cip1 at the Onset of Mitosis: a Role at the G2/M-Phase Transition

Cell cycle arrest in G₁ in response to ionizing radiation or senescence is believed to be provoked by inactivation of G₁ cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21^{Cip1/Waf1/Sdi1}. We provide evidence that in addition to exerting negative control of the G₁/S phase transition, p21 may play a role at the onset of mitosis. In nontransformed fibroblasts, p21 transiently reaccumulates in the nucleus near the G₂/M-phase boundary, concomitant with cyclin B1 nuclear translocation, and associates with a fraction of cyclin A-Cdk and cyclin B1-Cdk complexes. Premitotic nuclear accumulation of cyclin B1 is not detectable in cells with low p21 levels, such as fibroblasts expressing the viral human papillomavirus type 16 E6 oncoprotein, which functionally inactivates p53, or in tumor-derived cells. Moreover, synchronized E6-expressing fibroblasts show accelerated entry into mitosis compared to wild-type cells and exhibit higher cyclin A- and cyclin B1-associated kinase activities. Finally, primary embryonic fibroblasts derived from p21^−/− mice have significantly reduced numbers of premitotic cells with nuclear cyclin B1. These data suggest that p21 promotes a transient pause late in G₂ that may contribute to the implementation of late cell cycle checkpoint controls.     

Activation of DNA damage response signaling in mouse embryonic stem cells

Mouse embryonic stem cells (mESC) are characterized by high proliferation activity. mESC are highly sensitive to genotoxic stresses and do not undergo G1/S checkpoint upon DNA-damage. mESC are supposed to develop sensitive mechanisms to maintain genomic integrity provided by either DNA damage repair or elimination of defected cells by apoptosis. The issue of how mESC recognize the damages and execute DNA repair remains to be studied. We analyzed the kinetics of DNA repair foci marked by antibodies to phosphorylated ATM kinase and histone H2AX (γH2AX). We showed that mESC display non-induced DNA single-strand breaks (SSBs), as revealed by comet-assay, and a noticeable background of γH2AX staining. Exposure of mESC to γ-irradiation induced the accumulation of phosphorylated ATM-kinase in the nucleus as well as the formation of additional γH2AX foci, which disappeared thereafter. To decrease the background of γH2AX staining in control non-irradiated cells, we pre-synchronized mESC at the G2/M by low concentration of nocodazol for a short time (6 h). The cells were then irradiated and stained for γH2AX. Irradiation induced the formation of γH2AX foci both in G2-phase and mitotic cells, which evidenced for the active state of DNA-damage signaling at these stages of the cell cycle in mESC. Due to the G1/S checkpoint is compromised in mESCs, we checked, whether wild-type p53, a target for ATM kinase, was phosphorylated in response to γ-irradiation. The p53 was barely phosphorylated in response to irradiation, which correlated with a very low expression of p53-target p21/Waf1 gene. Thus, in spite of the dysfunction of the p53/Waf1 pathway and the lack of cell cycle checkpoints, the mESC are capable of activating ATM and inducing γH2AX foci formation, which are necessary for the activation of DNA damage response.     

Transfection with the E1A and E1B-19kDa Oncogenes Does Not Prevent Rat Embryo Fibroblasts from Cell-Cycle Arrest after γ-Irradiation

Introduction of the E1A early region of the human adenovirus type 5 impairs the ability of mammalian cells to stop in the cell cycle at G1/S after damage. Two-parameter fluorescence cell sorting with iododeoxyuridine revealed the radiation-induced G1/S arrest in rat embryo fibroblasts transformed with the complementing E1A and E1B-19kDa oncogenes. This was due to selective inhibition of CyclE/Cdk2-associated kinase activity, while activities of type 2 kinase and of CyclA/Cdk2 complexes remained unchanged. The inhibitor of G1-phase cyclin kinases, p21/Waf1, was accumulated and interacted with target kinases both in normal and in transformed cells after irradiation. As shown by immunoprecipitation, p21/Waf1 formed complexes with the E1A oncoproducts in the transformants, which possibly accounted for its functional inactivation. Kinase modification in cyclin–kinase complexes was assumed to play a key role in regulation of cyclin-dependent kinases in the transformants with inactivated p21/Waf1.