MDM2 Promotes Proteasomal Degradation of p21Waf1 via a Conformation Change

MDM2 plays a major role in cancer development and progression via both p53-dependent and -independent functions. One of its p53-independent functions is the induction of the ubiquitin-independent proteasomal degradation of p21^Waf1. The present study was designed to characterize the mechanism(s) by which MDM2 induces p21^Waf1 degradation. We first determined the regions of MDM2 required for p21^Waf1 degradation using pulldown assays and Western blotting and then examined the mechanisms using limited proteolysis and fluorescence resonance energy transfer assays. We found that the MDM2-p21^Waf1 interaction depended on the central domain of MDM2 and that nuclear localization of both proteins was necessary for p21^Waf1 degradation. Specifically, amino acids 226–250 of MDM2 were required for p21^Waf1 binding and degradation, and amino acids 251–260 were necessary for p21^Waf1 degradation. The latter region induced a conformation change in p21^Waf1, increasing its interaction with the C8 subunit of the proteasome, leading to its degradation. When MDM2 lacked either segment (aa 226–250 or aa 251–260), its capacity to promote p21^Waf1 degradation and cell cycle progression was significantly reduced. In summary, the present study elucidated a previously unknown mechanism by which MDM2 promotes the degradation of an intact protein (p21^Waf1) through an ubiquitin-independent proteasomal degradation pathway. Because MDM2 also increases the degradation of other proteins in a ubiquitin-independent manner, this mechanism may underlie part of its tumorigenic properties.     

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.     

Characterization of the p53-Dependent Postmitotic Checkpoint following Spindle Disruption

The p53 tumor suppressor gene product is known to act as part of a cell cycle checkpoint in G₁ following DNA damage. In order to investigate a proposed novel role for p53 as a checkpoint at mitosis following disruption of the mitotic spindle, we have used time-lapse videomicroscopy to show that both p53^+/+ and p53^−/− murine fibroblasts treated with the spindle drug nocodazole undergo transient arrest at mitosis for the same length of time. Thus, p53 does not participate in checkpoint function at mitosis. However, p53 does play a critical role in nocodazole-treated cells which have exited mitotic arrest without undergoing cytokinesis and have thereby adapted. We have determined that in nocodazole-treated, adapted cells, p53 is required during a specific time window to prevent cells from reentering the cell cycle and initiating another round of DNA synthesis. Despite having 4N DNA content, adapted cells are similar to G₁ cells in that they have upregulated cyclin E expression and hypophosphorylated Rb protein. The mechanism of the p53-dependent arrest in nocodazole-treated adapted cells requires the cyclin-dependent kinase inhibitor p21, as p21^−/− fibroblasts fail to arrest in response to nocodazole treatment and become polyploid. Moreover, p21 is required to a similar extent to maintain cell cycle arrest after either nocodazole treatment or irradiation. Thus, the p53-dependent checkpoint following spindle disruption functionally overlaps with the p53-dependent checkpoint following DNA damage.     

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.     

PNAS-4, an Early DNA Damage Response Gene,Induces S Phase Arrest and Apoptosis by Activating Checkpoint Kinases in Lung Cancer Cells

PNAS-4, a novel pro-apoptotic gene, was activated during the early response to DNA damage. Our previous study has shown that PNAS-4 induces S phase arrest and apoptosis when overexpressed in A549 lung cancer cells. However, the underlying action mechanism remains far from clear. In this work, we found that PNAS-4 expression in lung tumor tissues is significantly lower than that in adjacent lung tissues; its expression is significantly increased in A549 cells after exposure to cisplatin, methyl methane sulfonate, and mitomycin; and its overexpression induces S phase arrest and apoptosis in A549 (p53 WT), NCI-H460 (p53 WT), H526 (p53 mutation), and Calu-1 (p53^−/−) lung cancer cells, leading to proliferation inhibition irrespective of their p53 status. The S phase arrest is associated with up-regulation of p21^Waf1/Cip1 and inhibition of the Cdc25A-CDK2-cyclin E/A pathway. Up-regulation of p21^Waf1/Cip1 is p53-independent and correlates with activation of ERK. We further showed that the intra-S phase checkpoint, which occurs via DNA-dependent protein kinase-mediated activation of Chk1 and Chk2, is involved in the S phase arrest and apoptosis. Gene silencing of Chk1/2 rescues, whereas that of ATM or ATR does not affect, S phase arrest and apoptosis. Furthermore, human PNAS-4 induces DNA breaks in comet assays and γ-H2AX staining. Intriguingly, caspase-dependent cleavage of Chk1 has an additional role in enhancing apoptosis. Taken together, our findings suggest a novel mechanism by which elevated PNAS-4 first causes DNA-dependent protein kinase-mediated Chk1/2 activation and then results in inhibition of the Cdc25A-CDK2-cyclin E/A pathway, ultimately causing S phase arrest and apoptosis in lung cancer cells.     

The xeroderma pigmentosum group E gene product DDB2 activates nucleotide excision repair by regulating the level of p21Waf1/Cip1

The xeroderma pigmentosum group E gene product DDB2, a protein involved in nucleotide excision repair (NER), associates with the E3 ubiquitin ligase complex Cul4A-DDB1. But the precise role of these interactions in the NER activity of DDB2 is unclear. Several models, including DDB2-mediated ubiquitination of histones in UV-irradiated cells, have been proposed. But those models lack clear genetic evidence. Here we show that DDB2 participates in NER by regulating the cellular levels of p21^Waf1/Cip1. We show that DDB2 enhances nuclear accumulation of DDB1, which binds to a modified form of p53 containing phosphorylation at Ser18 (p53^S18P) and targets it for degradation in low-dose-UV-irradiated cells. DDB2^−/− mouse embryonic fibroblasts (MEFs), unlike wild-type MEFs, are deficient in the proteolysis of p53^S18P. Accumulation of p53^S18P in DDB2^−/− MEFs causes higher expression p21^Waf1/Cip1. We show that the increased expression of p21^Waf1/Cip1 is the cause NER deficiency in DDB2^−/− cells because deletion or knockdown of p21^Waf1/Cip1 reverses their NER-deficient phenotype. p21^Waf1/Cip1 was shown to bind PCNA, which is required for both DNA replication and NER. Moreover, an increased level of p21^Waf1/Cip1 was shown to inhibit NER both in vitro and in vivo. Our results provide genetic evidence linking the regulation of p21^Waf1/Cip1 to the NER activity of DDB2.     

The p53-p21WAF1 checkpoint pathway plays a protective role in preventing DNA rereplication induced by abrogation of FOXF1 function

We previously identified FOXF1 as a potential tumor suppressor gene with an essential role in preventing DNA rereplication to maintain genomic stability, which is frequently inactivated in breast cancer through the epigenetic mechanism. Here we further addressed the role of the p53-p21^WAF1 checkpoint pathway in DNA rereplication induced by silencing of FOXF1. Knockdown of FOXF1 by small interference RNA (siRNA) rendered colorectal p53-null and p21^WAF1-null HCT116 cancer cells more susceptible to rereplication and apoptosis than the wild-type parental cells. In parental HCT116 cells with a functional p53 checkpoint, the p53-p21^WAF1 checkpoint pathway was activated upon FOXF1 knockdown, which was concurrent with suppression of the CDK2-Rb cascade and induction of G₁ arrest. In contrast, these events were not observed in FOXF1-depleted HCT116-p53−/− and HCT116-p21−/− cells, indicating that the p53-dependent checkpoint function is vital for inhibiting CDK2 to induce G₁ arrest and protect cells from rereplication. The pharmacologic inhibitor (caffeine) of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases abolished activation of the p53-p21^WAF1 pathway upon FOXF1 knockdown, suggesting that suppression of FOXF1 function triggered the ATM/ATR-mediated DNA damage response. Cosilencing of p53 by siRNA synergistically enhanced the effect of FOXF1 depletion on the stimulation of DNA rereplication and apoptosis in wild-type HCT116. Finally, we show that FOXF1 expression is predominantly silenced in breast and colorectal cancer cell lines with inactive p53. Our study demonstrated that the p53-p21^WAF1 checkpoint pathway is an intrinsically protective mechanism to prevent DNA rereplication induced by silencing of FOXF1.     

Decreased translation of p21waf1 mRNA causes attenuated p53 signaling in some p53 wild-type tumors

DNA damage induces cell cycle arrest through both Chk1 and the p53 tumor suppressor protein, the latter arresting cells through induction of p21^waf1 protein. Arrest permits cells to repair the damage and recover. The frequent loss of p53 in tumor cells makes them more dependent on Chk1 for arrest and survival. However, some p53 wild type tumor cell lines, such as HCT116 and U2OS, are also sensitive to inhibition of Chk1 due to attenuated p21^waf1 induction upon DNA damage. The purpose of this study is to determine the cause of this attenuated p21^waf1 protein induction. We find that neither the induction of p21^waf1 mRNA nor protein half-life is sufficient to explain the low p21^waf1 protein levels in HCT116 and U2OS cells. The induced mRNA associates with polysomes but little protein is made suggesting these two cell lines have a reduced rate of p21^waf1 mRNA translation. This represents a novel mechanism for disruption of the p53-p21^waf1 pathway as currently known mechanisms involve either mutation of p53 or reduction of p53 protein levels. As a consequence, this attenuated p21^waf1 expression may render some p53 wild type tumors sensitive to a combination of DNA damage plus checkpoint inhibition.     

Decreased translation of p21waf1 mRNA causes attenuated p53 signaling in some p53 wild-type tumors

DNA damage induces cell cycle arrest through both Chk1 and the p53 tumor suppressor protein, the latter arresting cells through induction of p21^waf1 protein. Arrest permits cells to repair the damage and recover. The frequent loss of p53 in tumor cells makes them more dependent on Chk1 for arrest and survival. However, some p53 wild type tumor cell lines, such as HCT116 and U2OS, are also sensitive to inhibition of Chk1 due to attenuated p21^waf1 induction upon DNA damage. The purpose of this study is to determine the cause of this attenuated p21^waf1 protein induction. We find that neither the induction of p21^waf1 mRNA nor protein half-life is sufficient to explain the low p21^waf1 protein levels in HCT116 and U2OS cells. The induced mRNA associates with polysomes but little protein is made suggesting these two cell lines have a reduced rate of p21^waf1 mRNA translation. This represents a novel mechanism for disruption of the p53-p21^waf1 pathway as currently known mechanisms involve either mutation of p53 or reduction of p53 protein levels. As a consequence, this attenuated p21^waf1 expression may render some p53 wild type tumors sensitive to a combination of DNA damage plus checkpoint inhibition.     

p53 checkpoint-defective cells are sensitive to X rays, but not hypoxia

X-ray-induced damage leads to cell-cycle "checkpoint" arrest by p53-dependent induction of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1). Human tumor cells that lack this response fail to arrest after exposure to DNA-damaging agents, undergo multiple rounds of endoreduplicative DNA synthesis, and eventually commit to an apoptotic cell death. Since low oxygen tension can also induce p53 protein accumulation, and can lead to cell-cycle arrest or apoptosis, we examined the expression of p21 in tumor cells under normoxic and hypoxic conditions. In a survey of cells, mRNA for the p21 gene was induced two- to threefold in response to hypoxia in a seemingly p53-independent manner. We therefore examined genetically matched cells that differ in their p21 and p53 status for response to ionizing radiation and hypoxia. We found that both p21-deficient and p53-deficient cells exhibit an increase in chromosome instability, an increased level of apoptosis, and a failure to arrest after exposure to ionizing radiation. However, cells that lack either p21 or p53 exhibit no increase in chromosome instability or elevated apoptosis and still arrest in response to hypoxia. Thus, the mechanism responsible for the differential response to either hypoxia or X rays presumably lies in the control of cell-cycle progression in response to stress and its dependence on p21. Since the loss of a DNA-damage-dependent checkpoint does not sensitize cells to killing by stresses that elicit a DNA-damage-independent checkpoint, targeting the function of p21 pharmacologically will not kill tumor cells in situ in the absence of a DNA damage signal.     

Suppression of Polo like kinase 1 (PLK1) by p21(Waf1) mediates the p53-dependent prevention of caspase-independent mitotic death

Polo-like kinase 1 (Plk1) plays key roles in many aspects of mitosis. We have previously shown that induction of p21^Waf1 by p53 is responsible for protection of cells against adriamycin-induced polyploidy formation and mitotic catastrophe. Here we show that adriamycin treatment suppressed Plk1 expression in a p53- and p21^Waf1-dependent manner. Ablation of p21^Waf1 inhibited the adriamycin-induced p53 activation, and this inhibition was alleviated by knockdown of Plk1, suggesting that p21^Waf1-dependent suppression of Plk1 expression is responsible for maintaining p53 activation during stress response. Plk1 associated with p53 and disrupted its interaction with target gene promoters in cells treated with adriamycin. Overexpression of Plk1 inhibited the p53-mediated prevention of caspase-independent mitotic death, but not polyploidy formation, in adriamycin-treated cells. Together our results indicate that suppression of Plk1 by p21^Waf1 is responsible for p53-dependent protection against adriamycin-induced caspase-independent mitotic death.     

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.     

Nuclear translocation of Cyclin B1 marks the restriction point for terminal cell cycle exit in G2 phase

Upon DNA damage, cell cycle progression is temporally blocked to avoid propagation of mutations. While transformed cells largely maintain the competence to recover from a cell cycle arrest, untransformed cells past the G1/S transition lose mitotic inducers, and thus the ability to resume cell division. This permanent cell cycle exit depends on p21, p53, and APC/C^Cdh1. However, when and how permanent cell cycle exit occurs remains unclear. Here, we have investigated the cell cycle response to DNA damage in single cells that express Cyclin B1 fused to eYFP at the endogenous locus. We find that upon DNA damage Cyclin B1-eYFP continues to accumulate up to a threshold level, which is reached only in G2 phase. Above this threshold, a p21 and p53-dependent nuclear translocation required for APC/C^Cdh1-mediated Cyclin B1-eYFP degradation is initiated. Thus, cell cycle exit is decoupled from activation of the DNA damage response in a manner that correlates to Cyclin B1 levels, suggesting that G2 activities directly feed into the decision for cell cycle exit. Once Cyclin B1-eYFP nuclear translocation occurs, checkpoint inhibition can no longer promote mitotic entry or re-expression of mitotic inducers, suggesting that nuclear translocation of Cyclin B1 marks the restriction point for permanent cell cycle exit in G2 phase.     

Epimorphic regeneration in mice is p53-independent

The process of regeneration is most readily studied in species of sponge, hydra, planarian, and salamander (i.e., newt and axolotl). The closure of MRL mouse ear pinna through-and-through holes provides a model of unusual wound healing/regeneration in which a blastema-like structure closes the ear hole and cartilage and hair follicles are replaced. Recent studies, based on a broad level of DNA damage and a cell cycle pattern of G2/M "arrest," showed that p21^Cip1/Waf1 was missing from the MRL mouse ear and that a p21-null mouse could close its ear holes. Given the p53/p21 axis of control of DNA damage, cell cycle arrest, apoptosis, and senescence, we tested the role of p53 in the ear hole regenerative response. Using backcross mice, we found that loss of p53 in MRL mice did not show reduced healing. Furthermore, cross sections of MRL.p53^-/- mouse ears at 6 weeks post-injury showed an increased level of adipocytes and chondrocytes in the region of healing whereas MRL or p21^-/- mice show chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cycle-related mutant mice to determine how p21 was being regulated. We demonstrate that p16 and Gadd45 null mice show little healing capacity. Interestingly, a partial healing phenotype in mice with a dual Tgfb/Rag2 knockout mutation was seen. These data demonstrate an independence of p53 signaling for mouse appendage regeneration and suggest that the role of p21 in this process is possibly through the abrogation of the Tgfb/Smad pathway.