Genetic Characterization of the Role of the Cip/Kip Family of Proteins as Cyclin-Dependent Kinase Inhibitors and Assembly Factors

The Cip/Kip family, namely, p21^Cip1, p27^Kip1, and p57^Kip2, are stoichiometric cyclin-dependent kinase inhibitors (CKIs). Paradoxically, they have been proposed to also act as positive regulators of Cdk4/6-cyclin D by stabilizing these heterodimers. Loss of p21^Cip1 and p27^Kip1 reduces Cdk4/6-cyclin D complexes, although with limited phenotypic consequences compared to the embryonic lethality of Cdk4/6 or triple cyclin D deficiency. This milder phenotype was attributed to Cdk2 compensatory mechanisms. To address this controversy using a genetic approach, we generated Cdk2^−/− p21^−/− p27^−/− mice. Triple-knockout mouse embryonic fibroblasts (MEFs) displayed minimal levels of D-type cyclins and Cdk4/6-cyclin D complexes. p57^Kip2 downregulation in the absence of p21^Cip1 and p27^Kip1 aggravated this phenotype, yet MEFs lacking all Cip/Kip proteins exhibited increased retinoblastoma phosphorylation, together with enhanced proliferation and transformation capacity. In vivo, Cdk2 ablation induced partial perinatal lethality in p21^−/− p27^−/− mice, suggesting partial Cdk2-dependent compensation. However, Cdk2^−/− p21^−/− p27^−/− survivors displayed all phenotypes described for p27^−/− mice, including organomegalia and pituitary tumors. Thus, Cip/Kip deficiency does not impair interphasic Cdk activity even in the absence of Cdk2, suggesting that their Cdk-cyclin assembly function is dispensable for homeostatic control in most cell types.     

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.     

Involvement of p53 and p21 in Cellular Defects and Tumorigenesis in Atm−/− Mice

Disruption of the mouse Atm gene, whose human counterpart is consistently mutated in ataxia-telangiectasia (A-T) patients, creates an A-T mouse model exhibiting most of the A-T-related systematic and cellular defects. While ATM plays a major role in signaling the p53 response to DNA strand break damage, Atm^−/− p53^−/− mice develop lymphomas earlier than Atm^−/− or p53^−/− mice, indicating that mutations in these two genes lead to synergy in tumorigenesis. The cell cycle G₁/S checkpoint is abolished in Atm^−/− p53^−/− mouse embryonic fibroblasts (MEFs) following γ-irradiation, suggesting that the partial G₁ cell cycle arrest in Atm^−/− cells following γ-irradiation is due to the residual p53 response in these cells. In addition, the Atm^−/− p21^−/− MEFs are more severely defective in their cell cycle G₁ arrest following γ-irradiation than Atm^−/− and p21^−/− MEFs. The Atm^−/− MEFs exhibit multiple cellular proliferative defects in culture, and an increased constitutive level of p21 in these cells might account for these cellular proliferation defects. Consistent with this notion, Atm^−/− p21^−/− MEFs proliferate similarly to wild-type MEFs and exhibit no premature senescence. These cellular proliferative defects are also rescued in Atm^−/− p53^−/− MEFs and little p21 can be detected in these cells, indicating that the abnormal p21 protein level in Atm^−/− cells is also p53 dependent and leads to the cellular proliferative defects in these cells. However, the p21 mRNA level in Atm^−/− MEFs is lower than that in Atm^+/+ MEFs, suggesting that the higher level of constitutive p21 protein in Atm^−/− MEFs is likely due to increased stability of the p21 protein.     

p21Waf1/Cip1 Inhibition of Cyclin E/Cdk2 Activity Prevents Endoreduplication after Mitotic Spindle Disruption

During a normal cell cycle, entry into S phase is dependent on completion of mitosis and subsequent activation of cyclin-dependent kinases (Cdks) in G₁. These events are monitored by checkpoint pathways. Recent studies and data presented herein show that after treatment with microtubule inhibitors (MTIs), cells deficient in the Cdk inhibitor p21^Waf1/Cip1 enter S phase with a ≥4N DNA content, a process known as endoreduplication, which results in polyploidy. To determine how p21 prevents MTI-induced endoreduplication, the G₁/S and G₂/M checkpoint pathways were examined in two isogenic cell systems: HCT116 p21^+/+ and p21^−/− cells and H1299 cells containing an inducible p21 expression vector (HIp21). Both HCT116 p21^−/− cells and noninduced HIp21 cells endoreduplicated after MTI treatment. Analysis of G₁-phase Cdk activities demonstrated that the induction of p21 inhibited endoreduplication through direct cyclin E/Cdk2 regulation. The kinetics of p21 inhibition of cyclin E/Cdk2 activity and binding to proliferating-cell nuclear antigen in HCT116 p21^+/+ cells paralleled the onset of endoreduplication in HCT116 p21^−/− cells. In contrast, loss of p21 did not lead to deregulated cyclin D1-dependent kinase activities, nor did p21 directly regulate cyclin B1/Cdc2 activity. Furthermore, we show that MTI-induced endoreduplication in p53-deficient HIp21 cells was due to levels of p21 protein below a threshold required for negative regulation of cyclin E/Cdk2, since ectopic expression of p21 restored cyclin E/Cdk2 regulation and prevented endoreduplication. Based on these findings, we propose that p21 plays an integral role in the checkpoint pathways that restrain normal cells from entering S phase after aberrant mitotic exit due to defects in microtubule dynamics.     

DDB2, an Essential Mediator of Premature Senescence

Reactive oxygen species (ROS) is critical for premature senescence, a process significant in tumor suppression and cancer therapy. Here, we reveal a novel function of the nucleotide excision repair protein DDB2 in the accumulation of ROS in a manner that is essential for premature senescence. DDB2-deficient cells fail to undergo premature senescence induced by culture shock, exogenous oxidative stress, oncogenic stress, or DNA damage. These cells do not accumulate ROS following DNA damage. The lack of ROS accumulation in DDB2 deficiency results from high-level expression of the antioxidant genes in vitro and in vivo. DDB2 represses antioxidant genes by recruiting Cul4A and Suv39h and by increasing histone-H3K9 trimethylation. Moreover, expression of DDB2 also is induced by ROS. Together, our results show that, upon oxidative stress, DDB2 functions in a positive feedback loop by repressing the antioxidant genes to cause persistent accumulation of ROS and induce premature senescence.DDB2 is encoded by the nucleotide excision repair (NER) XPE gene (17, 24, 33). Unlike other NER gene-deficient cells or xeroderma pigmentosum (XP) cells, the XPE cells exhibit only a mild deficiency in NER (55). However, because of its high affinity for cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts, several studies implicated DDB2 in the early damaged-DNA recognition step of NER (61). However, a direct role of DDB2 in NER is a point of controversy (28, 41, 57). Lower organisms (yeasts), in which other XP genes are conserved, apparently do not encode a DDB2 homolog (55, 64). We showed that DDB2 associates with Cul4, a component of an E3 ubiquitin ligase complex that is now known to involve the DDB2 binding protein DDB1 as its adapter (48). The Cul4-DDB1 E3 ligase associates with a number of substrate-specific adapter proteins to target substrates for ubiquitination (30, 35). DDB2 is believed to be one of those substrate adapters, which allows Cul4-DDB1 to target specific proteins. Two studies suggested that the Cul4A-DDB1-DDB2 complex could participate in the ubiquitination of histones, indicating a role of DDB2 in chromatin remodeling (23, 59). Other investigators suggested a role of Cul4A-DDB1-DDB2 in the ubiquitination of XPC (15, 52). We recently found that DDB2 is involved also in targeting p21 for proteolysis and demonstrated that DDB2 stimulated NER by regulating the level of p21 (51).It was shown elsewhere that DDB2^−/− mouse embryonic fibroblasts (MEFs) are resistant to UV-induced apoptosis (20, 21). Recently, we extended those observations by demonstrating that DDB2^−/− MEFs or DDB2-deficient human cells are resistant to apoptosis induced by a variety of DNA-damaging agents (50). Moreover, DDB2^−/− MEFs are deficient in E2F1-induced apoptosis. The resistance to apoptosis is linked also to high-level accumulation of p21 because deletion of p21 restored apoptosis. The polyubiquitination of p21 is significantly reduced in DDB2-deficient cells (50), suggesting that after DNA damage DDB2 plays a key role in polyubiquitinating p21. Also, we observed evidence for a physical association between DDB2 and p21, which was increased in UV-irradiated cells (50), indicating that DDB2 plays a direct role in targeting p21 for proteolysis after DNA damage. These observations provided evidence that DDB2, in addition to stimulating NER, plays a significant role in terminating DNA damage checkpoint, allowing cells with extensive DNA damage to undergo apoptosis.In addition to its role in the inhibition of cell cycle and apoptosis, p21 has been implicated also in cellular senescence, as its level increases in senescent cells (7). Cellular senescence is defined as a proliferative arrest of a cell after a limited number of cell divisions while the cell remains metabolically and synthetically active (6, 63). Senescence can be triggered by both extrinsic factors such as oncogenic stress, DNA damage, oxidative stress, and culture shock and intrinsic factors such as telomere regression in human cells (19). When grown in cell culture medium, human diploid fibroblasts undergo 60 to 80 population doublings, after which they cease proliferation as a result of telomere erosion and enter into the stage of replicative senescence characterized by enlarged and flattened morphology, increased granularity, and enhanced senescence-associated β-galactosidase (SA-β-Gal) activity (13). In contrast, telomere length does not limit the ability of the murine fibroblasts to proliferate in culture. It was shown that the supraphysiological level of oxygen or reactive oxygen species (ROS) under which the cells are grown led murine fibroblasts to senesce (39). ROS accumulation or oxidative stress induces the senescent phenotype in response to oncogenic stress as well as in response to DNA-damaging agents (56). These pathways have been termed premature senescence, which recapitulates molecular features of replicative senescence. Premature senescence induced by oncogene expression is a significant mechanism of tumor suppression involving the Ink4a/Arf locus (47). Moreover, DNA damage-induced premature senescence is significant, as many anticancer drugs have been shown to induce premature senescence of tumor cells (12, 44).Because DDB2^−/− MEFs express p21 at a high level, we expected those cells to undergo premature senescence at an earlier passage than the wild-type (WT) MEFs. Surprisingly, we found that DDB2^−/− MEFs escape senescence at a very high frequency. Moreover, DDB2^−/− MEFs or DDB2-deficient human cells are resistant to premature senescence induced by a variety of agents, including oncogenic stress, exogenous oxidative stress, and DNA damage. The lack of premature senescence in the presence of high-level p21, especially after DNA damage, suggests that DDB2 functions in the senescence program through a mechanism that is downstream of the p21 pathway senescence. Here we show that DDB2 participates in the senescence program by inducing persistent accumulation of ROS.     

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.     

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.     

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 mammalian 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 showed chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cyclerelated 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 Tgfβ/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 Tgfβ/Smad pathway.Key words: mouse, regeneration, p53, p21, MRL, ear-hole, Tgfβ     

p38δ Regulates p53 to Control p21Cip1 Expression in Human Epidermal Keratinocytes

PKCδ suppresses keratinocyte proliferation via a mechanism that involves increased expression of p21^Cip1. However, the signaling mechanism that mediates this regulation is not well understood. Our present studies suggest that PKCδ activates p38δ leading to increased p21^Cip1 promoter activity and p21^Cip1 mRNA/protein expression. We further show that exogenously expressed p38δ increases p21^Cip1 mRNA and protein and that p38δ knockdown or expression of dominant-negative p38 attenuates this increase. Moreover, p53 is an intermediary in this regulation, as p38δ expression increases p53 mRNA, protein, and promoter activity, and p53 knockdown attenuates the activation. We demonstrate a direct interaction of p38δ with PKCδ and MEK3 and show that exogenous agents that suppress keratinocyte proliferation activate this pathway. We confirm the importance of this regulation using a stratified epidermal equivalent model, which mimics in vivo-like keratinocyte differentiation. In this model, PKCδ or p38δ knockdown results in reduced p53 and p21^Cip1 levels and enhanced cell proliferation. We propose that PKCδ activates a MEKK1/MEK3/p38δ MAPK cascade to increase p53 levels and p53 drives p21^Cip1 gene expression.     

Hepatitis C Virus Core Protein Down-Regulates p21Waf1/Cip1 and Inhibits Curcumin-Induced Apoptosis through MicroRNA-345 Targeting in Human Hepatoma Cells

Background

Hepatitis C virus (HCV) has been reported to regulate cellular microRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma, but HCV core-modulated cellular microRNAs are unknown. The HCV core protein regulates p21^Waf1/Cip1 expression. However, the mechanism of HCV core-associated p21^Waf1/Cip1 regulation remains to be further clarified. Therefore, we attempted to determine whether HCV core-modulated cellular microRNAs play an important role in regulating p21^Waf1/Cip1 expression in human hepatoma cells.

Methods

Cellular microRNA profiling was investigated in core-overexpressing hepatoma cells using TaqMan low density array. Array data were further confirmed by TaqMan real-time qPCR for single microRNA in core-overexpressing and full-length HCV replicon-expressing cells. The target gene of microRNA was examined by reporter assay. The gene expression was determined by real-time qPCR and Western blotting. Apoptosis was examined by annexin V-FITC apoptosis assay. Cell cycle analysis was performed by propidium iodide staining. Cell proliferation was analyzed by MTT assay.

Results

HCV core protein up- or down-regulated some cellular microRNAs in Huh7 cells. HCV core-induced microRNA-345 suppressed p21^Waf1/Cip1 gene expression through targeting its 3′ untranslated region in human hepatoma cells. Moreover, the core protein inhibited curcumin-induced apoptosis through p21^Waf1/Cip1-targeting microRNA-345 in Huh7 cells.

Conclusion and Significance

HCV core protein enhances the expression of microRNA-345 which then down-regulates p21^Waf1/Cip1 expression. It is the first time that HCV core protein has ever been shown to suppress p21^Waf1/Cip1 gene expression through miR-345 targeting.     

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.     

Amlodipine inhibits cell proliferation via PKD1-related pathway

Human coronary artery smooth muscle cell (hCASMC) proliferation is involved in the progression of coronary artery disease. Amlodipine, a widely used antihypertensive drug, exerts antiproliferative effects by increasing the expression of p21^(Waf1/Cip1). Polycystic kidney disease 1 (PKD1) is also involved in cell cycle inhibition via p21^(Waf1/Cip1) up-regulation. We clarified the involvement of PKD1-related signaling on hCASMCs. Cultured hCASMCs, which constitutively express PKD1, were stimulated with 5% serum. Amlodipine increased p21^(Waf1/Cip1) expression in a dose- and time-dependent manner, resulting in reduced hCASMC proliferation. The inhibitory effect of amlodipine was mimicked by overexpression of PKD1 and was reversed by a dominant-negative version of PKD1 (R4227X). Immunoblot analysis showed that phosphorylated JAK2 was increased by amlodipine treatment or PKD1 overexpression. A luciferase assay revealed that the overexpression of PKD1 induced STAT1 enhancer activity. These data suggest that PKD1 contributes to the antiproliferative effect of amlodipine on hCASMCs via JAK/STAT signaling and p21^(Waf1/Cip1) up-regulation.     

p53-independent induction of p21(waf1/cip1) contributes to the activation of caspases in GTP-depletion-induced apoptosis of insulin-secreting cells

We investigated the role of some key regulators of cell cycle in the activation of caspases during apoptosis of insulin-secreting cells after sustained depletion of GTP by a specific inosine 5'-monophosphate dehydrogenase inhibitor, mycophenolic acid (MPA). p21(Waf1/Cip1) was significantly increased following MPA treatment, an event closely correlated with the time course of caspase activation under the same conditions. MPA-induced p21(Waf1/Cip1) was not mediated by p53, since p53 mass was gradually reduced over time of MPA treatment. The increment of p21(Waf1/Cip1) by MPA was further enhanced in the presence of a pan-caspase inhibitor, indicating that the increased p21(Waf1/Cip1) may occur prior to caspase activation. This notion of association of p21(Waf1/Cip1) accumulation with caspase activation and apoptosis was substantiated by using mimosine, a selective p21(Waf1/Cip1) inducer independent of p53. Mimosine, like MPA, also increased p21(Waf1/Cip1), promoted apoptosis and simultaneously increased the activity of caspases. Furthermore, knocking down of p21(Waf1/Cip1) transfection of siRNA duplex inhibited caspase activation and apoptosis due to GTP depletion. In contrast to p21(Waf1/Cip1), a reduction in p27(Kip1) occurred in MPA-treated cells. These results indicate that p21(Waf1/Cip1) may act as an upstream signal to block mitogenesis and activate caspases which in turn contribute to induction of apoptosis.     

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.