5-aza-2'-deoxycytidine activates the p53/p21Waf1/Cip1 pathway to inhibit cell proliferation

In addition to its demethylating function, 5-aza-2'-deoxycytidine (5-aza-CdR) also plays an important role in inducing cell cycle arrest, differentiation, and cell death. However, the mechanism by which 5-aza-CdR induces antineoplastic activity is not clear. In this study, we found that 5-aza-CdR at limited concentrations (0.01-5 microm) induces inhibition of cell proliferation as well as increased p53/p21(Waf1/Cip1) expression in A549 cells (wild-type p53) but not in H1299 (p53-null) and H719 cells (p53 mutant). The p53-dependent p21(Waf1/Cip1) expression induced by 5-aza-CdR was not seen in A549 cells transfected with the wild-type human papilloma virus type-16 E6 gene that induces p53 degradation. Furthermore, deletion analysis and site-directed mutagenesis of the p21 promoter reveals that 5-aza-CdR induces p21(Waf1/Cip1) expression through two p53 binding sites in the p21 promoter. Finally, 5-aza-CdR-induced p21(Waf1/Cip1) expression was dependent on DNA damage but not on DNA demethylation as demonstrated by comet assay and bisulfite sequencing, respectively. Our data provide useful clues for judging the therapeutic efficacy of 5-aza-CdR in the treatment of human cancer cells.     

1,25-Dihydroxycholecalciferol enhances butyrate-induced p21(Waf1/Cip1) expression

Butyrate, a short-chain fatty acid produced in the colon, as well as its prodrug tributyrin, reduce proliferation and increase differentiation of colon cancer cells. p21(Waf1/Cip1) and p27(Kip1) are negative regulators of cell cycle and are thought to have a key function in the differentiation of various cell lines. We studied the effects of butyrate on differentiation, VDR expression, as well as on p21(Waf1/Cip1) and p27(Kip1) expression in human colon cancer cells (Caco-2). Butyrate induced cell differentiation, which was further enhanced after addition of 1,25-dihydroxycholecalciferol. Synergistic effect of butyrate and dihydroxycholecalciferol in Caco-2 cells was due to butyrate-induced overexpression of VDR. While butyrate as well as dihydroxycholecalciferol increased p21(Waf1/Cip1) and p27(Kip1) expression, in contrast combined exposure of butyrate and dihydroxycholecalciferol resulted in a synergistic amplification of p21(Waf1/Cip1), but not of p27(Kip1) expression. These data imply that butyrate selectively increases p21(Waf1/Cip1) expression via upregulation of VDR in Caco-2 cells.     

Age-dependent changes of p57(Kip2) and p21(Cip1/Waf1) expression in skeletal muscle and lung of mice

p57(Kip2) and p21(Cip1/Waf1) are members of cyclin-dependent kinase (Cdk) inhibitors which play critical roles in the terminal differentiation of skeletal muscle and lung. We investigated mRNA levels of p57(Kip2) and p21(Cip1/Waf1) in skeletal muscle and lung of mice during maturation and aging using Northern hybridization. The mRNA levels of p57(Kip2) and p21(Cip1/Waf1) decreased in skeletal muscle and lung of mice during maturation and aging except that the level of p21(Cip1/Waf1) mRNA in skeletal muscle of mice showed an increase only during maturation. The decrease of the p57(Kip2) mRNA level involved neither a change of DNA methylation at the promoter region nor an alteration of the imprinting status in aged mice. The decreases of p57(Kip2) and p21(Cip1/Waf1) mRNA levels during aging suggest that the process of tissue-specific terminal differentiation may be gradually downregulated with senescence in tissues where p57(Kip2) and p21(Cip1/Waf1) play key roles in differentiation. The downregulation of p57(Kip2) and p21(Cip1/Waf1) during aging is contrary to the upregulation of Cdk inhibitors during cellular replicative senescence, indicating that aging in an organismal level is mediated by mechanisms different from replicative senescence of cultured cells.     

Sustained activation of the JNK cascade and rapamycin-induced apoptosis are suppressed by p53/p21(Cip1)

Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces apoptosis of cells lacking functional p53. Cells expressing wild-type p53 or p21(Cip1)arrest in G1 and remain viable. In cells lacking functional p53, rapamycin or amino acid deprivation induces rapid and sustained activation of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase, and elevation of phosphorylated c-Jun that results in apoptosis. This stress response depends on expression of eukaryotic initiation factor 4E binding protein 1 and is suppressed by p21(Cip1) independent of cell cycle arrest. Rapamycin induces p21(Cip1) binding to ASK1, suppressing kinase activity and attenuating cellular stress. These results suggest that inhibition of mTOR triggers a potentially lethal response that is prevented only in cells expressing p21(Cip1).     

Protective role of p21(Waf1/Cip1) against prostaglandin A2-mediated apoptosis of human colorectal carcinoma cells.

Prostaglandin A2 (PGA2) suppresses tumor growth in vivo, is potently antiproliferative in vitro, and is a model drug for the study of the mammalian stress response. Our previous studies using breast carcinoma MCF-7 cells suggested that p21(Waf1/Cip1) induction enabled cells to survive PGA2 exposure. Indeed, the marked sensitivity of human colorectal carcinoma RKO cells to the cytotoxicity of PGA2 is known to be associated with a lack of a PGA2-mediated increase in p21(Waf1/Cip1) expression, inhibition of cyclin-dependent kinase activity, and growth arrest. To determine if cell death following exposure to PGA2 could be prevented by forcing the expression of p21(Waf1/Cip1) in RKO cells, we utilized an adenoviral vector-based expression system. We demonstrate that ectopic expression of p21(Waf1/Cip1) largely rescued RKO cells from PGA2-induced apoptotic cell death, directly implicating p21(Waf1/Cip1) as a determinant of the cellular outcome (survival versus death) following exposure to PGA2. To discern whether p21(Waf1/Cip1)-mediated protection operates through the implementation of cellular growth arrest, other growth-inhibitory treatments were studied for the ability to attenuate PGA2-induced cell death. Neither serum depletion nor suramin (a growth factor receptor antagonist) protected RKO cells against PGA2 cytotoxicity, and neither induced p21(Waf1/Cip1) expression. Mimosine, however, enhanced p21(Waf1/Cip1) expression, completely inhibited RKO cell proliferation, and exerted marked protection against a subsequent PGA2 challenge. Taken together, our results directly demonstrate a protective role for p21(Waf1/Cip1) during PGA2 cellular stress and provide strong evidence that the implementation of cellular growth arrest contributes to this protective influence.     

Stoichiometry of cyclin A-cyclin-dependent kinase 2 inhibition by p21Cip1/Waf1

Progression through the eukaryotic cell cycle is regulated by phosphorylation, which is catalyzed by cyclin-dependent kinases. Cyclin-dependent kinases are regulated through several mechanisms, including negative regulation by p21 (variously called CAP20, Cip1, Sdi1, and WAF1). It has been proposed that multiple p21 molecules are required to inhibit cyclin-dependent kinases, such that p21 acts as a sensitive buffer of cyclin-dependent kinase activity or as an assembly factor for the complexes formed by the cyclins and cyclin-dependent kinases. Using purified, full-length proteins of known concentration (determined by absorbance) and cyclin A-Cdk2 of known activity (calibrated with staurosporine), we find that a 1:1 molar ratio of p21 to cyclin A-Cdk2 is able to inhibit Cdk2 activity both in the binary cyclin A-Cdk2 complex and in the presence of proliferating cell nuclear antigen (PCNA). Our results indicate that the mechanism of p21 inhibition of cyclin A-Cdk2 does not involve multiple molecules of bound p21.     

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.     

Solution structure of p21(Waf1/Cip1/Sdi1) C-terminal domain bound to Cdk4.

Cyclin-dependent kinase (Cdk) inhibitor p21(Waf1/Cip1/Sdi1), a multifunctional protein, has a major role as tumor suppressor, mediating G1/S arrest through inhibition of Cdks. Recent biological studies of Cyclin D1/Cdk4 have proposed that p21 C-terminal domain (p21(CT)) plays a key role as a potent Cdk4 inhibitor. We report here solution structures of p21(CT) for both the free and Cdk4-bound forms using 2D transferred NOE spectroscopy and dynamical simulated annealing calculations. Even though p21(CT) peptide is very flexible in the free state, when it bound to Cdk4, the structure becomes well structured in the binding domain. Therefore we propose that p21(CT) experiences an extensive conformational change upon Cdk4 binding. This structural change of p21(CT) may suggest the molecular mechanism of p21 for specificity and inhibition mode to assemble different cyclin-Cdk complexes. Especially, our data suggests that the D(149)FYHSKRR(156) region of p21 is critical for Cdk4 binding, indicating that the major driving force for complex originates from hydrophobic interaction between p21 and Cdk4.     

Acetylation of p53 at lysine 373/382 by the histone deacetylase inhibitor depsipeptide induces expression of p21(Waf1/Cip1)

Generally, histone deacetylase (HDAC) inhibitor-induced p21(Waf1/Cip1) expression is thought to be p53 independent. Here we found that an inhibitor of HDAC, depsipeptide (FR901228), but not trichostatin A (TSA), induces p21(Waf1/Cip1) expression through both p53 and Sp1/Sp3 pathways in A549 cells (which retain wild-type p53). This is demonstrated by measuring relative luciferase activities of p21 promoter constructs with p53 or Sp1 binding site mutagenesis and was further confirmed by transfection of wild-type p53 into H1299 cells (p53 null). That p53 was acetylated after depsipeptide treatment was tested by sequential immunoprecipitation/Western immunoblot analysis with anti-acetylated lysines and anti-p53 antibodies. The acetylated p53 has a longer half-life due to a significant decrease in p53 ubiquitination. Further study using site-specific antiacetyllysine antibodies and transfection of mutated p53 vectors (K319/K320/K321R mutated and K373R/K382R mutations) into H1299 cells revealed that depsipeptide specifically induces p53 acetylation at K373/K382, but not at K320. As assayed by coimmunoprecipitation, the K373/K382 acetylation is accompanied by a recruitment of p300, but neither CREB-binding protein (CBP) nor p300/CBP-associated factor (PCAF), to the p53 C terminus. Furthermore, activity associated with the binding of the acetylated p53 at K373/K382 to the p21 promoter as well as p21(Waf1/Cip1) expression is significantly increased after depsipeptide treatment, as tested by chromatin immunoprecipitations and Western blotting, respectively. In addition, p53 acetylation at K373/K382 is confirmed to be required for recruitment of p300 to the p21 promoter, and the depsipeptide-induced p53 acetylation at K373/K382 is unlikely to be dependent on p53 phosphorylation at Ser15, Ser20, and Ser392 sites. Our data suggest that p53 acetylation at K373/K382 plays an important role in depsipeptide-induced p21(Waf1/Cip1) expression.     

EGF receptor-dependent JNK activation is involved in arsenite-induced p21Cip1/Waf1 upregulation and endothelial apoptosis

Arsenic exposure is associated with an increased risk of atherosclerosis and vascular diseases. Although endothelial cells have long been considered to be the primary targets of arsenic toxicity, the underlying molecular mechanism remains largely unknown. In this study, we sought to explore the signaling pathway triggered by sodium arsenite and its implication for endothelial phenotype. We found that sodium arsenite produced time- and dose-dependent decreases in human umbilical vein endothelial cell viability. This effect correlated with the induction of p21Cip1/Waf1 (up to 10-fold), a regulatory protein of cell cycle and apoptosis. We also found that arsenite-stimulated EGF (ErbB1) and ErbB2 receptor transactivation, manifest as receptor tyrosine phosphorylation, appeared to be a proximal signaling event leading to p21Cip1/Waf1 induction, because both pharmacological inhibitors and knockdown of receptors by RNA interference blocked arsenite-induced p21Cip1/Waf1 upregulation. Arsenite-induced activation of JNK and p38 MAPK was distinct, with only JNK as a downstream target of the EGF receptor. Moreover, inhibition of JNK with SP-600125 or dominant negative MKK7 inhibited only p21Cip1/Waf1 induction, whereas the p38 MAPK inhibitor SB-203580 or dominant negative MKK4 inhibited both p21Cip1/Waf1 and p53 induction. Functionally, inhibition of p21Cip1/Waf1 induction prevented endothelial apoptosis due to arsenite treatment. Insofar as endothelial dysfunction promotes vascular disease, these data provide a mechanism for the increased incidence of cardiovascular disease due to arsenite exposure.