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.     

HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells

Reendothelialization involves endothelial progenitor cell (EPC) homing, proliferation, and differentiation, which may be influenced by fluid shear stress and local flow pattern. This study aims to elucidate the role of laminar flow on embryonic stem (ES) cell differentiation and the underlying mechanism. We demonstrated that laminar flow enhanced ES cell-derived progenitor cell proliferation and differentiation into endothelial cells (ECs). Laminar flow stabilized and activated histone deacetylase 3 (HDAC3) through the Flk-1-PI3K-Akt pathway, which in turn deacetylated p53, leading to p21 activation. A similar signal pathway was detected in vascular endothelial growth factor-induced EC differentiation. HDAC3 and p21 were detected in blood vessels during embryogenesis. Local transfer of ES cell-derived EPC incorporated into injured femoral artery and reduced neointima formation in a mouse model. These data suggest that shear stress is a key regulator for stem cell differentiation into EC, especially in EPC differentiation, which can be used for vascular repair, and that the Flk-1-PI3K-Akt-HDAC3-p53-p21 pathway is crucial in such a process.     

Inhibition of c-Jun N-terminal kinase 2 expression suppresses growth and induces apoptosis of human tumor cells in a p53-dependent manner

c-Jun N-terminal kinase (JNK) plays a critical role in coordinating the cellular response to stress and has been implicated in regulating cell growth and transformation. To investigate the growth-regulatory functions of JNK1 and JNK2, we used specific antisense oligonucleotides (AS) to inhibit their expression. A survey of several human tumor cell lines revealed that JNKAS treatment markedly inhibited the growth of cells with mutant p53 status but not that of cells with normal p53 function. To further examine the influence of p53 on cell sensitivity to JNKAS treatment, we compared the responsiveness of RKO, MCF-7, and HCT116 cells with normal p53 function to that of RKO E6, MCF-7 E6, and HCT116 p53(-/-), which were rendered p53 deficient by different methods. Inhibition of JNK2 (and to a lesser extent JNK1) expression dramatically reduced the growth of p53-deficient cells but not that of their normal counterparts. JNK2AS-induced growth inhibition was correlated with significant apoptosis. JNK2AS treatment induced the expression of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) in parental MCF-7, RKO, and HCT116 cells but not in the p53-deficient derivatives. That p21(Cip1/Waf1) expression contributes to the survival of JNK2AS-treated cells was supported by additional experiments demonstrating that p21(Cip1/Waf1) deficiency in HCT116 cells also results in heightened sensitivity to JNKAS treatment. Our results indicate that perturbation of JNK2 expression adversely affects the growth of otherwise nonstressed cells. p53 and its downstream effector p21(Cip1/Waf1) are important in counteracting these detrimental effects and promoting cell survival.     

Histone deacetylase inhibitors differentially stabilize acetylated p53 and induce cell cycle arrest or apoptosis in prostate cancer cells

In LNCaP prostate cancer cells CG-1521, a new inhibitor of histone deacetylases, alters the acetylation of p53 in a site-specific manner. While p53 is constitutively acetylated at Lys320 in LNCaP cells, treatment with CG-1521, stabilizes the acetylation of p53 at Lys373, elevating p21 (and inducing cell cycle arrest). Treatment with CG-1521 also promotes Bax translocation to the mitochondria and cleavage, and apoptosis. TSA stabilizes the acetylation of p53 at Lys382, elevating p21 levels and inducing cell cycle arrest, but does not induce Bax translocation or apoptosis. In LNCaP cells CG-1521, but not TSA, promotes the rapid degradation of HDAC2. These data suggest that the acetylation of p53 at Lys373 is required for the p53-mediated induction of cell cycle arrest and apoptosis, while acetylation of p53 at Lys382 induces only cell cycle arrest. In p53(-/-) PC3 cells both compounds induce p21 and cell cycle arrest, but not Bax translocation or apoptosis, suggesting that both compounds can also induce p21 through a p53-independent mechanism.     

The HDAC inhibitor depsipeptide transactivates the p53/p21 pathway by inducing DNA damage

Histone deacetylase (HDAC) inhibitors have been proven to be effective therapeutic agents to kill cancer cells through inhibiting HDAC activity or altering the structure of chromatin. As a potent HDAC inhibitor, depsipeptide not only modulates histone deacetylation but also activates non-histone protein p53 to inhibit cancer cell growth. However, the mechanism of depsipeptide-induced p53 transactivity remains unknown. Here, we show that depsipeptide causes DNA damage through induction of reactive oxygen species (ROS) generation, as demonstrated by a comet assay and by detection of the phosphorylation of H2AX. Depsipeptide induced oxidative stress was confirmed to relate to a disturbance in reduction-oxidation (redox) reactions through inhibition of the transactivation of thioredoxin reductase (TrxR) in human cancer cells. Upon treatment with depsipeptide, p53 phosphorylation at threonine 18 (Thr18) was specifically induced. Furthermore, we also demonstrated that phosphorylation of p53 at Thr18 is required for p53 acetylation at lysine 373/382 and for p21 expression in response to depsipeptide treatment. Our results demonstrate that depsipeptide plays an anti-neoplastic role by generating ROS to elicit p53/p21 pathway activation.     

Inactivation of p21WAF1 sensitizes cells to apoptosis via an increase of both p14ARF and p53 levels and an alteration of the Bax/Bcl-2 ratio

p21(WAF1) appears to be a major determinant of the cell fate in response to anticancer therapy. It was shown previously that HCT116 human colon cancer cells growing in vitro enter a stable arrest upon DNA damage, whereas cells with a defective p21(WAF1) response undergo apoptosis. Here we report that the enhanced sensitivity of HCT116/p21(-/-) cells to chemotherapeutic drug-induced apoptosis correlates with an increased expression of p53 and a modification of their Bax/Bcl-2 ratio in favor of the pro-apoptotic protein Bax. Treatment of HCT116/p21(-/-) cells with daunomycin resulted in a reduction of the mitochondrial membrane potential and in activation of caspase-9, whereas no such changes were observed in HCT116/p21(+/+) cells, providing evidence that p21(WAF1) exerts an antagonistic effect on the mitochondrial pathway of apoptosis. Moreover, the role of p53 in activation of this pathway was demonstrated by the fact that inhibition of p53 activity by pifithrin-alpha reduced the sensitivity of HCT116/p21(-/-) cells to daunomycin-induced apoptosis and restored a Bax/Bcl-2 ratio similar to that observed in HCT116p21(+/+) cells. Enhancement of p53 expression after disruption of p21(WAF1) resulted from a stabilization of p53, which correlated with an increased expression of the tumor suppressor p14(ARF), an inhibitor of the ubiquitin ligase activity of Mdm2. In accordance with the role of p14(ARF) in p53 stabilization, overexpression of p14(ARF) in HCT116/p21(+/+) cells resulted in a strong increase in p53 activity. Our results identify a novel mechanism for the anti-apoptotic effect of p21(WAF1) consisting in maintenance of mitochondrial homeostasis that occurs in consequence of a negative control of p14(ARF) 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.     

Incompatible effects of p53 and HDAC inhibition on p21 expression and cell cycle progression

Nutlin-3 selectively activates p53 by inhibiting the interaction of this tumor suppressor with its negative regulator murine double minute 2 (mdm2), while trichostatin A (TSA) is one of the most potent histone deacetylase (HDAC) inhibitors currently available. As both Nutlin-3 and TSA increase the levels of the cell cycle inhibitor p21(cip1/waf1) in cells, we investigated whether a combination of these compounds would further augment p21 levels. Contrary to expectations, we found that short-term exposure to Nutlin-3 and TSA in combination did not have an additive effect on p21 expression. Instead, we observed that activation of p53 prevented the ability of TSA to increase p21 levels. Furthermore, TSA inhibited Nutlin-3-induced expression of p53-dependent mRNAs including P21. This negative effect of TSA on Nutlin-3 was significantly less pronounced in the case of hdm2, another p53 downstream target. Aside from suggesting a model to explain these incompatible effects of Nutlin-3 and TSA, we discuss the implications of our findings in cancer therapy and cell reprogramming.     

Role of p21 in apoptosis and senescence of human colon cancer cells treated with camptothecin

Treatment of cells with the anti-cancer drug camptothecin (CPT) induces topoisomerase I (Top1)-mediated DNA damage, which in turn affects cell proliferation and survival. In this report, we demonstrate that treatment of the wild-type HCT116 (wt HCT116) human colon cancer cell line and the isogenic p53(-/-) HCT116 and p21(-/-) HCT116 cell lines with a high concentration (250 nm) of CPT resulted in apoptosis, indicating that apoptosis occurred by a p53- and p21-independent mechanism. In contrast, treatment with a low concentration (20 nm) of CPT induced cell cycle arrest and senescence of the wt HCT116 cells, but apoptosis of the p53(-/-) HCT116 and p21(-/-) HCT116 cells. Further investigations indicated that p53-dependent expression of p21 blocked apoptosis of wt HCT116 cells treated with 20 nm, but not 250 nm CPT. Interestingly, blocking of the apoptotic pathway, by Z-VAD-FMK, in p21(-/-) HCT116 cells following treatment with 20 nm CPT did not permit the cells to develop properties of senescence. These observations demonstrated that p21 was required for senescence development of HCT116 cells following treatment with low concentrations of CPT.     

Mechanisms underlying the pro-survival pathway of p53 in suppressing mitotic death induced by adriamycin

The p53 tumor suppressor responds to chemotherapeutic stress by triggering apoptosis or eliciting pro-survival pathway through arresting cell cycle progression for DNA damage repair. Here we examined the pro-survival activity of p53 on the adriamycin-induced stress using H1299 cells stably expressing tsp53 V143A, a temperature-sensitive mutant activating only the subset of p53 target genes related to growth arrest and DNA repair, but not apoptosis. At 38 degrees C, cells evaded from adriamycin-induced G2 arrest and died of apoptosis and mitotic catastrophe, which could be inhibited by Cdk inhibitors. Activation of functional tsp53 V143A at 32 degrees C led to suppression of Cdk1/2 activities and Cyclin B1/Cdk1 expression, cells exhibited prolonged G2 arrest, regained reproductive potential and were protected from mitotic catastrophe induced by adriamycin. Inhibition of mitotic catastrophe and Cyclin B1/Cdk1 expression was ablated upon silencing p21 Waf1 expression in tsp53 V143A-H1299 cells or in HCT116 cells. Together we show that p21 Waf1 is a key component of G2 checkpoint necessary and sufficient for protecting tumor cells against adriamycin-induced mitotic catastrophe.     

Simvastatin induced HCT116 colorectal cancer cell apoptosis through p38MAPK-p53-survivin signaling cascade

Background

Statins, the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors with cholesterol-lowering properties, were recently shown to exhibit anti-cancer effects. However, the molecular mechanism underlying statin-induced cancer cell death remains to be elucidated. Elevated level of survivin is often found over-expressed in human cancers and has been implicated in the progression of tumorigenesis. Given its central role in cell division and action as an apoptosis suppressor, survivin represents a potential molecular target in cancer management.

Methods

In this study, we explored the underlying mechanisms in simvastatin-induced HCT116 colorectal cancer cell apoptosis.

Results

Simvastatin decreased cell viability and induced cell apoptosis in HCT116 cells. These results are associated with the modulation of p21^cip/Waf1 and survivin. Survivin knockdown using survivin siRNAs also decreased cell viability and induced cell apoptosis. Simvastatin's actions on p21^cip/Waf1, survivin and apoptosis were reduced in p53 null HCT116 cells. Simvastatin caused an increase in p53 phosphorylation and acetylation. In addition, simvastatin activated p38 mitogen-activated protein kinase (p38MAPK), whereas an inhibitor of p38MAPK signaling abrogated simvastatin's effects of increasing p53 and p21^cip/Waf1 promoter luciferase activity. Cell viability and survivin promoter luciferase activity in the presence of simvastatin were also restored by p38MAPK inhibitor. Furthermore, Sp1 binding to the survivin promoter region decreased while p53 and p63 binding to the promoter region increased after simvastatin exposure.

Conclusions

Simvastatin activates the p38MAPK-p53-survivin cascade to cause HCT116 colorectal cancer cell apoptosis.

General significance

This study delineates, in part, the underlying mechanisms of simvastatin in decreasing survivin and subsequent colorectal cancer cell apoptosis.