The lack of G1/S arrest of teratocarcinoma F9 cells is determinated by degradation of cyclin-dependent kinases inhibitor p21waf1/cip1

We have studied the ability of F9 teratocarcinoma cells to arrest in G1/S and G2/M checkpoints following gamma-irradiation. Wild-type p53 protein is rapidly accumulated in F9 cells after gamma-irradiation, however this is not followed by G1/S arrest; there is just a reversible delay of the cell cycle in G2/M. In order to elucidate the reasons of the lack of G1/S arrest in F9 cells we investigated the levels of regulatory cell cycle proteins: G1-cyclins, cyclin dependent kinases and kinase inhibitor p21WAF1/CIP1. We have shown that in spite of p53-dependent activation of p21WAF1/CIP1 promoter, p21WAF1/CIP1 protein is not revealed by different polyclonal and monoclonal antibodies, either by immunoblotting or by immunofluorescent staining. However, when cells are treated with specific proteasome inhibitor lactacystin, p21WAF1/CIP1 protein is revealed. We therefore suggest that p21WAF1/CIP1 protein is subjected to proteasome degradation in F9 cells and probably the lack of G1/S arrest after gamma-irradiation is due to this degradation. Thus, it is the combination of functionally active p53 with low level expression of p21WAF1/CIP1 that causes a short delay of the cell cycle progression in G2/M, rather than the G1-arrest after gamma-irradiation of F9 cells.     

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.     

TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.     

Hyperoxia induces macrophage cell cycle arrest by adhesion-dependent induction of p21Cip1 and activation of the retinoblastoma protein

Hyperoxia induces growth arrest, apoptosis, necrosis, and morphological changes (spreading and adhesion) in various types of cells. The mechanism of hyperoxia-induced cell growth arrest has not been well elucidated, especially in macrophages. One possible mechanism is a role of cell adhesion in hyperoxia-induced cell cycle arrest. To evaluate this finding, macrophages were cultured in normoxia (21% O2) or hyperoxia (95% O2) in adhesion or low adhesion conditions. Incubation of macrophages in hyperoxia induced cell cycle arrest. The hyperoxia-induced cell cycle arrest was prevented by low adhesion conditions. To evaluate pathways potentially involved in hyperoxia-induced growth arrest, we measured extracellular regulated kinase and retinoblastoma protein activation and p21Cip1 and p53 accumulation. Hyperoxia strongly induced activation of extracellular regulated kinase and retinoblastoma protein as well as up-regulation of p21Cip1. These effects of hyperoxia were attenuated under low adhesion conditions, suggesting a role for integrin-dependent signaling. The induction of p21Cip1 and activation of retinoblastoma protein occurred via a p53-independent mechanism. These results suggest that adhesion-dependent pathways are required for hyperoxia-induced cell cycle arrest in macrophages.     

Developmental arrest of T cells in Rpl22-deficient mice is dependent upon multiple p53 effectors

αβ and γδ lineage T cells are thought to arise from a common CD4(-)CD8(-) progenitor in the thymus. However, the molecular pathways controlling fate selection and maturation of these two lineages remain poorly understood. We demonstrated recently that a ubiquitously expressed ribosomal protein, Rpl22, is selectively required for the development of αβ lineage T cells. Germline ablation of Rpl22 impairs development of αβ lineage, but not γδ lineage, T cells through activation of a p53-dependent checkpoint. In this study, we investigate the downstream effectors used by p53 to impair T cell development. We found that many p53 targets were induced in Rpl22(-/-) thymocytes, including miR-34a, PUMA, p21(waf), Bax, and Noxa. Notably, the proapoptotic factor Bim, while not a direct p53 target, was also strongly induced in Rpl22(-/-) T cells. Gain-of-function analysis indicated that overexpression of miR-34a caused a developmental arrest reminiscent of that induced by p53 in Rpl22-deficient T cells; however, only a few p53 targets alleviated developmental arrest when individually ablated by gene targeting or knockdown. Co-elimination of PUMA and Bim resulted in a nearly complete restoration of development of Rpl22(-/-) thymocytes, indicating that p53-mediated arrest is enforced principally through effects on cell survival. Surprisingly, co-elimination of the primary p53 regulators of cell cycle arrest (p21(waf)) and apoptosis (PUMA) actually abrogated the partial rescue caused by loss of PUMA alone, suggesting that the G1 checkpoint protein p21(waf) facilitates thymocyte development in some contexts.     

Sustained nitric oxide delivery delays nitric oxide-dependent apoptosis in macrophages: contribution to the physiological function of activated macrophages

Treatment of the macrophage cell line RAW 264.7 with the short-lived NO donor S-nitrosoglutathione triggers apoptosis through the release of mitochondrial mediators. However, continuous supply of NO by long-lived NO donors protected cells from apoptosis through mechanisms that involved the maintenance or an increase in the levels of the inhibitor of apoptosis proteins (IAPs) cIAP-1, cIAP-2, and xIAP and decreases in the accumulation of p53 and in the levels and targeting of Bax to the mitochondria. As a result of these changes, the activation of caspases 9 and 3 was notably delayed, expanding the time of viability of the macrophages. Moreover, inhibition of NO synthase 2 activity after 8 h of stimulation of RAW 264.7 cells with LPS and IFN-gamma accelerated apoptosis via an increase in the processing and activation of caspases. These data suggest that NO exerts an important role in the autoregulation of apoptosis in macrophages.     

Apigenin causes G(2)/M arrest associated with the modulation of p21(Cip1) and Cdc2 and activates p53-dependent apoptosis pathway in human breast cancer SK-BR-3 cells

We studied the effects of apigenin on the cell cycle distribution and apoptosis of human breast cancer cells and explored the mechanisms underlying these effects. We first investigated the antiproliferative effects in SK-BR-3 cells exposed to between 1 and 100 microM apigenin for 24, 48 and 72 h. Apigenin significantly inhibited cell proliferation at concentrations over 50 microM, regardless of exposure time (P<.05), and resulted in significant cell cycle arrest in the G(2)/M phase after 48 h of treatment at high concentrations (50 and 100 microM; P<.05). To investigate the regulatory proteins of cell cycle arrest affected by apigenin, we treated cells with 50 and 100 microM apigenin for 72 h. Apigenin caused a slight decrease in cyclin D and cyclin E expression, with no change in CDK2 and CDK4. In addition, the apigenin-induced accumulation of the cell population in the G(2)/M phase resulted in a decrease in CDK1 together with cyclin A and cyclin B. In an additional study, apigenin also increased the accumulation of p53 and further enhanced the level of p21(Cip1), with no change in p27(Kip1). The expression of Bax and cytochrome c of p53 downstream target was increased markedly at high concentration treatment over 50 microM apigenin. Based on our findings, the mechanism by which apigenin causes cell cycle arrest via the regulation of CDK1 and p21(Cip1) and induction of apoptosis seems to be involved in the p53-dependent pathway.     

Stabilization and reactivation of the p53 tumor suppressor protein in nontumorigenic revertants of HeLa cervical cancer cells.

We demonstrated previously that loss of in vitro transformation and in vivo tumorigenicity in two independent revertant clones of HeLa cells (designated HA and HF) resulted from dominant-acting genetic changes. Analysis of the p53 tumor suppressor gene revealed stabilization and at least partial restoration of wild-type p53 transactivation properties pathways in both revertants of HPV-induced cell transformation. The half-lives of the p53 protein and both of the HA and HF clones were increased approximately 4 fold compared with the parental HeLa cells (16, 17, and 4 min, respectively). The levels of E6 viral protein expression were similar in the three cell lines, whereas the levels of the ubiquitin ligase protein, E6 associated protein (E6-AP), were elevated in the revertants. Western blot analysis of immunoaffinity-purified p53 demonstrated that stabilization of p53 in the revertants was correlated with a reduction in the in vivo formation of complexes involving the E6 oncoprotein and p53. Stabilization of p53 function in the revertants did not result from mutations in either the p53 or E6-AP genes. Despite the observed stabilization and restoration of p53 transactivation function in the revertants, exposure of the revertants to DNA-damaging agents did not result in elevated levels of p21(waf-1) protein and failed to induce growth arrest in the G1 phase of the cell cycle. However, p53-independent induction of p21(waf-1) protein also failed to induce the G1 phase of the cell cycle. Thus, restoration of wild-type p53 transactivation activity in the HA and HF revertants is insufficient to induce G1 arrest and reversion from HPV-induced cell transformation in our model system.     

Different levels of p53 induced either apoptosis or cell cycle arrest in a doxycycline-regulated hepatocellular carcinoma cell line <Emphasis Type="Italic">in vitro</Emphasis>

Induction of p53 gene expression in cancer cells can lead to both cell cycle arrest and apoptosis. To clarify whether the level of p53 expression determines the apoptotic response of hepatocellullar carcinoma (HCC) cells, we assessed the effect of various levels of expression of p53 gene on a p53-deficient HCC cell line, Hep3B, utilizing a doxycycline (Dox)-regulated inducible p53 expression system. Our results showed that apoptosis was induced in HCC cells with high levels of p53 expression. However, lower level of p53 expression induced only cell cycle arrest but not apoptosis. Bax expression was up-regulated following high levels of p53 expression, while bcl-2 expression was not altered by the level of p53 expression. Moreover, p21 expression was observed in both high and low expression of p53. These results suggest the level of p53 expression could determine if the HCC cells would go into cell cycle arrest or apoptosis. Bax may participate, at least in part, in inducing p53-dependent apoptosis and the induction of p21 alone was able to cause cell cycle arrest but not apoptosis.     

p53-inducible human homologue of Drosophila seven in absentia (Siah) inhibits cell growth: suppression by BAG-1.

The Drosophila seven in absentia (sina) gene is required for R7 photoreceptor cell formation during Drosophila eye development, where it functions within the Ras/Raf pathway and targets other proteins for degradation via associations with a ubiquitin-conjugating enzyme. Recently, a mammalian sina homologue was reported to be a p53-inducible gene in a myeloid leukemia cell line. To explore the function of human SINA-homologous (Siah) proteins, expression plasmids encoding Siah-1A were transiently transfected into 293 epithelial cells and GM701 fibroblast cells, resulting in growth arrest without induction of apoptosis. We discovered that BAG-1, a ubiquitin-like Hsp70/Hsc70-regulating protein, is a negative regulator of Siah-1A. Siah-1A was identified as a BAG-1-binding protein via yeast two-hybrid methods. Specific interaction of BAG-1 with Siah-1A was also demonstrated by in vitro binding experiments using glutathione S-transferase fusion proteins and co-immunoprecipitation studies. Siah-1A-induced growth arrest in 293 and GM701 cells was abolished by co-transfection of wild-type BAG-1 with Siah-1A but not by a C-terminal deletion mutant of BAG-1 that fails to bind Siah-1A. Over-expression of BAG-1 significantly inhibited p53-induced growth arrest in 293 cells without preventing p53 transactivation of reporter gene plasmids. BAG-1 also prevented growth arrest following UV-irradiation-induced genotoxic injury without interfering with accumulation of p53 protein or p21(waf-1) expression. BAG-1 functions downstream of p53-induced gene expression to inhibit p53-mediated suppression of cell growth, presumably by suppressing the actions of Siah-1A. We suggest that Siah-1A may be an important mediator of p53-dependent cell-cycle arrest and demonstrate that Siah-1A is directly inhibited by BAG-1.     

Nitric oxide nitrates tyrosine residues of tumor-suppressor p53 protein in MCF-7 cells

It has been reported that mammalian cells incubated with excess nitric oxide (NO) accumulate p53 protein but concomitantly this p53 loses its capacity for binding to its DNA consensus sequence. As nitration of tyrosine residues in various proteins has been shown to inhibit their functions, we examined whether NO nitrates tyrosine residues in p53 protein. MCF-7 cells expressing wild-type p53 were incubated with S-nitrosoglutathione for 4 h and cellular extracts were immunoprecipitated with an anti-p53 antibody. Western blot analyses of immunoprecipitates for p53 or for nitrotyrosine revealed low levels of nitrotyrosine in p53 from untreated cells. Incubation with 2 mM S-nitrosoglutathione induced a significant increase in the nitrotyrosine level in p53 protein compared to nontreated cells. These results suggest that excess NO produced in inflamed tissues could nitrate p53 protein, playing a role in carcinogenesis by impairing functions of this tumor-suppressor protein.     

Negative cross-talk between p53 and the glucocorticoid receptor and its role in neuroblastoma cells

The tumour suppressor p53 and the glucocorticoid receptor (GR) respond to different types of stress. We found that dexamethasone-activated endogenous and exogenous GR inhibit p53-dependent functions, including transactivation, up- (Bax and p21(WAF1/CIP1)) and down- (Bcl2) regulation of endogenous genes, cell cycle arrest and apoptosis. GR forms a complex with p53 in vivo, resulting in cytoplasmic sequestration of both p53 and GR. In neuroblastoma (NB) cells, cytoplasmic retention and inactivation of wild-type p53 involves GR. p53 and GR form a complex that is dissociated by GR antagonists, resulting in accumulation of p53 in the nucleus, activation of p53-responsive genes, growth arrest and apoptosis. These results suggest that molecules that efficiently disrupt GR-p53 interactions would have a therapeutic potential for the treatment of neuroblastoma and perhaps other diseases in which p53 is sequestered by GR.     

Overexpression of peptidylarginine deiminase IV features in apoptosis of haematopoietic cells

Peptidylarginine deiminases (PADIs) convert peptidylarginine into citrulline via posttranslational modification. One member of the family, PADI4, plays an important role in immune cell differentiation and cell death. To elucidate the participation of PADI4 in haematopoietic cell death, we examine whether inducible overexpression of PADI4 enhances the apoptotic cell death. PADI4 reduced the viability in a dose- and time-dependent manner of human leukemia HL-60 cells and human acute T leukemia Jurkat cells. The apoptosis-inducing activities were determined by nuclear condensation, DNA fragmentation, sub-G1 appearance, loss of mitochondrial membrane potential (Δψ_m), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Following PADI4 overexpression, cells arrest in G1 phase significantly before their entrance into apoptotic cell death. PADI4 increases tumor suppressor p53 and its downstream p21 to control cell cycle. In the detections of protein expression and kinase activity, all protein levels of cyclin-dependent kinases (CDKs) and cyclins are not reduced except cyclin D, however, CDK2 (G1 entry S phase) and CDK1 (G2 entry M phase) enzyme activities are inhibited by conditionally inducible PADI4. p53 also expands its other downstream Bax to induce cytochrome c release from mitochondria. According to these data, we suggest that PADI4 induces apoptosis mainly through cell cycle arrest and mitochondria-mediated pathway. Furthermore, p53 features in PADI4-induced apoptosis by increasing intracellular p21 to control cell cycle and by Bax accumulation to decline Bcl-2 function, destroy Δψ_m, release cytochrome c to cytoplasm and activate the caspase cascade.     

Phenotypic alterations of small cell lung carcinoma induced by different levels of wild-type p53 expression

p53 induces both growth arrest and apoptosis in cancer cells. To clarify whether the level of p53 expression determines the response of small cell lung carcinoma (SCLC) cells, we assessed the effect of various p53 levels on a p53-null SCLC cell line, N417, using a tetracycline (Tc)-regulated inducible p53 expression system. Apoptosis was induced in SCLC cells with high p53 expression. Although low levels of p53 induced G1 arrest accompanied by p21 expression, cells with G1 arrest seemed to undergo apoptosis after further cultivation. Expression of exogenous p21 induced G1 arrest but not apoptosis in SCLC cells, suggesting that p53-mediated G1 arrest was induced through p21 expression. Moreover, high level of p53 expression down-regulated Bcl-2 expression in SCLC cells, while Bax was consistently expressed irrespective to the level of p53 expression. These results suggest that p53-mediated apoptosis and G1 arrest depend on level of p53 expression in SCLC cells and that the relative dominancy of Bax to Bcl-2 is involved in the induction of apoptosis by high level of p53 expression.     

Nitric oxide protects thymocytes from gamma-irradiation-induced apoptosis in correlation with inhibition of p53 upregulation and mitochondrial damage

Apoptosis plays a crucial role in clonal deletion in the thymus, and NO has been shown to prevent apoptosis in some cell types. Therefore, we examined the effect of NO on gamma-irradiation-induced thymocyte apoptosis. Treatment of 5 Gy gamma-irradiated thymocytes with 1 mM SNAP reduced cell death from 78 to 49% after 8 h incubation (spontaneous cell death in medium control cells was 26%). Coincubation with ZVAD blocked both the spontaneous cell death and the cell death induced by SNAP or gamma-irradiation. The gamma-irradiation-induced increase in caspase 3 and 6 activities was inhibited in the presence of SNAP. The increase in cytosolic cytochrome c as well as the decrease in mitochondrial membrane potential after gamma-irradiation was inhibited in the presence of SNAP. SNAP treatment also decreased the p53 upregulation in gamma-irradiated cells. In summary, we found that NO exerts a protective effect on mouse thymocyte apoptosis induced by gamma-irradiation. The mechanism of this protective effect may involve inhibition of p53 upregulation and reduction in mitochondrial damage, with subsequent inhibition of downstream caspase activation.     

Apoptosis signal-regulating kinase 1 (ASK1) and HIF-1α protein are essential factors for nitric oxide-dependent accumulation of p53 in THP-1 human myeloid macrophages

Nitric oxide (NO) is a reactive secondary mediator, which has been found to participate in cell cycle regulation and apoptosis in myeloid macrophages, the key effectors of inflammatory and innate immune responses. However, the molecular mechanisms of nitric oxide-induced death of myeloid macrophages are not well understood. In this study we have found that NO derived from S-nitrosoglutathione (GSNO) activates ASK1 in THP-1 human myeloid macrophages in a concentration and time-dependent manner. It also induces accumulation of HIF-1α protein in a concentration-dependent manner, which peaks at 4 h of exposure to 1 mM GSNO. GSNO does not affect the level of HIF-1α mRNA as detected by the RT-PCR. In addition, GSNO was found to induce accumulation of p53 in normal but not HIF-1α knockdown THP-1 cells, where expression of this protein was silenced by specific siRNA. It has also been found that GSNO-mediated accumulation of p53 depends on activation of ASK1 since no GSNO-induced p53 stabilisation was observed in THP-1 cells transfected with dominant-negative form of this kinase. However, in both HIF-1α knockdown THP-1 cells and those transfected with the dominant-negative form of ASK1, GSNO was able to induce cell death as detected by the MTS cell viability assay leading to an increase in release of LDH.