p53-, SIRT1-, and PARP-1-independent downregulation of p21WAF1 expression in nicotinamide-treated cells

Nicotinamide at mM concentration is a potent inhibitor of certain key molecules involved in cell survival, such as SIRT1 and PARP-1, and affects cell survival in various conditions in vivo and in vitro. However, the effect of an acute treatment of nicotinamide on gene expression has rarely been closely examined. In our study, the treatment of 10 mM nicotinamide downregulated p21WAF1 expression in various human cells including p53-negative or SIRT1-knockdown cells indicating gene regulation not mediated by p53 or SIRT1. Meanwhile, in the nicotinamide-treated cells, Sp1 activity and protein level was substantially reduced due to increased proteasome-mediated degradation. Our results indicate that nicotinamide treatment attenuates p21WAF1 expression through Sp1 downregulation, and suggest a possible involvement of nicotinamide metabolism in cellular gene expression.     

Lithium increases expression of p21WAF/Cip1 and survivin in human glioblastoma cells

Lithium is the most widely prescribed mood stabilizer, but the precise molecular mechanisms underlying its therapeutic function are not yet fully elucidated. Recent preclinical and clinical evidence indicates its neuroprotective and neurotrophic effects. As a tight coupling of function and metabolism in the central nervous system between glial cells and neurons has recently been detected, lithium's effect on glial cells may participate also in the total beneficial effects of this drug. The aim of the present study was to analyze molecular mechanisms induced in human glioblastoma A1235 cells by the treatment with lithium, especially its influence on the expression of apoptosis-related genes. Lower levels of lithium (0.5 mmol/L and 2 mmol/L) did not cause any cytotoxicity or changes in the cell cycle phase distribution following 72 h incubation. However, a higher dose (20 mmol/L) was cytostatic for glioblastoma cells, and caused accumulation of cells in G₂/M phase of the cell cycle. The treatment with lithium did not alter the levels of Bcl-2 or procaspase-3 and did not cleave PARP, but increased the levels of p21^WAF/Cip1 and survivin. Thus, increased expression of p21^WAF/Cip1 (a protein with antiapoptotic function), and survivin (a protein that supports the growth of cells by suppression of apoptosis and promotion of cell proliferation) may be the early events in the long-term cell response to lithium that are involved in the beneficial effects of this drug.     

Loss of p21 expression in senescent cells after DNA damage accompanied with increase of miR-93 expression and reduced p53 interaction with p21 gene promoter

To answer what is a critical event for higher incidence of tumor development in old than young individuals, primary culture of human diploid fibroblasts were employed and DNA damage was induced by doxorubicin or X-ray irradiation. Response to the damage was different between young and old cells; loss of p21^sdi1 expression in spite of p53^S15 activation in old cells along with [³H]thymidine and BrdU incorporation, but not in young cells. The phenomenon was confirmed by other tissue fibroblasts obtained from different donor ages. Induction of miR-93 expression and reduced p53 binding to p21 gene promoter account for loss of p21^sdi1 expression in senescent cells after DNA damage, suggesting a mechanism of in vivo carcinogenesis in aged tissue without repair arrest.     

Tumor necrosis factor-alpha induces differentiation of human peripheral blood mononuclear cells into osteoclasts through the induction of p21(WAF1/Cip1)

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that mediates inflammation and induces bone loss caused by excessive bone resorption by osteoclasts. The interaction of TNF-alpha with its receptor activates several signal transduction pathways, including those of mitogen-activated protein (MAP) kinases (p38, JNK, and ERK) and NF-kappaB. Signaling from these molecules has been shown to play an important role in osteoclastogenesis. In the present study, we investigated the mechanism of TNF-alpha-induced osteoclast differentiation in human peripheral blood mononuclear cells (PBMCs). We found that TNF-alpha alone greatly induced differentiation of PBMCs into osteoclasts. The osteoclast differentiation induced by TNF-alpha was independent of RANKL binding to its receptor RANK on PBMCs. Furthermore, TNF-alpha potently activated p38 MAPK, JNK, and NF-kappaB. Western blotting analysis revealed that p21(WAF1/Cip1), a cyclin-dependent kinase (CDK) inhibitor, is significantly induced upon TNF-alpha stimulation. The induction of p21(WAF1/Cip1) during differentiation is responsible for arrest at G(0)/G(1) phase and associated with the JNK pathway. These results suggest that TNF-alpha regulates osteoclast differentiation through p21(WAF1/Cip1) expression and further shows that these events require JNK activity.     

Mitochondrial ribosomal protein L41 mediates serum starvation-induced cell-cycle arrest through an increase of p21(WAF1/CIP1)

Ribosomal proteins not only act as components of the translation apparatus but also regulate cell proliferation and apoptosis. A previous study reported that MRPL41 plays an important role in p53-dependent apoptosis. It also showed that MRPL41 arrests the cell cycle by stabilizing p27(Kip1) in the absence of p53. This study found that MRPL41 mediates the p21(WAF1/CIP1)-mediated G1 arrest in response to serum starvation. The cells were released from serum starvation-induced G1 arrest via the siRNA-mediated blocking of MRPL41 expression. Overall, these results suggest that MRPL41 arrests the cell cycle by increasing the p21(WAF1/CIP1) and p27(Kip1) levels under the growth inhibitory conditions.     

Let-7a elevates p21 levels by targeting of NIRF and suppresses the growth of A549 lung cancer cells

Down-regulation of let-7 microRNA (miRNA) is a key event in lung cancer. Despite recent advances in survival signaling, the roles of let-7 in the context of lung cancer are not fully clear. In this study, we showed that let-7a, a member of let-7 family, negatively regulated the expression of NIRF through NIRF 3′ UTR. We also showed that NIRF was required for the let-7a-mediated elevation of p21^WAF1. These findings suggest that growth-inhibitory effect of let-7a on the A549 cells in vitro and in vivo may be explained in part by le-7a-induced suppression of NIRF and elevation of p21^WAF1. This work reveals a novel regulatory mechanism for let-7a in the control of cellular proliferation and lung carcinogenesis.     

PKCdelta modulates p21WAF1/CIP1 ability to bind to Cdk2 during TNFalpha-induced apoptosis

Cyclin-dependent kinase 2 (Cdk2) activity is thought to be involved in cell death-associated chromatin condensation and other manifestations of apoptotic death. Here we show that during TNFalpha-induced apoptosis, PKCdelta is activated in a caspase-3-dependent manner and phosphorylates p21(WAF1/CIP1), a specific cyclin-dependent kinase inhibitor, on (146)Ser. This residue is located near a cyclin-binding motif (Cy2) that plays an important role in the interaction between p21(WAF1/CIP1) and Cdk2, and its phosphorylation modulates the ability of p21(WAF1/CIP1) to associate with Cdk2. The phosphorylation of p21(WAF1/CIP1) is temporally related to the activation kinetics of Cdk2 activity during the apoptosis. We propose that during TNFalpha-induced apoptosis, PKCdelta-mediated phosphorylation of p21(WAF1/CIP1) at (146)Ser attenuates the Cdk2 binding of p21(WAF1/CIP1) and thereby upregulates Cdk2 activity.     

Upregulation of p21(WAF1/Cip1) precedes tumor necrosis factor-induced necrosis-like cell death

The molecular mechanisms mediating death receptor-induced caspase-independent necrotic cell death are still largely unknown. We have previously reported that NIH3T3 cells are sensitized by caspase inhibition to death receptor-induced cytotoxicity leading to a necrosis-like cell death. In addition, we have identified an important role of cell cycle progression for this sensitization effect. Here, we report that tumor necrosis factor-induced necrotic death is preceded by an upregulation of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1). Increased expression of p21(WAF1/Cip1) occurs prior to cell death in the nucleus, where it binds to a cyclin-dependent kinase indicating its functionality. The use of specific pharmacological inhibitors revealed a partial involvement of p38 mitogen-activated protein kinase in the upregulation of p21(WAF1/Cip1). Inhibition of p21(WAF1/Cip1) upregulation prevents a previously observed delay of the cells in the G2/M phase of the cell cycle thereby augmenting, not inhibiting cell death.     

p21WAFl/CIPl、PCNA在骨巨细胞瘤中的表达及意义

目的探讨p21WAF1/CIP1、PCNA在骨巨细胞瘤中的表达特点及其与骨巨细胞瘤的分化和复发的关系.方法应用LSAB免疫组织化学方法对38例骨巨细胞瘤中的p21WAF1/CIP1、PCNA的表达进行检测.结果66.8%的骨巨细胞瘤中可检测到p21WAF1/CIP1的阳性表达,其阳性表达主要位于分化好的多核巨细胞的细胞核内,分化好的骨巨细胞瘤(Ⅰ级)中p21WAF1/CIP1的表达明显高于分化差组(Ⅱ、Ⅲ级)(P<0.01).38例骨巨细胞瘤均可检测到PCNA的阳性表达,其阳性表达主要位于单核基质细胞的细胞核内;未复发组及复发组中p21WAF1/CIP1、PCNA的表达无显著性差异(P>0.05).结论骨巨细胞瘤中p21WAF1/CIP1的表达与骨巨细胞瘤的分化相关,可作为骨巨细胞瘤分化的参考指标.     

IL-1-induced ERK1/2 activation up-regulates p21 protein by inhibition of degradation via ubiquitin-independent pathway in human melanoma cells A375

IL-1 inhibits the proliferation of human melanoma cells A375 by arresting the cell cycle at G0/G1 phase, which accompanies the increase of p21^Waf1/Cip1 (p21) protein. Here, we demonstrate that IL-1 induces the stabilization of p21 protein via ERK1/2 pathway. The degradation of p21 was inhibited by IL-1, however the ubiquitination level of p21 was not affected. In addition, the degradation of non-ubiquitinated form of lysine less mutant p21-K6R was also inhibited by IL-1, suggesting that IL-1 stabilized p21 protein via ubiquitin-independent pathway. Furthermore, the inhibition of p21 protein degradation was prevented by a selective inhibitor of ERK1/2 pathway, PD98059. These results suggest that IL-1-induced ERK1/2 activation leads to the up-regulation of p21 by inhibiting degradation via ubiquitin-independent pathway in human melanoma cells A375.