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.     

Expression of p21WAF1/CIP1 in bladder cancer: relation to schistosomiasis

Cell cycle regulation is mediated in part through expression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1. Loss of p21WAF1/CIP1 expression may, therefore, contribute partially to schistosomal carcinogenesis in the urinary bladder. We compared p21WAF1/CIP1 expression in schistosomal and nonschistosomal bladder cancer to explore possible differences in p21WAF1/CIP1 expression between the two subtypes and the possible association between schistosomiasis and loss of p21WAF1/CIP1 expression. Tumor specimens were obtained from 130 patients who underwent transurethral biopsy or cystectomy. p21WAF1/CIP1 was determined by immunodot blot, Western blot, and enzyme immunoassay (EIA). We validated a highly sensitive quantitative EIA assay for determination of p21WAF1/CIP1 in cell lysates. Precision, analytical recovery, and linearity were all excellent. Our results did not show any correlation between p21WAF1/CIP1 expression and most clinicopathologic variables. Lower expression of p21WAF1/CIP1 was evident in squamous cell carcinoma (SCC) and schistosomal subtype than in transitional cell carcinoma and nonschistosomal tumors. Our data suggest a potential role for p21WAF1/CIP1 alteration in schistosomal carcinogenesis.     

Involvement of MINK, a Ste20 family kinase, in Ras oncogene-induced growth arrest in human ovarian surface epithelial cells

The ability of activated Ras to induce growth arrest of human ovarian surface epithelial (HOSE) cells via induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) has been used to screen for Ras pathway signaling components using a library of RNA interference (RNAi) vectors targeting the kinome. Two known Ras-regulated kinases were identified, phosphoinositide 3-kinase p110alpha and ribosomal protein S6 kinase p70(S6K1), plus the MAP kinase kinase kinase kinase MINK, which had not previously been implicated in Ras signaling. MINK is activated after Ras induction via a mechanism involving reactive oxygen species and mediates stimulation of the stress-activated protein kinase p38 MAPK downstream of the Raf/ERK pathway. p38 MAPK activation is essential for Ras-induced p21(WAF1/CIP1) upregulation and cell cycle arrest. MINK is thus a distal target of Ras signaling in the induction of a growth-arrested, senescent-like phenotype that may act to oppose oncogenic transformation in HOSE cells.     

Oxidative stress response results in increased p21WAF1/CIP1 degradation in cystic fibrosis lung epithelial cells

Lung epithelium in cystic fibrosis (CF) patients is characterized by structural damage and altered repair due to oxidative stress. To gain insight into the oxidative stress-related damage in CF, we studied the effects of hyperoxia in CF and normal lung epithelial cell lines. In response to a 95% O2 exposure, both cell lines exhibited increased reactive oxygen species. Unexpectedly, the cyclin-dependent kinase inhibitor p21WAF1/CIP1 protein was undetectable in CF cells under hyperoxia, contrasting with increased levels of p21WAF1/CIP1 in normal cells. In both cell lines, exposure to hyperoxia led to S-phase arrest. Apoptotic features including nuclear condensation, DNA laddering, Annexin V incorporation, and elevated caspase-3 activity were not readily observed in CF cells in contrast to normal cells. Interestingly, treatment of hyperoxia-exposed CF cells with two proteasome inhibitors, MG132 and lactacystin, restored p21WAF1/CIP1 protein and was associated with an increase of caspase-3 activity. Moreover, transfection of p21WAF1/CIP1 protein in CF cells led to increased caspase-3 activity and was associated with increased apoptotic cell death, specifically under hyperoxia. Taken together, our data suggest that modulating p21WAF1/CIP1 degradation may have the therapeutic potential of reducing lung epithelial damage related to oxidative stress in CF patients.     

DNA损伤生物学反应中ATM对p21~(WAF1/CIP1)蛋白的直接磷酸化

毛细血管扩张性共济失调症突变蛋白 (mutatedinataxiatelangiectasia ,ATM)是直接感受DNA双链断裂损伤并起始诸多DNA损伤信号反应通路的主开关分子 .已有研究发现 ,DNA损伤生物学反应中 ,ATM可通过磷酸化活化p5 3,继而转录活化细胞周期检查点蛋白p2 1WAF1 CIP1的表达 ,而对于ATM是否直接参与p2 1WAF1 CIP1的早期活化迄今尚无实验证明 .通过免疫共沉淀反应 ,检测到细胞电离辐射 (ionizingradiation ,IR)反应早期ATM与p2 1WAF1 CIP1蛋白存在相互作用 .将p2 1WAF1 CIP1蛋白编码基因全长克隆入原核表达载体pGEX4T 2 ,经诱导表达及亲和层析纯化获取GST p2 1融合蛋白作为磷酸化底物 .体外磷酸化实验检测证明 ,IR活化的ATM具磷酸化p2 1WAF1 CIP1蛋白的功能 ,并且此磷酸化功能可被PI3K家族特异性抑制剂Wortmannin所抑制 .结果揭示了IR后ATM可通过直接磷酸化p2 1WAF1 CIP1蛋白 ,在IR致DNA损伤生物学反应早期调控p2 1WAF1 CIP1蛋白的快速活化过程     

Protein p21(WAF1/CIP1) is phosphorylated by protein kinase CK2 in vitro and interacts with the amino terminal end of the CK2 beta subunit

Protein kinase CK2 is a ubiquitous protein that phosphorylates multiple substrates and is composed of catalytic (alpha, alpha') and regulatory (beta) subunits. Abundant evidence relates CK2 to the regulation of cell division. p21(WAF1/CIP1) is a potent inhibitor of cyclin-dependent kinases and of DNA replication and acts as a key inhibitor of cell cycle progression. In this work we examine the relation between these two important proteins. The interaction between the CK2 beta regulatory subunit of CK2 and p21(WAF1/CIP1) has been confirmed. Using a pull-down assay and fusion constructs of glutathione transferase with fragments of CK2 beta and other mutants, it was possible to define that the N-terminal (1-44) portion of CK2 beta contains a p21(WAF1/CIP1) binding site. CK2 reconstituted from recombinant alpha and beta subunits can phosphorylate p21(WAF1/CIP1) in vitro. This phosphorylation is greatly enhanced by histone H1. p21(WAF1/CIP1) can inhibit the phosphorylation of substrate casein by CK2. This inhibition, however, seems to be due to competition by p21(WAF1/CIP1) as an alternate substrate since in order to observe inhibition it is necessary that the concentration of p21 be of the same order of magnitude as the casein substrate concentration. This competition is not related to the binding of p21(WAF1/CIP1) to CK2 beta because it can also be observed when, in the absence of CK beta, CK alpha is used to phosphorylate casein in the presence of the p21.     

The immunosuppressive agent tacrolimus induces p21WAF/CIP1WAF1/CIP1 via TGF-beta secretion

Tacrolimus (Tac) is more immunosuppressive drug compared to cyclosporine (CsA). Our previous studies have demonstrated that CsA induces the expression of p21WAF/CIP1 expression. In this study we explored if like CsA, Tac also induces expression of p21WAF/CIP1. We also determined if induction of p21WAF/CIP1 by Tac is dependent on TGF-beta. Using RT-PCR and Western blot analysis, we studied the induction of p21WAF/CIP1 mRNA and protein in human T cells and A-549 cells (human lung adenocarcinoma cells) by Tac. The stimulation of p21WAF/CIP1 promoter activity was studied by luciferase assay using p21WAF/CIP1-luc, chimeric plasmid DNA containing a p21WAF/CIP1 promoter segment and luciferase reporter gene. Using anti-TGF-beta antibody, we studied if induction of p21WAF/CIP1 by tacrolimus is dependent on TGF-beta. The results demonstrate that Tac induced p21WAF/CIP1 mRNA and protein expression as well as stimulated its promoter activity in T cells and A-549 cells. The induction of p21WAF/CIP1 expression by tacrolimus was dependent on TGF-beta since a neutralizing anti-TGF-beta antibody inhibited induction of p21WAF/CIP1in A-549 cells. These data support the hypothesis that cyclin inhibitor p21WAF/CIP1 might represent a unified mediator of the anti-proliferative effects of Tac and other immunosuppressive agents. Strategies involving p21WAF/CIP1 induction should be considered a viable alternative strategy to achieve immunosuppression possibly with reduced toxicity associated with current immunosuppression.     

Caspase 3-mediated cleavage of p21WAF1/CIP1 associated with the cyclin A-cyclin-dependent kinase 2 complex is a prerequisite for apoptosis in SK-HEP-1 cells

Apoptosis of SK-HEP-1 human hepatoma cells induced by treatment with ginsenoside Rh2 (G-Rh2) is associated with rapid and selective activation of cyclin A-associated cyclin-dependent kinase 2 (Cdk2). Here, we show that in apoptotic cells, the Cdk inhibitory protein p21(WAF1/CIP1), which is associated with the cyclin A-Cdk2 complex, undergoes selective proteolytic cleavage. In contrast, another Cdk inhibitory protein, p27(KIP1), which is associated with cyclin A-Cdk2 and cyclin E-Cdk2 complexes, remained unaltered during apoptosis. Ectopic overexpression of p21(WAF1/CIP1) suppressed apoptosis as well as cyclin A-Cdk2 activity induced by treatment of SK-HEP-1 cells with G-Rh2. The suppressive effects of p21(WAF1/CIP1) were much higher in the cells transfected with p21D112N, an expression vector that encodes a p21(WAF1/CIP1) mutant resistant to caspase 3 cleavage. Overexpression of cyclin A in SK-HEP-1 cells dramatically up-regulated cyclin A-Cdk2 activity and accordingly enhances apoptosis induced by treatment with G-Rh2. These up-regulating effects were blocked by coexpression of a dominant negative allele of cdk2. Furthermore, olomoucine, a specific inhibitor of Cdks, also blocked G-Rh2-induced apoptosis. These data suggest that the induction of apoptosis in human hepatoma cells treated with G-Rh2 occurs by a mechanism that involves the activation of cyclin A-Cdk2 by caspase 3-mediated cleavage of p21(WAF1/CIP1).     

Critical role of both p27KIP1 and p21CIP1/WAF1 in the antiproliferative effect of ZD1839 ('Iressa'), an epidermal growth factor receptor tyrosine kinase inhibitor,in head and neck squamous carcinoma cells

High expression of the epidermal growth factor receptor (EGFR) has been implicated in the development of squamous-cell carcinomas of head and neck (SCCHN). ZD1839 ('Iressa') is an orally active, selective EGFR-TKI (EGFR-tyrosine kinase inhibitor) that blocks signal transduction pathways implicated in proliferation and survival of cancer cells, and other host-dependent processes promoting cancer growth. We have demonstrated that ZD1839 induces growth arrest in SCCHN cell lines by inhibiting EGFR-mediated signaling. Cell cycle kinetic analysis demonstrated that ZD1839 induces a delay in cell cycle progression and a G1 arrest together with a partial G2/M block; this was associated with increased expression of both p27(KIP1) and p21(CIP1/WAF1) cyclin-dependent kinase (CDK) inhibitors. The activity of CDK2, the main target of CIP/KIP CDK inhibitors, was reduced in a dose-dependent fashion after 24 h of ZD1839 treatment and this effect correlated to the increased amount of p27(KIP1) and p21(CIP1/WAF1) proteins associated with CDK2-cyclin-E and CDK2-cyclin-A complexes. In addition, ZD1839-induced growth inhibition was significantly reduced in cell transfectants expressing p27(KIP1) or p21(CIP1/WAF1) antisense constructs. Overall, these results as well as the timing of the effect of ZD1839 on G1 arrest and p27(KIP1) and p21(CIP1/WAF1) upregulation, suggest a mechanistic connection between these events.     

Osteoclast differentiation is associated with transient upregulation of cyclin-dependent kinase inhibitors p21(WAF1/CIP1) and p27(KIP1)

Osteoclasts, bone-resorbing multinucleated cells, develop from monocyte-macrophage lineage cells in the presence of osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) and macrophage colony-stimulating factor (M-CSF). M-CSF-dependent bone marrow macrophages (M-BMMPhis) from mouse bone marrow cells have been shown to differentiate into osteoclast-like multinucleated cells (OCLs) in the presence of soluble ODF/RANKL (sODF/RANKL) and M-CSF within 3 days. In this study, we found that stimulation of M-BMMPhis with sODF/RANKL induced a transient expression of cyclin-dependent kinase inhibitors (CDK inhibitors) p21(WAF1/CIP1) and p27(KIP1) by 24 h. The CDK inhibitor proteins disappeared by 48 h. Tumor necrosis factor alpha (TNF-alpha), which is reported to stimulate OCL differentiation, stimulated p21(WAF1/CIP1) and p27(KIP1) expression in M-BMMPhis as well. However, M-CSF alone did not stimulate the expression of the two CDK inhibitors. To clarify the role of p21(WAF1/CIP1) and p27(KIP1) in osteoclastogenesis, accumulation of these CDK inhibitors was aborted by antisense oligonucleotides. Treatment with p21(WAF1/CIP1) antisense oligonucleotide alone, or p27(KIP1) antisense oligonucleotide alone, showed a limited inhibitory effect on OCL formation. However, treatment with a mixture of these two antisense oligonucleotides strongly inhibited OCL formation. These results suggest that a combined modulation of the CDK inhibitors p21(WAF1/CIP1) and p27(KIP1) may be involved in osteoclast differentiation induced by ODF/RANKL.     

8-chloroadenosine induced HL-60 cell growth inhibition, differentiation, and G(0)/G(1) arrest involves attenuated cyclin D1 and telomerase and up-regulated p21(WAF1/CIP1)

8-Chloroadenosine, an active dephosphorylated metabolite of the antineoplastic agent 8-chloroadenosine 3',5'-monophosphate (8-Cl-cAMP), induces growth inhibition in multiple carcinomas. Here we report that 8-chloroadenosine inhibits growth in human promyelocytic leukemia HL-60 cells by a G(0)/G(1) phase arrest and terminates cell differentiation along the granulocytic lineage. The mechanism of 8-chloroadenosine-induced G(0)/G(1) arrest is independent of apoptosis. The expressions of cyclin D1 and c-myc in HL-60 are suppressed by 8-chloroadenosine, whereas the cyclin-dependent kinases inhibitor p21(WAF1/CIP1) is up-regulated. 8-Chloroadenosine has less effect on the expressions of cyclin-dependent kinase (cdk)2 and cdk4, G(1) phase cyclin-dependent kinases, and only moderately induces the expression of transforming growth factor beta1 (TGFbeta1) and the mitotic inhibitor p27(KIP1). Telomerase activity is reduced in extracts of 8-chloroadenosine treated HL-60 cells, but 8-chloroadenosine does not directly inhibit the catalytic activity of telomerase in vitro. Therefore, anti-proliferation of HL-60 cells by 8-chloroadenosine involves coordination of cyclin D1 suppression, reduction of telomerase activity, and up-regulation of p21(WAF1/CIP1) that arrest cell-cycle progression at G(0)/G(1) phase and terminate cell differentiation.     

Alphavirus M1 induces apoptosis of malignant glioma cells via downregulation and nucleolar translocation of p21WAF1/CIP1 protein

Alphavirus, a genus of arthropod-borne togavirus, is well-known for its pro-apoptotic capability. However, the underlying mechanism remains to be further clarified. Here, we have identified that M1, an alphavirus isolated in 1960s, targeted C6 malignant glioma cells for apoptosis. Flow cytometry analysis showed that more cells enter S-phase post M1 infection, and subsequently undergo a classic apoptosis. To elucidate the mechanism of S-phase arrest and its relationship to apoptosis, we tested the expression of several critical cell cycle regulatory proteins and found elevated phosphorylation of cyclin-dependent kinase 2 (CDK2), decreased expression of cyclin A and proliferating cell nuclear antigen (PCNA). Notably, the protein level of p21^WAF1/CIP1 was downregulated earliest and most effectively among all tested changes of cell cycle regulators, though its mRNA level was strongly upregulated. To evaluate the role of p21^WAF1/CIP1 in S-phase accumulation and subsequent apoptosis, we confirmed that exogenous p21^WAF1/CIP1 overexpression or treatment with roscovitine (a selective chemical inhibitor of CDK2) efficiently protected against apoptosis with a reduced S-phase accumulation Thus, it is indicated that the downregulation of p21^WAF1/CIP1 mediated C6 apoptosis via overactivation of CDK2. In addition, confocal microscopy showed that p21^WAF1/CIP1 totally translocated to nucleolus during M1-induced C6 apoptosis. Altogether, downregulation and nucleolar translocation of the p21^WAF1/CIP1 protein played an active role in M1-induced C6 apoptosis.