The p21(WAF1/CIP1) promoter is methylated in Rat-1 cells: stable restoration of p53-dependent p21(WAF1/CIP1) expression after transfection of a genomic clone containing the p21(WAF1/CIP1) gene

Rat-1 cells are used in many studies on transformation, cell cycle, and apoptosis. Whereas UV treatment of Rat-1 cells results in apoptosis, X-ray treatment does not induce either apoptosis or a cell cycle block. X-ray treatment of Rat-1 cells results in both an increase of p53 protein and expression of the p53-inducible gene MDM2 but not the protein or mRNA of the p53-inducible p21(WAF1/CIP1) gene, which in other cells plays an important role in p53-mediated cell cycle block. The lack of p21(WAF1/CIP1) expression appears to be the result of hypermethylation of the p21(WAF1/CIP1) promoter region, as p21(WAF1/CIP1) protein expression could be induced by growth of Rat-1 cells in the presence of 5-aza-2-deoxycytidine. Furthermore, sequence analysis of bisulfite-treated DNA demonstrated extensive methylation of cytosine residues in CpG dinucleotides in a CpG-rich island in the promoter region of the p21(WAF1/CIP1) gene. Stable X-ray-induced p53-dependent p21(WAF1/CIP1) expression and cell cycle block were restored to a Rat-1 clone after transfection with a P1 artificial chromosome (PAC) DNA clone containing a rat genomic copy of the p21(WAF1/CIP1) gene. The absence of expression of the p21(WAF1/CIP1) gene may contribute to the suitability of Rat-1 cells for transformation, cell cycle, and apoptosis studies.     

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.     

Platelet-derived growth factor (PDGF) receptor-alpha-activated c-Jun NH2-terminal kinase-1 is critical for PDGF-induced p21WAF1/CIP1 promoter activity independent of p53

Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cells. PDGF AA functions as a "competent factor" that stimulates cell cycle entry but requires additional (progression) factors in serum to transit the cell cycle beyond the G1/S checkpoint. Unlike PDGF AA, PDGF B-chain (c-sis) homodimer (PDGF BB) and its viral counterpart v-sis can serve as both competent and progression factors. PDGF BB activates alpha- and beta-receptor subunits (alpha-PDGFR and beta-PDGFR) and induces phenotypic transformation in NIH 3T3 cells, whereas PDGF AA activates alpha-PDGFR only and fails to induce transformation. We showed previously that alpha-PDGFR antagonizes beta-PDGFR-mediated transformation through activation of stress-activated protein kinase-1/c-Jun NH2-terminal kinase-1, whereas both alpha-PDGFR and beta-PDGFR induce mitogenic signals. These studies revealed a striking feature of PDGF signaling; the specificity and the strength of the PDGF growth signal is modulated by alpha-PDGFR-mediated simultaneous activation of growth stimulatory and inhibitory signals, whereas beta-PDGFR mainly induces a growth-promoting signal. Here we demonstrate that PDGF BB activation of beta-PDGFR alone results in more efficient cell cycle transition from G1 to S phase than PDGF BB activation of both alpha-PDGFR and beta-PDGFR. PDGF AA activation of alpha-PDGFR or PDGF BB activation of both alpha- and beta-PDGFRs up-regulates expression of p21WAF1/CIP1, an inhibitor of cell cycle-dependent kinases and a downstream mediator of the tumor suppressor gene product p53. However, beta-PDGFR activation alone fails to induce p21WAF1/CIP1 expression. We also demonstrate that alpha-PDGFR-activated JNK-1 is a critical signaling component for PDGF induction of p21WAF1/CIP1 promoter activity. The ability of PDGF/JNK-1 to induce p21WAF1/CIP1 promoter activity is independent of p53, although the overall p21WAF1/CIP1 promoter activities are greatly reduced in the absence of p53. These results provide a molecular basis for differential regulation of the cell cycle and transformation by alpha- and beta-PDGFRs.     

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.     

DNA methyltransferase controls stem cell aging by regulating BMI1 and EZH2 through microRNAs

Epigenetic regulation of gene expression is well known mechanism that regulates cellular senescence of cancer cells. Here we show that inhibition of DNA methyltransferases (DNMTs) with 5-azacytidine (5-AzaC) or with specific small interfering RNA (siRNA) against DNMT1 and 3b induced the cellular senescence of human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs) and increased p16(INK4A) and p21(CIP1/WAF1) expression. DNMT inhibition changed histone marks into the active forms and decreased the methylation of CpG islands in the p16(INK4A) and p21(CIP1/WAF1) promoter regions. Enrichment of EZH2, the key factor that methylates histone H3 lysine 9 and 27 residues, was decreased on the p16(INK4A) and p21(CIP1/WAF1) promoter regions. We found that DNMT inhibition decreased expression levels of Polycomb-group (PcG) proteins and increased expression of microRNAs (miRNAs), which target PcG proteins. Decreased CpG island methylation and increased levels of active histone marks at genomic regions encoding miRNAs were observed after 5-AzaC treatment. Taken together, DNMTs have a critical role in regulating the cellular senescence of hUCB-MSCs through controlling not only the DNA methylation status but also active/inactive histone marks at genomic regions of PcG-targeting miRNAs and p16(INK4A) and p21(CIP1/WAF1) promoter regions.     

利用RNA干扰抑制p21WAF1/CIP1基因的表达及周期阻滞效应

目的：构建p21WAF1/CIP1基因小干扰RNA（siRNA）的真核表达载体,观察其对p21WAF1/CIP1表达的影响和细胞周期的变化。方法：合成了针对p21WAF1/CIP1基因的siRNA,将其克隆到siRNA表达载体pSliencer2.1-U6neo上,将重组质粒和带FLAG标签的p21WAF1/CIP1共转染293T人胚肾细胞,通过Westernblot检验RNA干扰（RNAi）敲低外源p21WAF1/CIP1的效果;将重组质粒单独转染293T人胚肾细胞,利用p21WAF1/CIP1抗体检测RNAi敲低内源p21WAF1/CIP1的效果;利用流式细胞仪检测敲低后细胞周期的变化。结果：测序证明构建了p21WAF1/CIP1siRNA真核表达载体;Westernblot和流式细胞分析证明,构建的siRNA能有效降低p21WAF1/CIP1基因的表达,并且使G1期细胞数减少14.03%,S期细胞增多13.45%。结论：构建了p21WAF1/CIP1siRNA的真核表达载体,该siRNA能有效抑制p21WAF1/CIP1基因的表达并部分解除了G1期阻滞。     

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.     

FOXO1 is a direct target of EWS-Fli1 oncogenic fusion protein in Ewing's sarcoma cells

Ewing’s family tumors are characterized by a specific t(11;22) chromosomal translocation that results in the formation of EWS-Fli1 oncogenic fusion protein. To investigate the effects of EWS-Fli1 on gene expression, we carried out DNA microarray analysis after specific knockdown of EWS-Fli1 through transfection of synthetic siRNAs. EWS-Fli1 knockdown increased expression of genes such as DKK1 and p57 that are known to be repressed by EWS-Fli1 fusion protein. Among other potential EWS-Fli1 targets identified by our microarray analysis, we have focused on the FOXO1 gene since it encodes a potential tumor suppressor and has not been previously reported in Ewing’s cells. To better understand how EWS-Fli1 affects FOXO1 expression, we have established a doxycycline-inducible siRNA system to achieve stable and reversible knockdown of EWS-Fli1 in Ewing’s sarcoma cells. Here we show that FOXO1 expression in Ewing’s cells has an inverse relationship with EWS-Fli1 protein level, and FOXO1 promoter activity is increased after doxycycline-induced EWS-Fli1 knockdown. In addition, we have found that direct binding of EWS-Fli1 to FOXO1 promoter is attenuated after doxycycline-induced siRNA knockdown of the fusion protein. Together, these results suggest that suppression of FOXO1 function by EWS-Fli1 fusion protein may contribute to cellular transformation in Ewing’s family tumors.     

Dissociation of bone morphogenetic protein-mediated growth arrest and apoptosis of mouse B cells by HPV-16 E6/E7

We have previously found that bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta family, induces cell-cycle arrest in the G1 phase and apoptotic cell death of HS-72 mouse hybridoma cells. In this study, we show that BMP-2 did not alter expression of cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4, p27KIP1, p16INK4a, or p15INK4b, but enhanced expression of p21(CIP1/WAF1). Accumulation of p21(CIP1/WAF1) resulted in increased binding of p21(CIP1/WAF1) to CDK4 and concomitantly caused a profound decrease in the in vitro retinoblastoma protein (Rb) kinase activity of CDK4. Furthermore, the ectopic expression of human papilloma virus type-16 E7, an inhibitor of p21(CIP1/WAF1) and Rb, reverted G1 arrest induced by BMP-2. Expression of E6/E7, without increasing the p53 level, blocked inhibition of Rb phosphorylation and G1 arrest, but did not attenuate cell death in BMP-treated HS-72 cells. Taken together, these results suggest that inhibition of Rb phosphorylation by p21(CIP1/WAF1) is responsible for BMP-2-mediated G1 arrest and that BMP-2-induction of apoptosis might be independent of Rb hypophosphorylation.     

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