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.     

Induction of apoptosis in p53-deficient human hepatoma cell line by wild-type p53 gene transduction: inhibition by antioxidant

We investigated the role of wild-type (wt)-p53 as an inducer of apoptotic cell death in human hepatoma cell lines. Following the retrovirus-mediated transduction of the wt-p53 gene, Hep3B cells lacking the endogenous p53 expression began to die through apoptosis in 4 h. They showed a maximal apoptotic death at 12 h, whereas HepG2 cells expressing endogenous p53 did not. However, the transduction of the wt-p53 gene elicited growth suppression of both Hep3B and HepG2 cells. P21(WAF1/CIP1), a p53-inducible cell cycle inhibitor, was induced, not only in Hep3B cells undergoing apoptosis, but also in HepG2 cells. The kinetics of the p21(WAF1/CIP1) induction, DNA fragmentation, and growth suppression of the Hep3B cells showed that DNA fragmentation and growth suppression progressed rapidly following p21(WAF1/CIP1) accumulation. N-acetyl-cysteine or glutathione, potent antioxidants, strongly inhibited the DNA fragmentation, but did not reduce the elevated level of p21(WAF1/CIP1). These findings suggested that p21(WAF1/CIP1) was not a critical mediator for the execution of p53-mediated apoptosis, although it contributed to the growth inhibition of cells undergoing apoptosis. Furthermore, p53-mediated apoptosis could be repressed by antioxidants.     

Mechanisms of differential activation of target gene promoters by p53 hinge domain mutants with impaired apoptotic function

Suppression of tumor cell growth by p53 results from the activation of both apoptosis and cell cycle arrest functions that have been shown to be separable activities of p53. We report here that some mutants in the p53 hinge domain, a short linker between the DNA binding and tetramerization domains, differentially activated the promoters of p53 target genes and possessed an impaired apoptotic function. Our results indicate that the hinge domain may play an important role in differentially regulating p53 cell cycle arrest and apoptotic functions. However, the mechanisms by which p53 hinge domain mutants differentially activate its target genes, e.g. p21(WAF1/CIP1) and Bax, remain unknown. To investigate the possible mechanisms, recombinant p21(WAF1/CIP1) and Bax promoters were constructed, resulting in rearrangement of the existing p53 binding sites within a given promoter or actually swapping p53 binding sites between the two promoters. Our results suggest that multiple mechanisms of differential transactivation occur, depending on the molecular nature of the relevant hinge domain mutant, such as the possibility that dual separate DNA binding sites in the p21(WAF1/CIP1) promoter are responsible for the selective transactivation activity of p53 hinge domain mutant del300-327, which has a large deletion in the hinge domain. Lack of ideal p53 binding sites in the Bax promoter results in less potent activation than that seen with the p21(WAF1/CIP1) promoter when it is transactivated by hinge domain point mutant mutR306P or short deletion mutant del300-308 proteins. How the single mutation or the short deletion affect the conformation of p53 and consequently the transactivation of the Bax promoter will require further investigation of the relevant p53 protein: DNA-binding domain by NMR and x-ray crystallographic techniques.     

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.     

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.     

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.     

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.     

Adriamycin (ADM) induced apoptosis in Transitional Cell Cancer (TCC) cell lines accompanied by p21 WAF1/CIP1 induction

Genotoxic stimuli, including anticancer drugs, induce apoptosis in cancer cells through increase of p53, p21WAF1/CIP1 , at least in part. Bcl-2 and Bax modify this pathway or directly regulated by p53. Here we studied Adriamycin (ADM)-induced apoptosis in four human bladder cancer cell lines in respect of p53, p21WAF1/CIP1 and Bcl-2 family proteins. After ADM, treatment bladder cancer cells underwent dose-dependent cell death with typical morphologic features of apoptosis. Among four cell lines RT4 with wt p53, low ratio of Bcl-2 to Bax and induction of p21WAF1/CIP1 after ADM treatment, was the most sensitive to induction of apoptosis. Thus, p53, p21WAF1/CIP1 , Bcl-2 and Bax status might determine susceptibility of bladder cancer cells to ADM induced apoptosis.     

T-cadherin-mediated cell growth regulation involves G2 phase arrest and requires p21(CIP1/WAF1) expression

Members of the cadherin family have been implicated as growth regulators in multiple tumor types. Based on recent studies from our laboratory implicating T-cadherin expression in mouse brain tumorigenesis, we examined the role of T-cadherin in astrocytoma growth regulation. In this report, we show that T-cadherin expression increased during primary astrocyte physiologic growth arrest in response to contact inhibition and serum starvation in vitro, suggesting a function for T-cadherin in astrocyte growth regulation. We further demonstrate that transient and stable reexpression of T-cadherin in deficient C6 glioma cell lines results in growth suppression. In addition, T-cadherin-expressing C6 cell lines demonstrated increased homophilic cell aggregation, increased cell attachment to fibronectin, and decreased cell motility. Cell cycle flow cytometry demonstrated that T-cadherin reexpression resulted in G2 phase arrest, which was confirmed by mitotic index analysis. This growth arrest was p53 independent, as T-cadherin could still mediate growth suppression in p53(-/-) mouse embryonic fibroblasts. T-cadherin-expressing C6 cell lines exhibited increased p21(CIP1/WAF1), but not p27(Kip1), expression. Lastly, T-cadherin-mediated growth arrest was dependent on p21(CIP1/WAF1) expression and was eliminated in p21(CIP1/WAF1)-deficient fibroblasts. Collectively, these observations suggest a novel mechanism of growth regulation for T-cadherin involving p21(CIP1/WAF1) expression and G2 arrest.     

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.     

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。     

The OGF-OGFr axis utilizes the p16INK4a and p21WAF1/CIP1 pathways to restrict normal cell proliferation

Opioid growth factor (OGF) is an endogenous opioid peptide ([Met⁵]enkephalin) that interacts with the OGF receptor (OGFr) and serves as a tonically active negative growth factor in cell proliferation of normal cells. To clarify the mechanism by which OGF inhibits cell replication in normal cells, we investigated the effect of the OGF–OGFr axis on cell cycle activity in human umbilical vein endothelial cells (HUVECs) and human epidermal keratinocytes (NHEKs). OGF markedly depressed cell proliferation of both cell lines by up to 40% of sterile water controls. Peptide treatment induced cyclin-dependent kinase inhibitor (CKI) p16^INK4a protein expression and p21^WAF1/CIP1 protein expression in HUVECs and NHEKs, but had no effect on p15, p18, p19, or p27 protein expression in either cell type. Inhibition of either p16^INK4a or p21^WAF1/CIP1 activation by specific siRNAs blocked OGF inhibitory action. Human dermal fibroblasts and mesenchymal stem cells also showed a similar dependence of OGF action on p16^INK4a and p21^WAF1/CIP1. Collectively, these results indicate that both p16^INK4a and p21^WAF1/CIP1 are required for the OGF–OGFr axis to inhibit cell proliferation in normal cells.     

CREB represses p53-dependent transactivation of MDM2 through the complex formation with p53 and contributes to p53-mediated apoptosis in response to glucose deprivation

Recently, we have described that CREB (cAMP-responsive element-binding protein) has the ability to transactivate tumor suppressor p53 gene in response to glucose deprivation. In this study, we have found that CREB forms a complex with p53 and represses p53-mediated transactivation of MDM2 but not of p21^WAF1. Immunoprecipitation analysis revealed that CREB interacts with p53 in response to glucose deprivation. Forced expression of CREB significantly attenuated the up-regulation of the endogenous MDM2 in response to p53. By contrast, the mutant form of CREB lacking DNA-binding domain (CREBΔ) had an undetectable effect on the expression level of the endogenous MDM2. During the glucose deprivation-mediated apoptosis, there existed an inverse relationship between the expression levels of MDM2 and p53/CREB. Additionally, p53/CREB complex was dissociated from MDM2 promoter in response to glucose deprivation. Collectively, our present results suggest that CREB preferentially down-regulates MDM2 and thereby contributing to p53-mediated apoptosis in response to glucose deprivation.