P21 Promoter Activity Research Articles

Progesterone receptor-NFκB complex formation is required for progesterone-induced NFκB nuclear translocation and binding onto the p53 promoter.

We previously demonstrated that progesterone (P4) up-regulates p53 expression in human umbilical venous endothelial cells (HUVECs) through P4 receptor (PR) activation of extranuclear signaling pathways. However, the involvement of nuclear PR in P4-increased p53 expression is still unclear. Here, the molecular mechanism underlying PR-regulated p53 expression in HUVECs was investigated. Treatment with P4 increased nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α phosphorylation (IκBα and nuclear factor-κB (NFκB) nuclear translocation. Interestingly, P4 also increased PR-A, but not PR-B, nuclear translocation in HUVECs. Immunoprecipitation assay illustrated that P4 increased the formation of PR-A-NFκB complex in both the cytosol and the nucleus of HUVEC. Chromatin immunoprecipitation assay showed an interaction between PR and the NFκB binding motif on the p53 promoter. Ablation of the NFκB binding motif in the p53 promoter completely abolished P4-increased p53 promoter activity. In the absence of P4, overexpression of NFκB did not increase NFκB nuclear translocation. In contrast, treatment of NFκB-overexpressing HUVECs with P4 for only 4 hours, which is much shorter than the time (21.5 h) required for P4-induced IκBα phosphorylation, increased NFκB nuclear translocation. Blockade of PR activity abolished this effect. Taken together, these results uncover a novel role of PR for P4-induced NFκB nuclear translocation and suggest that PR-A-NFκB complex formation is required for NFκB nuclear translocation and binding onto the p53 promoter in HUVECs. Our data indicate that both nuclear and extranuclear signaling pathways of PR are involved in P4-regulated p53 expression in HUVECs.

Identification of two promoters for human D-amino acid oxidase gene: implication for the differential promoter regulation mediated by PAX5/PAX2.

D-amino acid oxidase (DAO) is a flavoenzyme that metabolizes d-amino acids. Until now, the DAO expression mechanism is still unclear. Our assessment of human DAO (hDAO) promoter activity using luciferase reporter system indicated the proximal upstream region of exon1 (-237/+1) has promoter activity (P1). Interestingly, we identified an alternative promoter in the proximal upstream region of exon2 (+4,126/+4,929) (P2). This alternative promoter has stronger activity than that of P1. Our results also revealed a negative regulatory segment (+1,163/+1,940) in intron1; that would act in concert with P1 and P2. Bioinformatics analyses elucidated the conservation of transcription factor PAX5 family binding sites among species. These sites (-60/-31) and (+4,464/+4,493), locate in P1 and P2 of hDAO, respectively. Gel shift assays demonstrated P1 contains a site (-60/-31) for PAX5 binding while P2 has three sites for both paired box gene 2 (PAX2) and paired box gene 5 (PAX5) binding. The dual roles of PAX5 family in regulating hDAO transcription by modulating promoter activity of P1 and activating promoter activity of P2 were implicated based on the site-directed mutagenesis experiment. Altogether, our data suggested the differential regulation of hDAO expression by two promoters whose activities may be modulated by the binding of PAX2 and PAX5.

Hypofractionated radiotherapy induces miR-34a expression and enhances apoptosis in human nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a relatively radiosensitive disease. However, the therapeutic effects of radiotherapy are not always satisfactory due to radioresistance. The hypofractionated schema is currently widely used in clinical practice. In the present study, we investigated the effects of hypofractionated radiotherapy on NPC cells and explored the mechanisms involved. In addition, we aimed to determine the role of miR-34a in the effects of hypofractionated radiotherapy and whether these effects occur in a p53-dependent manner. For this purpose, we used CNE1 and CNE2 NPC cells which were subjected to hyperfractionated and hypofractionated radiotherapy. The viability of the cells was measured by MTT assay and acridine orange (AO) and ethidium bromide (EB) staining was used to observe morphological changes. In addition, AnnexinV-propidium iodide (PI) staining and flow cytometry were used to determine the number of apoptotic cells and mRNA and protein expression was measured by qPCR and western blot analysis, respectively. The results revealed that hypofractionated radiotherapy enhanced apoptosis and increased the expression of miR-34a and p53 in the NPC cells. In addition, it stimulated p53 promoter activity and downregulated the protein expression of c-Myc in the human NPC cells. Furthermore, the knockdown of miR-34a suppressed the growth inhibitory effects induced by hypofractionated radiotherapy. Thus, our results suggest that the enhanced apoptosis of NPC cells may be associated with the miR-34a-mediated suppression of c-Myc in a p53-dependent manner.

Long non-coding RNA urothelial carcinoma associated1 induces cell replication by inhibiting BRG1 in 5637 cells.

Long non-coding RNA urothelial carcinoma associated 1 (UCA1) was first identified in bladder cancer tissues. High expression of UCA1 in bladder cancer has suggested it may serve as a potential diagnostic molecular marker for bladder cancer. Subsequent research in bladder cancer cell lines showed that UCA1 can promote cell proliferation, but the underlying mechanism remains unknown. In the present study, we identified BRG1 as a UCA1 binding partner using an in vitro RNA pull-down assay, and RNA-binding protein immunoprecipitation assay confirmed UCA1-BRG binding in bladder cancer cells in vivo. BRG1 is a chromatin remodeling factor with reported tumor suppressor activities that directly upregulates levels of the p21 cell cycle inhibitor by binding sequences in the p21 promoter. Depletion of UCA1 by RNAi resulted in upregulated p21 levels and inhibition of cell replication, while overexpressed UCA1 reduced p21 protein and promoted cell growth. Notably, UCA1 downregulation of p21 and induction of cell proliferation antagonized the function of BRG1. UCA1 highly expressed tissue samples are often with BRG1 high expression. Furthermore, we found that UCA1 impairs both binding of BRG1 to the p21 promoter and chromatin remodeling activity of BRG1. Collectively, these results demonstrate that UCA1 promotes bladder cancer cell proliferation by inhibiting BRG1.

P38δ Regulates p53 to Control p21Cip1 Expression in Human Epidermal Keratinocytes

PKCδ suppresses keratinocyte proliferation via a mechanism that involves increased expression of p21(Cip1). However, the signaling mechanism that mediates this regulation is not well understood. Our present studies suggest that PKCδ activates p38δ leading to increased p21(Cip1) promoter activity and p21(Cip1) mRNA/protein expression. We further show that exogenously expressed p38δ increases p21(Cip1) mRNA and protein and that p38δ knockdown or expression of dominant-negative p38 attenuates this increase. Moreover, p53 is an intermediary in this regulation, as p38δ expression increases p53 mRNA, protein, and promoter activity, and p53 knockdown attenuates the activation. We demonstrate a direct interaction of p38δ with PKCδ and MEK3 and show that exogenous agents that suppress keratinocyte proliferation activate this pathway. We confirm the importance of this regulation using a stratified epidermal equivalent model, which mimics in vivo-like keratinocyte differentiation. In this model, PKCδ or p38δ knockdown results in reduced p53 and p21(Cip1) levels and enhanced cell proliferation. We propose that PKCδ activates a MEKK1/MEK3/p38δ MAPK cascade to increase p53 levels and p53 drives p21(Cip1) gene expression.

P21-Driven Multifusion Gene System for Evaluating the Efficacy of Histone Deacetylase Inhibitors by In Vivo Molecular Imaging and for Transcription Targeting Therapy of Cancer Mediated by Histone Deacetylase Inhibitor

Overexpressed histone deacetylase (HDAC) activity has been linked with tumor initiation and progression that prompt the development of histone deacetylase inhibitors (HDACIs) as anticancer agents. HDACI was reported to be able to activate p21 promoter through the SP1 binding sites in the proximal region of p21(WAF1/CIP1) promoter. In this study, we established a p21(WAF1/CIP1) promoter-driven triple-fused reporter gene system (p21-3H) to evaluate the efficacy of HDACI and the ganciclovir (GCV)-mediated anticancer effect contributed by HDACI-induced and p21-driven truncated herpes simplex virus-1 thymidine kinase sr39 mutant (ttksr39) in vitro and in vivo. The p21-3H construct was generated and stably or transiently transfected into H1299 cell lines. These cells were treated with trichostatin A or vorinostat (suberoylanilide hydroxamic acid [SAHA]) to evaluate the activation of p21 promoter-driven reporter gene expression by in vitro confocal fluorescence microscopy, luciferase assay, 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil ((3)H-FEAU) cellular uptake, in vivo bioluminescence imaging, and 9-(4-(18)F-fluoro-3-hydroxymethylbutyl) guanine ((18)F-FHBG) small-animal PET imaging. The therapeutic efficacy on p21-3H-expressing tumor xenografts was assessed by daily administration with SAHA (100 mg/kg intraperitoneally) or GCV (20 mg/kg) for 9 d, followed by tumor volume measurement. On treatment with trichostatin A or SAHA, H1299 cells carrying p21-3H showed a significant increase of luciferase activity, cellular uptake of (3)H-FEAU (Moravek), and DsRed expression. In vivo tumor xenografts carrying p21-3H also showed increased luciferase activity by luminescent imaging and enhanced accumulation of (18)F-FHBG by small-animal PET imaging. Furthermore, when cells transfected with p21-3H or p21/PstI-3H (which lacks p53-binding sites) were treated, the increase of luciferase activity was similar in both groups, indicating that HDACI-induced p21 promoter activation is independent of p53. Both in vitro and in vivo results showed improved therapeutic effect by combined treatment of GCV and HDACI. We have established an HDACI-inducible, p21-driven reporter system that has the potential for evaluating the anticancer effect of HDACIs on cancer cells by multiple molecular imaging modalities. Furthermore, ttksr39 in a p21-3H reporter construct provides a potential combination with thymidine kinase-mediated gene therapy to optimize the therapeutic benefit of HDACI.

Dux4 induces cell cycle arrest at G1 phase through upregulation of p21 expression

It has been implicated that Dux4 plays crucial roles in development of facioscapulohumeral dystrophy. But the underlying myopathic mechanisms and related down-stream events of this retrogene were far from clear. Here, we reported that overexpression of Dux4 in a cell model TE671 reduced cell proliferation rate, and increased G1 phase accumulation. We also determined the impact of Dux4 on p53/p21 signal pathway, which controls the checkpoint in cell cycle progression. Overexpression of Dux4 increased p21 mRNA and protein level, while expression of p53, phospho-p53 remained unchanged. Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. Furthermore, we demonstrated that enhanced Dux4 expression increased p21 promoter activity and elevated expression of Sp1 transcription factor. Mutation of Sp1 binding site decreased dux4 induced p21 promoter activation. Chromatin immunoprecipitation (ChIP) assays confirmed the Dux4-induced binding of Sp1 to p21 promoter in vivo. These results suggest that Dux4 might induce proliferation inhibition and G1 phase arrest through upregulation of p21.

Hepatitis B virus X protein promotes P3 transcript expression of the insulin‐like growth factor 2 gene via inducing hypomethylation of P3 promoter in hepatocellular carcinoma

Hepatitis B virus (HBV) X protein (HBx) contributes to hepatocarcinogenesis. The overexpression of transcripts from P3 and P4 promoters of the insulin-like growth factor 2 (IGF2) gene is observed in hepatocellular carcinoma (HCC). Here, we aimed to explore the involvement of HBx in P3-driven mRNA overexpression and underlying epigenetic mechanism. P3 mRNA, P3 methylation status, HBx mRNA and HBx protein were analysed in human HCC samples with and without HBV infection using quantitative RT-PCR, bisulphite sequencing and Western blotting. The effects of HBx on P3 mRNA expression, and P3 transcriptional activity and methylation were further evaluated in HCC cell lines. P3 mRNA level was higher and P3 methylation level was lower in HBV-positive HCC specimens compared with those of HBV-negative HCC specimens. P3 transcript abundance was positively correlated with HBx expression and negatively correlated with P3 methylation in HCC specimens. The stable expression of HBx upregulated P3 mRNA expression and reduced P3 methylation level in HepG2-HBx cells. The transient expression of HBx stimulated P3 promoter activity and decreased P3 methylation level of P3 promoter-luciferase construct in a dose-dependent manner in HepG2 and Huh-7 cells. Furthermore, HBx mRNA expression was found to be independent predictive factors for both shorter disease-free survival time and shorter overall survival time of HCC patients. HBx may promote IGF2-P3 transcript expression by inducing hypomethylation of P3 promoter and may be associated with an inferior clinical outcome of HBV-related HCC patients. This study provides useful information for understanding the mechanism of HBx-mediated HCC.

Differential Promoter Usage and Regulation Of The Human Interleukin-5 Receptor α (IL-5Rα) Gene In Developing Eosinophil Progenitors

IL-5Rα is a critical component of the IL-5 signaling pathway responsible for regulating the expansion of the eosinophil lineage for development of eosinophilia in allergic diseases, including asthma. IL-5Rα has two promoters, P1 and P2, both functionally active in eosinophilic cell lines. However, their differential roles and regulation during the development of authentic eosinophil progenitors remain unexplored. Human umbilical cord blood-derived CD34+progenitors under IL-5-induced eosinophil differentiation were transfected with IL-5Rα-P1 or -P2 promoter luciferase reporter vectors at various time points to assess their differential promoter activity. Chromatin immunoprecipitation (ChIP) was performed at corresponding time points to determine the dynamic occupancy of these promoters by transcription factors implicated in IL-5Rα regulation during eosinophil development. The IL-5Rα-P1 promoter is active throughout IL-5-induced eosinophil differentiation of CD34+/IL-5Rα+progenitors. However, P2 promoter activity is suddenly induced on day 7, overtaking P1 activity, before being attenuated during continued eosinophil development. Peak P2 activity coincides with a dramatic induction in expression of the predominant soluble IL-5Rα isoform mRNA. Correspondingly, quantitative ChIPs revealed that the two promoters exhibit differential occupancy by transcription factors GATA-1, PU.1 and/or the C/EBP family of transcription factors (α, β and ε). Human IL-5Rα-P1 and -P2 promoters are under differential regulation, mediated in part through dynamic and combinatorial interactions of stage-specific transcription factors. The coincidental peak in P2 promoter activity and IL-5Rα soluble isoform mRNA suggest that the IL-5-binding soluble receptor isoform is induced during eosinophil development to regulate or prevent excessive expansion of the eosinophil lineage in parasitic and allergic diseases.

Transcriptional activation of p21WAF1/CIP1 is mediated by increased DNA binding activity and increased interaction between p53 and Sp1 via phosphorylation during replicative senescence of human embryonic fibroblasts

Although p21(WAF1/CIP1) is known to be elevated during replicative senescence of human embryonic fibroblasts (HEFs), the mechanism for p21 up-regulation has not been elucidated clearly. In order to explore the mechanism, we analyzed expression of p21 mRNA and protein and luciferase activity of full-length p21 promoter. The result demonstrated that p21 up-regulation was accomplished largely at transcription level. The promoter assay using serially-deleted p21 promoter constructs revealed that p53 binding site was the most important site and Sp1 binding sites were necessary but not sufficient for transcriptional activation of p21. In addition, p53 protein was shown to interact with Sp1 protein. The interaction was increased in aged fibroblasts and was regulated by phosphorylation of p53 and Sp1. DNA binding activity of p53 was significantly elevated in aged fibroblasts but that of Sp1 was not. DNA binding activities of p53 and Sp1 were also regulated by phosphorylation. Phosphorylation of p53 at serine-15 and of Sp1 at serines appears to be involved. Taken together, the result demonstrated that p21 transcription during replicative senescence of HEFs is up-regulated by increase in DNA binding activity and interaction between p53 and Sp1 via phosphorylation.

Identification of Primary Equilibrative Nucleoside Transporter 1 mRNA Isoforms Resulting from Alternative Promoter Usage in Human Hepatocytes

Human equilibrative nucleoside transporter 1 (hENT1) transports various nucleoside analogues into cells. Although the single hENT1 promoter region (P1) and the mRNA isoform (a1) have been characterized previously, we have recently identified additional promoter regions P2 and P3 (which primarily generate c1/2/3 mRNAs and d1/2/3/4 mRNAs, respectively) in the human liver. Therefore, this study aimed at identifying the primary hENT1 mRNA isoforms expressed in human hepatocytes, while simultaneously obtaining functional evidence of alternative hENT1 promoter usage. Our results showed that the expressions of hENT1c1, d3, and (to a lesser extent) c2 mRNAs were strikingly predominant over the other mRNA isoforms in human hepatocytes, that the abundant expression of these mRNAs was consistent with the high levels of P2 and P3 promoter activity, and that these promoters were significantly marked by transcriptionally active histone modification in hepatic cells. To summarize, our results demonstrate that, resulting from the manipulated alternative promoter usage, hENT1c1 and d3 (and c2) mRNAs are primarily expressed in human hepatocytes, which suggests that they may play important roles in controlling hENT1 expression levels in those cells. Our findings are expected to provide significant insights into the molecular machinery of hENT1 expression control.

Lappaol F, a Novel Anticancer Agent Isolated from PlantArctium LappaL.

In an effort to search for new cancer-fighting therapeutics, we identified a novel anticancer constituent, Lappaol F, from plant Arctium Lappa L. Lappaol F suppressed cancer cell growth in a time- and dose-dependent manner in human cancer cell lines of various tissue types. We found that Lappaol F induced G(1) and G(2) cell-cycle arrest, which was associated with strong induction of p21 and p27 and reduction of cyclin B1 and cyclin-dependent kinase 1 (CDK1). Depletion of p21 via genetic knockout or short hairpin RNA (shRNA) approaches significantly abrogated Lappaol F-mediated G(2) arrest and CDK1 and cyclin B1 suppression. These results suggest that p21 seems to play a crucial role in Lappaol F-mediated regulation of CDK1 and cyclin B1 and G(2) arrest. Lappaol F-mediated p21 induction was found to occur at the mRNA level and involved p21 promoter activation. Lappaol F was also found to induce cell death in several cancer cell lines and to activate caspases. In contrast with its strong growth inhibitory effects on tumor cells, Lappaol F had minimal cytotoxic effects on nontumorigenic epithelial cells tested. Importantly, our data also demonstrate that Lappaol F exhibited strong growth inhibition of xenograft tumors in nude mice. Lappaol F was well tolerated in treated animals without significant toxicity. Taken together, our results, for the first time, demonstrate that Lappaol F exhibits antitumor activity in vitro and in vivo and has strong potential to be developed as an anticancer therapeutic.

Shikonin induces cell cycle arrest in human gastric cancer (AGS) by early growth response 1 (Egr1)-mediated p21 gene expression

Ethnopharmacological relevanceLithospermum erythrorhizon, a naphthoquinone compound derived from a shikonin, has long been used as traditional Chinese medicine for treatment of various diseases, including cancer. To evaluate the cytotoxic effects of shikonin on AGS gastric cancer cells via induction of cell cycle arrest. Materials and methodsWe observed the effects of 12.5–100ng/mL dosage of shikonin treatment on AGS cancer cell line with the incubation time of 6h. Cytotoxic effects were assessed by measuring the changes in the intracellular ROS, appearance of senescence phenotype, cell cycle progression, CDK and cyclins expression levels upon shikonin treatment. We also examined upon the activation of Egr1-mediated p21 expression, by siRNA transfection, Luciferase assay, and ChIP assay. ResultsIn this study, we found that shikonin inhibits cell proliferation by arresting cell cycle progression at the G2/M phase via modulation of p21 in AGS cells. Also, our results revealed that the p21 gene was transactivated by early growth response1 (Egr1) in response to the shikonin treatment. Transient Egr1 expression enhanced shikonin-induced p21 promoter activity, whereas the suppression of Egr1 expression by small interfering RNA attenuated the ability of shikonin to induce p21 promoter activity. ConclusionOur results suggested that the anti-proliferative activity of shikonin was due to its ability to induce cell cycle arrest via Egr1–p21 signaling pathway. Thus, the work stated here validates the traditional use of shikonin in the treatment of cancer.

Transcription factor C/EBPβ and 17β-estradiol promote transcription of the porcine p53 gene

The tumor protein 53 (p53) gene played a crucial role in maternal reproduction except its classic roles in maintaining genomic stability and preventing tumorigenesis. However, little is known concerning the regulatory elements which control the expression of p53 gene. In this study, we predicted two binding sites (−490/−477 and −405/−392) of transcription factor CCAAT/enhancer binding protein beta (C/EBPβ) within the core promoter (−985/−273) determined by promoter deletion analysis, and discovered that the second site (−405/−392) was important for p53 promoter activity by site-directed mutagenesis. Then the binding of C/EBPβ to the p53 promoter was identified by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP). Moreover, evidence from C/EBPβ overexpression and RNAi studies showed C/EBPβ regulated p53 promoter activity and endogenous p53 expression. Meanwhile, we observed p53 mRNA at the peak in 10−6mol/L 17β-estradiol treated cells for 24h via enhancing its core promoter activity. Taken together, our study indicates that C/EBPβ and 17β-estradiol are the essential regulatory factors for p53 transcription.

Lithium Down-regulates Histone Deacetylase 1 (HDAC1) and Induces Degradation of Mutant Huntingtin

Lithium is an effective mood stabilizer that has been clinically used to treat bipolar disorder for several decades. Recent studies have suggested that lithium possesses robust neuroprotective and anti-tumor properties. Thus far, a large number of lithium targets have been discovered. Here, we report for the first time that HDAC1 is a target of lithium. Lithium significantly down-regulated HDAC1 at the translational level by targeting HDAC1 mRNA. We also showed that depletion of HDAC1 is essential for the neuroprotective effects of lithium and for the lithium-mediated degradation of mutant huntingtin through the autophagic pathway. Our studies explain the multiple functions of lithium and reveal a novel mechanism for the function of lithium in neurodegeneration.

Identification of TSG101 Functional Domains and p21 Loci Required for TSG101-Mediated p21 Gene Regulation

TSG101 (tumor susceptibility gene 101) is a multi-domain protein known to act in the cell nucleus, cytoplasm, and periplasmic membrane. Remarkably, TSG101, whose location within cells varies with the stage of the cell cycle, affects biological events as diverse as cell growth and proliferation, gene expression, cytokinesis, and endosomal trafficking. The functions of TSG101 additionally are recruited for viral and microvesicle budding and for intracellular survival of invading bacteria. Here we report that the TSG101 protein also interacts with and down-regulates the promoter of the p21CIP1/WAF1tumor suppressor gene, and identify a p21 locus and TSG101 domains that mediate this interaction. TSG101 deficiency in Saos-2 human osteosarcoma cells was accompanied by an increased abundance of p21 mRNA and protein and the retardation of cell proliferation. A cis-acting element in the p21 promoter that interacts with TSG101 and is required for promoter repression was located using chromatin immunoprecipitation (ChIP) analysis and p21-driven luciferase reporter gene expression, respectively. Additional analysis of TSG101 deletion mutants lacking specific domains established the role of the central TSG101 domains in binding to the p21 promoter and demonstrated the additional essentiality of the TSG101 C-terminal steadiness box (SB) in the repression of p21 promoter activity. Neither binding of TSG101 to the p21 promoter nor repression of this promoter required the TSG101 N-terminal UEV domain, which mediates the ubiquitin-recognition functions of TSG101 and its actions as a member of ESCRT endocytic trafficking complexes, indicating that regulation of the p21 promoter by TSG101 is independent of its role in such trafficking.

Exposure to Nerve Growth Factor Worsens Nephrotoxic Effect Induced by Cyclosporine A in HK-2 Cells

Nerve growth factor is a neurotrophin that promotes cell growth, differentiation, survival and death through two different receptors: TrkANTR and p75NTR. Nerve growth factor serum concentrations increase during many inflammatory and autoimmune diseases, glomerulonephritis, chronic kidney disease, end-stage renal disease and, particularly, in renal transplant. Considering that nerve growth factor exerts beneficial effects in the treatment of major central and peripheral neurodegenerative diseases, skin and corneal ulcers, we asked whether nerve growth factor could also exert a role in Cyclosporine A-induced graft nephrotoxicity. Our hypothesis was raised from basic evidence indicating that Cyclosporine A-inhibition of calcineurin-NFAT pathway increases nerve growth factor expression levels. Therefore, we investigated the involvement of nerve growth factor and its receptors in the damage exerted by Cyclosporine A in tubular renal cells, HK-2. Our results showed that in HK-2 cells combined treatment with Cyclosporine A + nerve growth factor induced a significant reduction in cell vitality concomitant with a down-regulation of Cyclin D1 and up-regulation of p21 levels respect to cells treated with Cyclosporine A alone. Moreover functional experiments showed that the co-treatment significantly up-regulated human p21promoter activity by involvement of the Sp1 transcription factor, whose nuclear content was negatively regulated by activated NFATc1. In addition we observed that the combined exposure to Cyclosporine A + nerve growth factor promoted an up-regulation of p75 NTR and its target genes, p53 and BAD leading to the activation of intrinsic apoptosis. Finally, the chemical inhibition of p75NTR down-regulated the intrinsic apoptotic signal. We describe two new mechanisms by which nerve growth factor promotes growth arrest and apoptosis in tubular renal cells exposed to Cyclosporine A.

Activation of p53 by spermine mediates induction of autophagy in HT1080 cells

The recent evidences indicate that autophagy is associated with a number of pathological processes including cancer, muscular disorder and neurodegeneration in addition to longevity. The efficacy of spermine was investigated on induction of autophagy through histone deacetylation and p53 activation in human fibrosarcoma cell line, HT1080. In this study, it was discovered that spermine increases the activity of HAT and autophagy. It was also identified that the transcriptional activation of p53 and the activation of p21 promoter by spermine are related to the induction of autophagy in reporter gene assay. Furthermore, western blot analyses demonstrated that spermine modulates the expression of proteins related to autophagy and apoptosis. The expression levels of Ac-histone H3, HDAC1, HAT1, p300 and SIRT1 were increased in HT1080 cells treated with spermine. In addition, the expression levels of protein such as acetyl-p53, p-p53, Bcl-2 and caspase-9 inducing apoptosis were increased in the presence of spermine. Moreover, the levels of Mdm2 and caspase-3 expression were reduced in the cells exposed to spermine compared to blank group. These results suggest that activation of HAT in the presence of spermine promotes the induction of autophagy in HT1080 cells through the enhanced activity of p-p53 and acetyl p53.

CtIP is required for DNA damage-dependent induction of P21

DNA endonuclease CtIP is involved in both DNA double-strand break (DSB) repair and transcriptional repression/activation. The cyclin-dependent kinase inhibitor P21, which is induced at transcription level in response to a variety of stresses, controls G1/S transition. In this report, we found that CtIP bound to the P21 promoter, and this binding was enhanced in response to DNA damage. Concomitantly, ectopic expression of CtIP increased P21 promoter activity, and this increment was enhanced upon camptothecin treatment. Conversely, DNA damage failed to induce P21 gene expression in CtIP-deficient cells. Taken together, our data demonstrate that CtIP is required for DNA damage-induced P21 induction.

Forkhead Box O1 (FOXO1) Protein, but Not p53, Contributes to Robust Induction of p21 Expression in Fasted Mice

Reporter mice that enable the activity of the endogenous p21 promoter to be dynamically monitored in real time in vivo and under a variety of experimental conditions revealed ubiquitous p21 expression in mouse organs including the brain. Low light bioluminescence microscopy was employed to localize p21 expression to specific regions of the brain. Interestingly, p21 expression was observed in the paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus, regions that detect nutrient levels in the blood stream and signal metabolic actions throughout the body. These results suggested a link between p21 expression and metabolic regulation. We found that short-term food deprivation (fasting) potently induced p21 expression in tissues involved in metabolic regulation including liver, pancreas and hypothalamic nuclei. Conditional reporter mice were generated that enabled hepatocyte-specific expression of p21 to be monitored in vivo. Bioluminescence imaging demonstrated that fasting induced a 7-fold increase in p21 expression in livers of reporter mice and Western blotting demonstrated an increase in protein levels as well. The ability of fasting to induce p21 expression was found to be independent of p53 but dependent on FOXO1. Finally, occupancy of the endogenous p21 promoter by FOXO1 was observed in the livers of fasted but not fed mice. Thus, fasting promotes loading of FOXO1 onto the p21 promoter to induce p21 expression in hepatocytes.