Cytoplasmic p21(Cip1/WAF1) regulates neurite remodeling by inhibiting Rho-kinase activity.
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ABSTRACT:
SUBMITTER: Tanaka H
PROVIDER: S-EPMC2173114 | biostudies-other | 2002 Jul
REPOSITORIES: biostudies-other
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Project description:Background & aimsMist1 is a basic helix-loop-helix (bHLH) transcription factor that is important to the proper development of the exocrine pancreas. The aim of this study was to investigate the role of Mist1 in modulating acinar cell proliferation.MethodsDuctal and acinar pancreatic cell lines were engineered to express an inducible Mist1 complementary DNA or to express a short hairpin RNA that targeted endogenous Mist1. Alterations in RNA and protein levels were detected by real-time reverse-transcription polymerase chain reaction and immunoblots. Chromatin immunoprecipitation and reporter gene assays were performed to map Mist1-responsive elements on target genes; the overall proliferation index of acinar cells from Mist1 null pancreata was evaluated by immunohistochemistry.ResultsExpression of Mist1 resulted in a significant decrease in the proliferative potential of cells that was associated with induced expression of p21(CIP1/WAF1). Short hairpin RNA-directed knockdown of p21(CIP1/WAF1) generated cells that were refractory to Mist1 expression, whereas knockdown of Mist1 transcripts or deletion of Mist1 from the mouse genome led to increased cell proliferation and a concomitant decrease in p21(CIP1/WAF1) protein levels. Surprisingly, Mist1-dependent activation of the p21(CIP1/WAF1) promoter was independent of classic basic helix-loop-helix protein binding sites. Instead, Sp1 binding sites were essential for Mist1-dependent transcription, suggesting that Mist1 activates p21(CIP1/WAF1) expression through a unique Sp1 pathway. Indeed, coimmunoprecipitation studies demonstrated that Mist1 and Sp1 were found within the same transcription complex.ConclusionsOur results show that Mist1 has a dual role in the development of the exocrine pancreas: controlling cell proliferation and promoting terminal differentiation.
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Project description: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.
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Project description:Although the role of p21(Waf1/Cip1) gene expression is well documented in various cell culture studies, its in vivo roles are poorly understood. To gain further insight into the role of p21(Waf1/Cip1) gene expression in vivo, we attempted to visualize the dynamics of p21(Waf1/Cip1) gene expression in living animals. In this study, we established a transgenic mice line (p21-p-luc) expressing the firefly luciferase under the control of the p21(Waf1/Cip1) gene promoter. In conjunction with a noninvasive bioluminescent imaging technique, p21-p-luc mice enabled us to monitor the endogenous p21(Waf1/Cip1) gene expression in vivo. By monitoring and quantifying the p21(Waf1/Cip1) gene expression repeatedly in the same mouse throughout its entire lifespan, we were able to unveil the dynamics of p21(Waf1/Cip1) gene expression in the aging process. We also applied this system to chemically induced skin carcinogenesis and found that the levels of p21(Waf1/Cip1) gene expression rise dramatically in benign skin papillomas, suggesting that p21(Waf1/Cip1) plays a preventative role(s) in skin tumor formation. Surprisingly, moreover, we found that the level of p21(Waf1/Cip1) expression strikingly increased in the hair bulb and oscillated with a 3-week period correlating with hair follicle cycle progression. Notably, this was accompanied by the expression of p63 but not p53. This approach, together with the analysis of p21(Waf1/Cip1) knockout mice, has uncovered a novel role for the p21(Waf1/Cip1) gene in hair development. These data illustrate the unique utility of bioluminescence imaging in advancing our understanding of the timing and, hence, likely roles of specific gene expression in higher eukaryotes.
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Project description:The MiT/TFE family of transcription factors (MITF, TFE3, and TFEB), which control transcriptional programs for autophagy and lysosome biogenesis have emerged as regulators of energy metabolism in cancer. Thus, their activation increases lysosomal catabolic function to sustain cancer cell growth and survival in stress conditions. Here, we found that TFEB depletion dramatically reduces basal expression levels of the cyclin-dependent kinase (CDK) inhibitor p21/WAF1 in various cell types. Conversely, TFEB overexpression increases p21 in a p53-dependent manner. Furthermore, induction of DNA damage using doxorubicin induces TFEB-mediated activation of p21, delays G2/M phase arrest, and promotes cell survival. Pharmacological inhibition of p21, instead, abrogates TFEB-mediated protection during the DNA damage response. Together, our findings uncover a novel and direct role of TFEB in the regulation of p21 expression in both steady-state conditions and during the induction of DNA-damage response (DDR). Our observations might open novel therapeutic strategies to promote cancer cell death by targeting the TFEB-p21 pathway in the presence of genotoxic agents.
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Project description:Decitabine (DAC) and 5-azacitidine have recently been approved for the treatment of myelodysplastic syndrome. The pharmacodynamic effects of DAC and 5-azacitidine outside their known activity as inhibitors of DNA methyltransferases (DNMTs) require further investigation. The purpose of this study was to investigate the effect of DAC on the expression of p21(WAF1/CIP1), a gene with a putative CpG island surrounding its promoter region. Promoter methylation analysis of p21(WAF1/CIP1) in leukemia cells revealed the absence of CpG methylation. However, DAC upregulated p21(WAF1/CIP1) expression in a dose-dependent manner (ED(50)=103.34 nM) and induced G2/M cell cycle arrest in leukemia cells. Sequential application of DAC followed by different histone deacetylase inhibitors induced expression of p21(WAF1/CIP1) synergistically. Upregulation of p21(WAF1/CIP1) paralleled DAC-induced apoptosis (ED(50)=153 nM). Low doses of DAC induced gamma-H2AX expression (ED(50)=16.5 nM) and upregulated p21(WAF1/CIP1) in congenic HCT 116 colon cancer cells in a DNMT-independent and p53-dependent fashion. Inhibition of p53 transactivation by pifithrin-alpha or the kinase activity of ATM by either the specific ATM inhibitor KU-5593 or caffeine abrogated p21(WAF1/CIP1) upregulation, indicating that DAC upregulation of p21(WAF1/CIP1) was p53- and ATM-dependent in leukemia cells. In conclusion, DAC upregulates p21(WAF1/CIP1) in DNMT-independent manner via the DNA damage/ATM/p53 axis.
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Project description:Microrchidia (MORC) family CW-type zinc-finger 2 (MORC2) regulates chromatin remodeling during the DNA-damage response, represses gene transcription, promotes lipogenesis. Here, we found that MORC2 down-regulated p21 by recruiting HDAC1 to the p21 promoter, in a p53-independent manner. MORC2-mediated down-regulation of p21 in turn promoted cell cycle progression in gastric cancer cells. Furthermore, MORC2 expression correlated negatively with p21 expression in gastric tumors in patients. We suggest that MORC2 may be a potential therapeutic target in cancer.
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Project description:Some individuals remain HIV-1 antibody and PCR negative after repeated exposures to the virus, and are referred to as HIV-exposed seronegatives (HESN). However, the causes of resistance to HIV-1 infection in cases other than those with a homozygous CCR5?32 deletion are unclear. We hypothesized that human p21WAF1/CIP1 (a cyclin-dependent kinase inhibitor) could play a role in resistance to HIV-1 infection in HESN, as p21 expression has been associated with suppression of HIV-1 in elite controllers and reported to block HIV-1 integration in cell culture. We measured p21 RNA expression in PBMC from 40 HESN and 40 low exposure HIV-1 seroconverters (LESC) prior to their infection using a real-time PCR assay. Comparing the 20 HESN with the highest exposure risk (median = 111 partners/2.5 years prior to the 20 LESC with the lowest exposure risk (median = 1 partner/2.5 years prior), p21 expression trended higher in HESN in only one of two experiments (P = 0.11 vs. P = 0.80). Additionally, comparison of p21 expression in the top 40 HESN (median = 73 partners/year) and lowest 40 LESC (median = 2 partners/year) showed no difference between the groups (P = 0.84). There was a weak linear trend between risk of infection after exposure and increasing p21 gene expression (R2 = 0.02, P = 0.12), but again only in one experiment. Hence, if p21 expression contributes to the resistance to viral infection in HESN, it likely plays a minor role evident only in those with extremely high levels of exposure to HIV-1.
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Project description:The role of glucocorticoids in the regulation of apoptosis remains incongruous. Here, we demonstrate that corticosterone protects neurons from apoptosis by a mechanism involving the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). In primary cortical neurons, corticosterone leads to a dose- and Akt-kinase-dependent upregulation with enhanced phosphorylation and cytoplasmic appearance of p21(Waf1/Cip1) at Thr 145. Exposure of neurons to the neurotoxin ethylcholine aziridinium (AF64A) results in activation of caspase-3 and a dramatic loss of p21(Waf1/Cip1) preceding apoptosis in neurons. These effects of AF64A are reversed by pretreatment with corticosterone. Corticosterone-mediated upregulation of p21(Waf1/Cip1) and neuroprotection are completely abolished by glucocorticoid and mineralocorticoid receptor antagonists as well as inhibitors of PI3- and Akt-kinase. Both germline and somatically induced p21(Waf1/Cip1) deficiency abrogate the neuroprotection by corticosterone, whereas overexpression of p21(Waf1/Cip1) suffices to protect neurons from apoptosis. We identify p21(Waf1/Cip1) as a novel antiapoptotic factor for postmitotic neurons and implicate p21(Waf1/Cip1) as the molecular target of neuroprotection by high-dose glucocorticoids.
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Project description:Platinum-based chemotherapies such as cisplatin are used as first-line treatment for the paediatric tumour neuroblastoma. Although the majority of neuroblastoma tumours respond to therapy, there is a high fraction of high-risk neuroblastoma patients that eventually relapse with increased resistance. Here, we show that one key determinant of cisplatin sensitivity is phosphorylation of the cyclin-dependent kinase inhibitor p21Cip1/Waf1. A panel of eight neuroblastoma cell lines and a TH-MYCN mouse model were investigated for the expression of p21Cip1/Waf1 using RT-qPCR, Western blot, and immunofluorescence. This was followed by investigation of sensitivity towards cisplatin and the p21Cip1/Waf1 inhibitor UC2288. Whereas the cell lines and the mouse model showed low levels of un-phosphorylated p21Cip1/Waf1, the phosphorylated p21Cip1/Waf1 (Thr145) was highly expressed, which in the cell lines correlated to cisplatin resistance. Furthermore, the neuroblastoma cell lines showed high sensitivity to UC2288, and combination treatment with cisplatin resulted in considerably decreased cell viability and delay in regrowth in the two most resistant cell lines, SK-N-DZ and BE(2)-C. Thus, targeting p21Cip1/Waf1 can offer new treatment strategies and subsequently lead to the design of more efficient combination treatments for high-risk neuroblastoma.
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Project description:Loss of cell cycle control is characteristic of tumorigenesis. The protein p21 is the founding member of cyclin-dependent kinase inhibitors and an important versatile cell cycle protein. p21 is transcriptionally controlled by p53 and p53-independent pathways. Its expression is increased in response to various intra- and extracellular stimuli to arrest the cell cycle ensuring genomic stability. Apart from its roles in cell cycle regulation including mitosis, p21 is involved in differentiation, cell migration, cytoskeletal dynamics, apoptosis, transcription, DNA repair, reprogramming of induced pluripotent stem cells, autophagy and the onset of senescence. p21 acts either as a tumor suppressor or as an oncogene depending largely on the cellular context, its subcellular localization and posttranslational modifications. In the present review, we briefly mention the general functions of p21 and summarize its roles in differentiation, migration and invasion in detail. Finally, regarding its dual role as tumor suppressor and oncogene, we highlight the potential, difficulties and risks of using p21 as a biomarker as well as a therapeutic target.
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