scholarly journals AMF1 (GPS2) Modulates p53 Transactivation

2001 ◽  
Vol 21 (17) ◽  
pp. 5913-5924 ◽  
Author(s):  
Yu-Cai Peng ◽  
Felix Kuo ◽  
David E. Breiding ◽  
Yu-Fang Wang ◽  
Claire P. Mansur ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Transcriptional Activation ◽  
Nuclear Factor ◽  
P53 Response ◽  
U2os Cells ◽  
Stress Signals ◽  
Cellular Transcription

ABSTRACT We have reported that the papillomavirus E2 protein binds the nuclear factor AMF1 (also called G-protein pathway suppressor 2 or GPS2) and that their interaction is necessary for transcriptional activation by E2. It has also been shown that AMF1 can influence the activity of cellular transcription factors. These observations led us to test whether AMF1 regulates the functions of p53, a critical transcriptional activator that integrates stress signals and regulates cell cycle and programmed cell death. We report that AMF1 associates with p53 in vivo and in vitro and facilitates the p53 response by augmenting p53-dependent transcription. Overexpression of AMF1 in U2OS cells increases basal level p21WAF1/CIP1 expression and causes a G1 arrest. U2OS cells stably overexpressing AMF1 show increased apoptosis upon exposure to UV irradiation. These data demonstrate that AMF1 modulates p53 activities.

scholarly journals p53 upregulated by HIF-1α promotes hypoxia-induced G2/M arrest and renal fibrosis in vitro and in vivo

2018 ◽  
Vol 11 (5) ◽  
pp. 371-382 ◽  
Author(s):  
Limin Liu ◽  
Peng Zhang ◽  
Ming Bai ◽  
Lijie He ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Renal Fibrosis ◽  
Intraperitoneal Administration ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
P53 Expression ◽  
Tubular Cells

Abstract Hypoxia plays an important role in the genesis and progression of renal fibrosis. The underlying mechanisms, however, have not been sufficiently elucidated. We examined the role of p53 in hypoxia-induced renal fibrosis in cell culture (human and rat renal tubular epithelial cells) and a mouse unilateral ureteral obstruction (UUO) model. Cell cycle of tubular cells was determined by flow cytometry, and the expression of profibrogenic factors was determined by RT-PCR, immunohistochemistry, and western blotting. Chromatin immunoprecipitation and luciferase reporter experiments were performed to explore the effect of HIF-1α on p53 expression. We showed that, in hypoxic tubular cells, p53 upregulation suppressed the expression of CDK1 and cyclins B1 and D1, leading to cell cycle (G2/M) arrest (or delay) and higher expression of TGF-β, CTGF, collagens, and fibronectin. p53 suppression by siRNA or by a specific p53 inhibitor (PIF-α) triggered opposite effects preventing the G2/M arrest and profibrotic changes. In vivo experiments in the UUO model revealed similar antifibrotic results following intraperitoneal administration of PIF-α (2.2 mg/kg). Using gain-of-function, loss-of-function, and luciferase assays, we further identified an HRE3 region on the p53 promoter as the HIF-1α-binding site. The HIF-1α–HRE3 binding resulted in a sharp transcriptional activation of p53. Collectively, we show the presence of a hypoxia-activated, p53-responsive profibrogenic pathway in the kidney. During hypoxia, p53 upregulation induced by HIF-1α suppresses cell cycle progression, leading to the accumulation of G2/M cells, and activates profibrotic TGF-β and CTGF-mediated signaling pathways, causing extracellular matrix production and renal fibrosis.

scholarly journals Anticancer Activity of Cynomorium coccineum

Cancers ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 354 ◽  
Author(s):  
Mouna Sdiri ◽  
Xiangmin Li ◽  
William Du ◽  
Safia El-Bok ◽  
Yi-Zhen Xie ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Anticancer Activity ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cell Cycle Progression ◽  
Cycle Progression

The extensive applications of Cynomorium species and their rich bioactive secondary metabolites have inspired many pharmacological investigations. Previous research has been conducted to examine the biological activities and numerous interesting pharmaceutical activities have been reported. However, the antitumor activities of these species are unclear. To understand the potential anticancer activity, we screened Cynomorium coccineum and Cynomorium songaricum using three different extracts of each species. In this study, the selected extracts were evaluated for their ability to decrease survival rates of five different cancer cell lines. We compared the cytotoxicity of the three different extracts to the anticancer drug vinblastine and one of the most well-known medicinal mushrooms Amaurederma rude. We found that the water and alcohol extracts of C. coccineum at the very low concentrations possessed very high capacity in decreasing the cancer cells viability with a potential inhibition of tumorigenesis. Based on these primitive data, we subsequently tested the ethanol and the water extracts of C. coccineum, respectively in in vitro and in vivo assays. Cell cycle progression and induction of programmed cell death were investigated at both biological and molecular levels to understand the mechanism of the antitumor inhibitory action of the C. coccineum. The in vitro experiments showed that the treated cancer cells formed fewer and smaller colonies than the untreated cells. Cell cycle progression was inhibited, and the ethanol extract of C. coccineum at a low concentration induced accumulation of cells in the G1 phase. We also found that the C. coccineum’s extracts suppressed viability of two murine cancer cell lines. In the in vivo experiments, we injected mice with murine cancer cell line B16, followed by peritoneal injection of the water extract. The treatment prolonged mouse survival significantly. The tumors grew at a slower rate than the control. Down-regulation of c-myc expression appeared to be associated with these effects. Further investigation showed that treatment with C. coccineum induced the overexpression of the tumor suppressor Foxo3 and other molecules involved in inducing autophagy. These results showed that the C. coccineum extract exerts its antiproliferative activity through the induction of cell death pathway. Thus, the Cynomorium plants appear to be a promising source of new antineoplastic compounds.

190 CELL CYCLE REGULATION IN RHESUS MONKEY OOCYTES AND EMBRYOS OF DIFFERENT DEVELOPMENTAL POTENTIAL

2009 ◽  
Vol 21 (1) ◽  
pp. 194
Author(s):  
N. Mtango ◽  
K. Latham
Keyword(s):  
Cell Cycle ◽  
Rhesus Monkey ◽  
Transcriptional Activation ◽  
Cell Cycle Regulation ◽  
Technical Assistance ◽  
Regulatory Gene ◽  
Developmental Potential ◽  
Cycle Regulation

After fertilization, cell division is required for development during the transition from a zygote to an embryo. Degradation of oocyte transcripts, transcriptional activation of the nucleus, and chromatin remodeling occur during early cleavage divisions. Defects in cell cycle regulation decrease the ability of embryo to grow and can be detrimental. In the rhesus monkey, embryos derived by fertilization of oocytes from prepubertal females or oocytes collected during the non-breeding season undergo cleavage arrest (Schramm and Bavister 1994; Zheng et al. 2001). We employed the Primate Embryo Gene Expression Resource (PREGER; www.Preger.org) to examine the expression pattern of 70 mRNAs involved in cell cycle regulation in rhesus monkey oocytes and embryos derived from different stimulation protocols (non-stimulated, FSH stimulated-in vitro matured, and FSH and hCG stimulated-in vivo matured; Mtango and Latham 2007, 2008; Zheng et al. 2005). The resource encompasses a large, biologically rich set of more than 170 samples with 1 to 4 oocytes or embryos which were constructed using the quantitative amplification and dot blotting method. This method entails the direct lysis of small numbers of oocytes or embryos in a reverse transcription buffer supplemented with nonionic detergent, thereby avoiding RNA losses associated with organic extractions (Brady and Iscove 1993). We find that aberrant regulation of cell cycle regulatory gene mRNAs is a prominent feature of oocytes and embryos of compromised developmental potential (FSH stimulated-moderate reduced potential and NS-severely compromised potential). Of the 56 mRNAs for which expression was detected, there was significant aberrations related to oocyte and embryo quality in the expression of more than half (n = 30), P < 0.05), 26 of 30 display significant differences in metaphase II stage oocytes, 20 being altered in FSH stimulated females and 24 of 30 being altered in NS females. The comparison between monkey and previously reported mouse array expression data (Zeng et al. 2004) revealed striking differences between 2 species. These data provide novel information about disruptions in the expression of genes controlling the cell cycle in oocytes and embryos of compromised developmental potential. We thank Bela Patel, Malgorzata McMenamin, and Ann Marie Paprocki for their technical assistance. We also thank R. Dee Schramm for his contribution to the development of the PREGER resource. This work was supported by National Centers for Research Resources Grant RR-15253.

scholarly journals C1orf109L promote R-loop accumulation induced DNA damage to inhibit cell growth

2019 ◽  
Author(s):  
Dou Peng ◽  
Li Yiqun ◽  
Xie Wanqiu ◽  
Zhang Xiaoqing ◽  
Zhang Dandan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Death ◽  
Cell Growth ◽  
Genome Instability ◽  
Cell Death Pathway ◽  
Unknown Gene ◽  
Lower Expression

AbstractAs a function unknown gene, C1orf109 is lower expression in various cells. Here, we reported that C1orf109L, the longest variant of C1orf109, which interacted with R-loop-regulating proteins to trigger R-loop, a three-stranded nucleic acid structure frequently mediated genome instability, accumulation. C1orf109L induce chronic DNA damage to promote P21 upregulation and strongly inhibits cell growth in vitro and in vivo by arresting the cell cycle in the G2 phase. With camptothecin (CPT), an R-loop activator, treatment, C1orf109L further triggers R-loop accumulation-induced DNA damage and promotes cell death by activating cell-death pathway. Furthermore, CPT treatment increases C1orf109L ubiquitination and turnover, which inhibits cell death and promotes the G0/G1 phase of the cell cycle. Therefore, our data illustrated the mechanisms underlying C1orf109L-related cell growth inhibition and provide feasibility and limitations for C1orf109L as a potential target for cancer therapy.

Small Molecule Inhibition of Cdc7, a Key Cell Cycle Regulator and Novel Therapeutic Target, Successfully Inhibits Leukemia Cell Growth in Vitro and in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2668-2668
Author(s):  
Mark G. Frattini ◽  
David Shum ◽  
Kristen M O’Dwyer ◽  
Renier J. Brentjens ◽  
Ray Yeh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Small Molecule ◽  
Leukemia Cell ◽  
Standard Cell ◽  
Cdc7 Kinase ◽  
Kinase Inhibitory Activity ◽  
Novel Therapeutic

Abstract Cdc7 is a heterodimeric serine/threonine protein kinase that is a key regulator in the process of initiation of DNA replication and the G1 to S phase transition. Both the kinase and its known substrates are over-expressed in the majority of human cancers. As a result of the recent progress in the areas of pharmacogenetics and high throughput screening technology, identifying specific small molecule inhibitors of cell cycle regulated protein kinases has provided a means not only to study these signal transduction pathways but also to identify potential novel therapeutic agents. To this end, we have developed an assay for Cdc7 kinase inhibitory activity using a highthroughput screening (HTS) approach, screening over 250,000 natural and synthetic small molecules. As a result, we have identified and confirmed seventeen compounds, representing nine different chemical scaffolds, with Cdc7 kinase inhibitory activity. Based on potency, we selected the lead compound (CKI-7) which was further characterized using kinase profiling, microarray experiments, and standard cell based cytotoxicity assays. These latter studies demonstrated that CKI-7 induced cytotoxicity of established leukemia and lymphoma cell lines in culture with inhibitory concentrations (IC50s) in the low nanomolar range. Significantly, CKI-7 likewise induced cytotoxicity of MDR1 overexpressing cell lines with similar IC50s, demonstrating that this novel compound can overcome a major mechanism of chemotherapy resistence in human tumor cells. We additonally demonstrate that CKI-7 induces cytotoxicity of patient-derived primary acute leukemia tumor cells (both chemotherapy naïve and relapsed/refractory samples) in vitro at similarly low nanomolar concentrations. In vivo dose-dependent anti-tumor activity of CKI-7 was subsequently demonstrated in a SCID-Beige mouse systemic tumor model utilzing a recently isolated Philadelphia chromosome positive acute lymphoblastic leukemia cell line (PhALL3.1). Standard cell cycle synchronization studies established that exposure to CKI-7 results in cell cycle dependent caspase 3 activation and apoptotic cell death. This cell death is the direct result of Cdc7 kinase inhibition by CKI-7 as demonstrated using a substrate biomarker assay. In conclusion, our data confirm that Cdc7 is a new promising target for cancer therapy, and that CKI-7, a selective small molecule inhibitor of this enzyme, is an equally promising novel cancer therapeutic agent.

scholarly journals PRMT4-Mediated Arginine Methylation Negatively Regulates Retinoblastoma Tumor Suppressor Protein and Promotes E2F-1 Dissociation

2014 ◽  
Vol 35 (1) ◽  
pp. 238-248 ◽  
Author(s):  
Kevin Y. Kim ◽  
Don-Hong Wang ◽  
Mel Campbell ◽  
Steve B. Huerta ◽  
Bogdan Shevchenko ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Suppressor ◽  
Posttranslational Modifications ◽  
Transcriptional Activation ◽  
Cell Cycle Control ◽  
Arginine Methylation ◽  
Reporter System ◽  
Suppressor Activity

The retinoblastoma protein (pRb/p105) tumor suppressor plays a pivotal role in cell cycle regulation by blockage of the G1-to-S-phase transition. pRb tumor suppressor activity is governed by a variety of posttranslational modifications, most notably phosphorylation by cyclin-dependent kinase (Cdk) complexes. Here we report a novel regulation of pRb through protein arginine methyltransferase 4 (PRMT4)-mediated arginine methylation, which parallels phosphorylation. PRMT4 specifically methylates pRb at the pRb C-terminal domain (pRb Cterm) on arginine (R) residues R775, R787, and R798in vitroand R787in vivo. Arginine methylation is important for efficient pRb Ctermphosphorylation, as manifested by the reduced phosphorylation of a methylation-impaired mutant, pRb (R3K). A methylmimetic form of pRb, pRb (R3F), disrupts the formation of the E2F-1/DP1-pRb complex in cells as well as in an isolated system. Finally, studies using a Gal4–E2F-1 reporter system show that pRb (R3F) expression reduces the ability of pRb to repress E2F-1 transcriptional activation, while pRb (R3K) expression further represses E2F-1 transcriptional activation relative to that for cells expressing wild-type pRb. Together, our results suggest that arginine methylation negatively regulates the tumor suppressor function of pRb during cell cycle control, in part by creating a better substrate for Cdk complex phosphorylation and disrupting the interaction of pRb with E2F-1.

scholarly journals The Typhoid Toxin Produced by the NontyphoidalSalmonella entericaSerotype Javiana Is Required for Induction of a DNA Damage ResponseIn Vitroand Systemic SpreadIn Vivo

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Rachel A. Miller ◽  
Michael I. Betteken ◽  
Xiaodong Guo ◽  
Craig Altier ◽  
Gerald E. Duhamel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Death ◽  
Salmonella Enterica ◽  
Biologically Active ◽  
Genes Encoding ◽  
Typhoid Toxin ◽  
Binding Subunit

ABSTRACTTheSalmonellacytolethal distending toxin (S-CDT), first described as the “typhoid toxin” inSalmonella entericasubsp.entericaserotype Typhi, induces DNA damage in eukaryotic cells. Recent studies have shown that more than 40 nontyphoidalSalmonella(NTS) serotypes carry genes that encode S-CDT, yet very little is known about the activity, function, and role of S-CDT in NTS. Here we show that deletion of genes encoding the binding subunit (pltB) and a bacteriophage muramidase predicted to play a role in toxin export (ttsA) does not abolish toxin activity in the S-CDT-positive NTSSalmonella entericasubsp.entericaserotype Javiana. However,S.Javiana strains harboring deletions of bothpltBand its homologartB, had a complete loss of S-CDT activity, suggesting thatS.Javiana carries genes encoding two variants of the binding subunit. S-CDT-mediated DNA damage, as determined by phosphorylation of histone 2AX (H2AX), producing phosphorylated H2AX (γH2AX), was restricted to epithelial cells in S and G2/M phases of the cell cycle and did not result in apoptosis or cell death. Compared to mice infected with a ΔcdtBstrain, mice infected with wild-typeS.Javiana had significantly higher levels ofS.Javiana in the liver, but not in the spleen, ileum, or cecum. Overall, we show that production of active S-CDT by NTS serotypeS.Javiana requires different genes (cdtB,pltA, and eitherpltBorartB) for expression of biologically active toxin than those reported for S-CDT production byS.Typhi (cdtB,pltA,pltB, andttsA). However, as inS.Typhi, NTS S-CDT influences the outcome of infection bothin vitroandin vivo.IMPORTANCENontyphoidalSalmonella(NTS) are a major cause of bacterial food-borne illness worldwide; however, our understanding of virulence mechanisms that determine the outcome and severity of nontyphoidal salmonellosis is incompletely understood. Here we show that S-CDT produced by NTS plays a significant role in the outcome of infection bothin vitroandin vivo, highlighting S-CDT as an important virulence factor for nontyphoidalSalmonellaserotypes. Our data also contribute novel information about the function of S-CDT, as S-CDT-mediated DNA damage occurs only during certain phases of the cell cycle, and the resulting damage does not induce cell death as assessed using a propidium iodide exclusion assay. Importantly, our data support that, despite having genetically similar S-CDT operons, NTS serotypeS.Javiana has different genetic requirements thanS.Typhi, for the production and export of active S-CDT.

Involvement of P-TEFb/CDK9 in Cooperative Transcriptional Activation of Megakaryocytic Genes by GATA-1 and RUNX1 and in Programming of Megakaryocytic Development In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1227-1227
Author(s):  
Kamaleldin E. Elagib ◽  
Ivailo S. Mihaylov ◽  
Lorrie L. Delehanty ◽  
Grant C. Bullock ◽  
Kevin D. Ouma ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Program Analysis ◽  
Dominant Negative ◽  
In Vivo Model ◽  
Megakaryocytic Differentiation ◽  
Human Cd34 ◽  
Unique Cell

Abstract Programming of megakaryocytic differentiation requires precise coordination of multiple signal transduction and transcription pathways. Previous in vivo and in vitro studies have implicated RUNX1 and GATA-1 as transcription factors that collaborate in the execution of this program. Analysis of the mechanism for the synergy of these two factors revealed induction of RUNX1 hyperphosphorylation by GATA-1 coexpression. A pharmacologic screen identified roscovitine as an inhibitor of the transcriptional cooperation, implicating a cyclin-dependent kinase (Cdk). A screen employing a panel of dominant-negative Cdk mutants identified Cdk9 as a critical component of the GATA-1-RUNX1 cooperation. In addition, HEXIM1, an endogenous Cdk9 inhibitor, similarly blocked transcriptional synergy. Furthermore, two kinase inhibitory compounds, DRB and flavopiridol, also blocked GATA-1-RUNX1 cooperation at concentrations specific for Cdk9 inhibition. Regarding the mechanism for GATA-1 induction of RUNX1 phosphorylation, coimmunoprecipitation experiments showed GATA-1 binding to both Cdk9 and cyclinT1. To examine the role of P-TEFb in primary megakaryocytic differentiation, human CD34+ cells in megakaryocytic cultures underwent treatment with 50 nM flavopiridol, a dose selective for Cdk9 inhibition. This treatment blocked megakaryocytic polyploidization while having no effect on the cell cycle properties of the non-megakaryocytic cells in the cultures. The treatment also impaired upregulation of CD41. Extending these findings to an in vivo model system, mice underwent treatment with daily low dose flavopiridol (5–7 mg/kg/day), a regimen previously shown to have no toxicity. Wild type C57BL/6 (wt BL/6) mice were compared with the ΔneoΔHS strain (GATA-1Lo) which has diminished GATA-1 expression in megakaryocytes. After only 1 week of treatment, the GATA-1Lo mice developed worsening thrombocytopenia associated with new-onset anemia, with several dying after 2 weeks of treatment. Flow cytometry on marrow from the treated GATA-1Lo mice revealed a marked expansion of abnormal megakaryocytes showing coexpression of CD71 plus CD41 and loss of polyploidization. Marrow and spleen histology showed extensive replacement by immature-appearing megakaryocytes with hypolobulated nuclei, as well as frequent pyknotic megakaryocytes. The control mice, flavopiridol treated wt BL/6 and saline treated GATA-1Lo, displayed none of these abnormalities. Additional experiments determined the flavopiridol effect on the GATA-1Lo mice to be completely reversible, with normalization of all parameters 2 weeks after ending treatment. In aggregate, these data implicate P-TEFb recruitment by GATA-1 in mediating cooperative activation of megakaryocytic promoters with RUNX1. This pathway may depend in part on the direct phosphorylation of RUNX1 by Cdk9. In mice, a synthetic lethal relationship between megakaryocytic GATA-1 deficiency and Cdk9 inhibition exists, manifesting as a fulminant but reversible megakaryocytic proliferative disorder reminiscent of the Down syndrome-associated megakaryocyte proliferations. A model is proposed in which P-TEFb, as a component of GATA-1-RUNX1 transcriptional complexes, plays an integral role in the specific programming of megakaryocytic differentiation, with particular importance in the unique cell cycle changes associated with this lineage.

Overexpression of the nuclear factor-κB subunit c-Rel protects against human islet cell death in vitro

2009 ◽  
Vol 297 (5) ◽  
pp. E1067-E1077 ◽  
Author(s):  
Dariush Mokhtari ◽  
Andreea Barbu ◽  
Ilir Mehmeti ◽  
Chantal Vercamer ◽  
Nils Welsh
Keyword(s):  
Cell Death ◽  
Islet Cell ◽  
Nuclear Translocation ◽  
Islet Cells ◽  
Human Islet ◽  
Nuclear Fraction ◽  
Nuclear Factor ◽  
Subunit C

The transcription factor nuclear factor (NF)-κB is known to modulate rates of apoptosis and may therefore play a role in the increased β-cell death that occurs in type 1 and type 2 diabetes. The aim of the present investigation was to study the expression of NF-κB subunits in human islet cells and whether overexpression of the NF-κB subunit c-Rel affects islet cell survival. We detected expression of p65, Rel-B, p50, p105, p52, and the ribosomal protein S3 (rpS3) in human islet cells. Among these, only p65 and rpS3 were translocated from the cytosolic to the nuclear fraction in response to cytokines. Interestingly, rpS3 participated in p65 binding to the κB-element in gel shift analysis experiments. We observed cytoplasmic c-Rel expression in vivo in 6J mice, and signs of nuclear translocation in β-cells of infiltrated nonobese diabetic islets. Human islet cells were also dispersed by trypsin treatment and transduced with a c-Rel adenoviral vector. This resulted in increased expression of c-Rel and inhibitory factor κB, increased κB-binding activity, and augmented protein levels of Bcl-XL, c-IAP2, and heat shock protein 72. c-Rel expression in human islet cells protected against cytokine-induced caspase 3 activation and cell death. c-Rel protected also against streptozotocin- and H2O2-induced cell death, in both intact rat islets and human islet cells. We conclude that rpS3 participates in NF-κB signaling and that a genetic increase in the activity of the NF-κB subunit c-Rel results in protection against cell death in human islets.

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