scholarly journals Chk2/hCds1 functions as a DNA damage checkpoint in G1 by stabilizing p53

2000 ◽  
Vol 14 (3) ◽  
pp. 278-288 ◽  
Author(s):  
Nabil H. Chehab ◽  
Asra Malikzay ◽  
Michael Appel ◽  
Thanos D. Halazonetis
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Dna Damage Checkpoint ◽  
Wild Type ◽  
Cycle Arrest ◽  
P53 Tumor Suppressor Protein

Chk2/hcds1, the human homolog of theSaccharomyces cerevisiae RAD53/SPK1 andSchizosaccharomyces pombe cds1 DNA damage checkpoint genes, encodes a protein kinase that is post-translationally modified after DNA damage. Like its yeast homologs, the Chk2/hCds1 protein phosphorylates Cdc25C in vitro, suggesting that it arrests cells in G2 in response to DNA damage. We expressed Chk2/hCds1 in human cells and analyzed their cell cycle profile. Wild-type, but not catalytically inactive, Chk2/hCds1 led to G1 arrest after DNA damage. The arrest was inhibited by cotransfection of a dominant-negative p53 mutant, indicating that Chk2/hCds1 acted upstream of p53. In vitro, Chk2/hCds1 phosphorylated p53 on Ser-20 and dissociated preformed complexes of p53 with Mdm2, a protein that targets p53 for degradation. In vivo, ectopic expression of wild-type Chk2/hCds1 led to increased p53 stabilization after DNA damage, whereas expression of a dominant-negative Chk2/hCds1 mutant abrogated both phosphorylation of p53 on Ser-20 and p53 stabilization. Thus, in response to DNA damage, Chk2/hCds1 stabilizes the p53 tumor suppressor protein leading to cell cycle arrest in G1.

scholarly journals DIPG-53. CHARACTERIZING THE ROLE OF PPM1D MUTATIONS IN THE PATHOGENESIS OF DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPGS)

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii297-iii297
Author(s):  
Prasidda Khadka ◽  
Zachary Reitman ◽  
Sophie Lu ◽  
Graham Buchan ◽  
Rachel Hartley ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Radiation Treatment ◽  
Ectopic Expression ◽  
Loss Of Function ◽  
In Vivo Models ◽  
Cycle Arrest ◽  
Diffuse Intrinsic Pontine Gliomas

Abstract INTRODUCTION We have previously found that up to 15% of all DIPGs harbor mutations in PPM1D, resulting in the expression of an activated and truncated PPM1D (PPM1Dtr). Here we evaluate the mechanisms through which PPM1Dtr enhances glioma formation and identify its associated therapeutic vulnerabilities. METHODS We have developed multiple in vitro and in vivo models of PPM1D-mutant DIPGs and applied quantitative proteomic and functional genomic approaches to identify pathways altered by PPM1Dtr and associated dependencies. RESULTS PPM1D mutations are clonal events that are anti-correlated to TP53 mutations. We find ectopic expression of PPM1Dtr to be sufficient to enhance glioma formation and to be necessary in PPM1D-mutant DIPG cells. In addition, endogenous truncation of PPM1D is sufficient to enhance glioma formation in the presence of mutant H3F3A and PDGFRA. PPM1Dtr overexpression attenuates g-H2AX formation and suppresses apoptosis and cell-cycle arrest in response to radiation treatment. Deep scale phosphoproteomics analyses reveal DNA-damage and cell cycle pathways to be most significantly associated with PPM1Dtr. Furthermore, preliminary analysis of genome-wide loss-of-function CRISPR/Cas9 screens in isogenic GFP and PPM1Dtr overexpressing mouse neural stem cells reveal differential dependency on DNA-damage response genes in the PPM1Dtr overexpressing cells. Consistent with PPM1D’s role in stabilizing MDM2, PPM1D-mutant DIPG models are sensitive to a panel of MDM2 inhibitors (Nutlin-3a, RG7388, and AMG232). CONCLUSION Our study shows that PPM1Dtr is both an oncogene and a dependency in PPM1D- mutant DIPG, and there are novel therapeutic vulnerabilities associated with PPM1D that may be exploited.

A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage

2019 ◽  
Vol 19 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Yang Liu ◽  
Jingyin Zhang ◽  
Shuyun Feng ◽  
Tingli Zhao ◽  
Zhengzheng Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Cyclin D ◽  
Dna Relaxation ◽  
Cycle Arrest ◽  
H460 Cell ◽  
H1975 Cell

Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.

scholarly journals Growth Suppression by Acute PromyelocyticLeukemia-Associated Protein PLZF Is Mediated by Repression ofc-mycExpression

2003 ◽  
Vol 23 (24) ◽  
pp. 9375-9388 ◽  
Author(s):  
Melanie J. McConnell ◽  
Nathalie Chevallier ◽  
Windy Berkofsky-Fessler ◽  
Jena M. Giltnane ◽  
Rupal B. Malani ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Cycle Arrest ◽  
Target Genes ◽  
Ectopic Expression ◽  
Growth Suppression ◽  
Quiescent State ◽  
Cycle Arrest

ABSTRACT The transcriptional repressor PLZF was identified by its translocation with retinoic acid receptor alpha in t(11;17) acute promyelocytic leukemia (APL). Ectopic expression of PLZF leads to cell cycle arrest and growth suppression, while disruption of normal PLZF function is implicated in the development of APL. To clarify the function of PLZF in cell growth and survival, we used an inducible PLZF cell line in a microarray analysis to identify the target genes repressed by PLZF. One prominent gene identified was c-myc. The array analysis demonstrated that repression of c-myc by PLZF led to a reduction in c-myc-activated transcripts and an increase in c-myc-repressed transcripts. Regulation of c-myc by PLZF was shown to be both direct and reversible. An interaction between PLZF and the c-myc promoter could be detected both in vitro and in vivo. PLZF repressed the wild-type c-myc promoter in a reporter assay, dependent on the integrity of the binding site identified in vitro. PLZF binding in vivo was coincident with a decrease in RNA polymerase occupation of the c-myc promoter, indicating that repression occurred via a reduction in the initiation of transcription. Finally, expression of c-myc reversed the cell cycle arrest induced by PLZF. These data suggest that PLZF expression maintains a cell in a quiescent state by repressing c-myc expression and preventing cell cycle progression. Loss of this repression through the translocation that occurs in t(11;17) would have serious consequences for cell growth control.

Induction of G1 Cell Cycle Arrest in Human Glioma Cells by Salinomycin Through Triggering ROS-Mediated DNA Damage In Vitro and In Vivo

2016 ◽  
Vol 42 (4) ◽  
pp. 997-1005 ◽  
Author(s):  
Shi-Jun Zhao ◽  
Xian-Jun Wang ◽  
Qing-Jian Wu ◽  
Chao Liu ◽  
Da-Wei Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Glioma Cells ◽  
Human Glioma ◽  
Human Glioma Cells ◽  
Cycle Arrest ◽  
G1 Cell Cycle Arrest

Phosphorylation-deficient Stat1 inhibits retinoic acid–induced differentiation and cell cycle arrest in U-937 monoblasts

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2870-2878
Author(s):  
Anna Dimberg ◽  
Kenneth Nilsson ◽  
Fredrik Öberg
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
Cell Cycle Arrest ◽  
Nuclear Translocation ◽  
Ectopic Expression ◽  
Leukemic Cell ◽  
Potent Inducer ◽  
Cycle Arrest

All-trans retinoic acid (ATRA) is a potent inducer of terminal differentiation of immature leukemic cell lines in vitro and of acute promyelocytic leukemia (APL) cells in vivo. Recent reports have shown that ATRA induces the expression of several interferon-regulated genes, including signal transducer and activator of transcription (Stat)1. To investigate the role of Stat1 activation in ATRA signaling, sublines were established for the human monoblastic cell line U-937 constitutively expressing wild-type or phosphorylation-defective Stat1, mutated in the conserved tyrosine 701 required for dimerization and nuclear translocation. Results showed that ATRA induction leads to activation of Stat1 by the phosphorylation of tyrosine 701 and subsequent nuclear translocation. Consistent with a functional importance of this activation, ectopic expression of Stat1Y701F suppressed ATRA-induced morphologic differentiation and expression of the monocytic surface markers CD11c and the granulocyte colony-stimulating factor receptor. Moreover, ATRA-induced growth arrest in the G0/G1phase of the cell cycle was inhibited by phosphorylation-deficient Stat1. Taken together, these results indicate that Stat1 is a key mediator of ATRA-induced cell cycle arrest and differentiation of U-937 cells.

scholarly journals A RAD9-dependent cell cycle arrest in response to unresolved recombination intermediates in Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Hardeep Kaur ◽  
GN Krishnaprasad ◽  
Michael Lichten
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Cell Division ◽  
Cell Cycle Arrest ◽  
Mitotic Cell ◽  
Cycle Arrest ◽  
Associated Lesions

AbstractIn Saccharomyces cerevisiae, the conserved Sgs1-Top3-Rmi1 helicase-decatenase regulates homologous recombination by limiting accumulation of recombination intermediates that are precursors of crossovers. In vitro studies have suggested that the dissolution of double-Holliday junction joint molecules by Sgs1-driven convergent junction migration and Top3-Rmi1 mediated strand decatenation could be responsible for this. To ask if dissolution occurs in vivo, we conditionally depleted Sgs1 and/or Rmi1 during return to growth, a procedure where recombination intermediates formed during meiosis are resolved when cells resume the mitotic cell cycle. Sgs1 depletion during return to growth delayed joint molecule resolution, but ultimately most were resolved and cells divided normally. In contrast, Rmi1 depletion resulted in delayed and incomplete joint molecule resolution, and most cells did not divide. rad9Δ mutation restored cell division in Rmi1-depleted cells, indicating that the DNA damage checkpoint caused this cell cycle arrest. Restored cell division in rad9Δ, Rmi1-depleted cells frequently produced anucleate cells, consistent with the suggestion that persistent recombination intermediates prevented chromosome segregation. Our findings indicate that Sgs1-Top3-Rmi1 acts in vivo, as it does in vitro, to promote recombination intermediate resolution by dissolution. They also indicate that, in the absence of Top3-Rmi1 activity, unresolved recombination intermediates persist and activate the DNA damage response, which is usually thought to be activated by much earlier DNA damage-associated lesions.

scholarly journals The G(2) DNA damage checkpoint targets both Wee1 and Cdc25

2000 ◽  
Vol 113 (10) ◽  
pp. 1727-1736 ◽  
Author(s):  
J.M. Raleigh ◽  
M.J. O'Connell
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Progression ◽  
Dna Damage Checkpoint ◽  
Checkpoint Control ◽  
Down Regulation ◽  
Cycle Progression ◽  
Cycle Arrest ◽  
Checkpoint Pathway

The onset of mitosis is controlled by the cyclin dependent kinase Cdc2p. Cdc2p activity is controlled through the balance of phosphorylation and dephosphorylation of tyrosine-15 (Y15) by the Wee1p kinase and Cdc25p phosphatase. In the fission yeast Schizosaccharomyces pombe, detection of DNA damage in G(2) activates a checkpoint that prevents entry into mitosis through the maintenance of Y15 phosphorylation of Cdc2p, thus ensuring DNA repair precedes chromosome segregation. The protein kinase Chk1p is the endpoint of this checkpoint pathway. We have previously reported that overexpression of Chk1p causes a wee1(+)-dependent G(2) arrest, and this or irradiation leads to hyperphosphorylation of Wee1p. Moreover, Chk1p directly phosphorylates Wee1p in vitro. These data suggested that Wee1p is a key target of Chk1p action in checkpoint control. However, cells lacking wee1(+) are checkpoint proficient and sustained Chk1p overexpression arrests cell cycle progression independently of Wee1p. Therefore, up-regulation of Wee1p alone cannot enforce a checkpoint arrest. Chk1p can also phosphorylate Cdc25p in vitro. These phosphorylation events are thought to promote the interaction with 14–3-3 proteins the cytoplasmic retention of the 14–3-3/Cdc25p complexes. However, we show here that the G(2) DNA damage checkpoint is intact in cells that regulate mitotic entry independently of Cdc25p. Further, these cells are still sensitive to Chk1p-mediated arrest, and so down-regulation of Cdc25p is also insufficient to regulate checkpoint arrest. Conversely, inactivation of both wee1(+) and cdc25(+)abolishes checkpoint control. We also show that activation of the G(2) DNA damage checkpoint induces a transient increase in Wee1p levels. We conclude that the G(2) DNA damage checkpoint simultaneously signals via both up-regulation of Wee1p and down-regulation of Cdc25p, thus providing a double-lock mechanism to ensure cell cycle arrest and genomic stability.

scholarly journals In Vitro and In Vivo Activity of Fomitopsis Pinicola (Sw. Ex Fr.) Karst Chloroform (Fpkc) Extract Against S180 Tumor Cells

2017 ◽  
Vol 44 (5) ◽  
pp. 2042-2056 ◽  
Author(s):  
Ye Gao ◽  
Pan Wang ◽  
Yaqin Wang ◽  
Lijie Wu ◽  
Xiaobing Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Survival Time ◽  
Tumor Cells ◽  
Propidium Iodide ◽  
Cell Morphology ◽  
Fomitopsis Pinicola ◽  
Cycle Arrest

Background/Aims: Non-toxic fomitopsis is has been traditionally used in folk medicine in many countries for its anti-inflammatory and anti-vascular disease activities. The present study investigates the antitumor effect of Fomitopsis pinicola (Sw. Ex Fr.) Karst chloroform extract (FPKc) on S180 tumor cells in vitro and in vivo and we determined the underlying mechanisms. Methods: HPLC was employed to analyze the constituents of FPKc. In-vitro 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to quantify the growth inhibition of FPKc; Propidium iodide (PI) exclusion assay and scanning electron microscopy (SEM) were used to observe the damage on the cell membrane and the changes of the cell morphology; Staining with Hoechst 33342/propidium iodide (HO/PI) and the application of the Annexin V-FITC/PI analysis permitted to observe the cell death triggered by FPKc; DNA damage and cell cycle arrest were detected by flow cytometry; Rhodamine 123 (RH123) and Cytochrome C were used as dyes to investigate the alterations of the mitochondria. In-vivo tumor inhibition and mice survival time were determined. Results: The results of the HPLC assay indicated that FPKc might contain DA (dehydroeburiconic acid), PA (pachymic acid), and ES (ergosterol), at percentages of 0.25%, 17.8%, and 10.5%, respectively. Concerning the study of the biological function, the results showed that FPKc exhibited preferential and significant suppression of proliferation on specific cell lines including S180, HL-60, U937, K562, SMMC-7721, and Eca-109 cells, which induced plasma membrane and cell morphology damages, triggering S180 tumor-cells late apoptosis and leading to DNA damage and S phase arrest. Mitochondria were suspected to play a vital role in these changes. In vivo data indicated that FPKc inhibited the solid tumor growth and prolonged the survival time of tumor-bearing mice. Moreover, FPKc provoked only little damage on normal cells in vitro and also on normal tissues in vivo. Conclusion: FPKc inhibited S180 tumor cells growth and prolonged the lifespan of mice. In vitro, we found that FPKc induced S180 tumor cells apoptosis and cell cycle arrest, possibly via the mitochondrial pathway.

Phosphorylation-deficient Stat1 inhibits retinoic acid–induced differentiation and cell cycle arrest in U-937 monoblasts

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2870-2878 ◽  
Author(s):  
Anna Dimberg ◽  
Kenneth Nilsson ◽  
Fredrik Öberg
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
Cell Cycle Arrest ◽  
Nuclear Translocation ◽  
Ectopic Expression ◽  
Leukemic Cell ◽  
Potent Inducer ◽  
Cycle Arrest

Abstract All-trans retinoic acid (ATRA) is a potent inducer of terminal differentiation of immature leukemic cell lines in vitro and of acute promyelocytic leukemia (APL) cells in vivo. Recent reports have shown that ATRA induces the expression of several interferon-regulated genes, including signal transducer and activator of transcription (Stat)1. To investigate the role of Stat1 activation in ATRA signaling, sublines were established for the human monoblastic cell line U-937 constitutively expressing wild-type or phosphorylation-defective Stat1, mutated in the conserved tyrosine 701 required for dimerization and nuclear translocation. Results showed that ATRA induction leads to activation of Stat1 by the phosphorylation of tyrosine 701 and subsequent nuclear translocation. Consistent with a functional importance of this activation, ectopic expression of Stat1Y701F suppressed ATRA-induced morphologic differentiation and expression of the monocytic surface markers CD11c and the granulocyte colony-stimulating factor receptor. Moreover, ATRA-induced growth arrest in the G0/G1phase of the cell cycle was inhibited by phosphorylation-deficient Stat1. Taken together, these results indicate that Stat1 is a key mediator of ATRA-induced cell cycle arrest and differentiation of U-937 cells.

scholarly journals Proteolytic cleavage of human p53 by calpain: a potential regulator of protein stability.

1997 ◽  
Vol 17 (1) ◽  
pp. 460-468 ◽  
Author(s):  
M H Kubbutat ◽  
K H Vousden
Keyword(s):  
Dna Damage ◽  
P53 Protein ◽  
Calpain Inhibitor ◽  
Cycle Arrest ◽  
A Cell ◽  
P53 Tumor Suppressor Protein ◽  
E6 Protein ◽  
Protein Treatment

The p53 tumor suppressor protein is activated in cells in response to DNA damage and prevents the replication of cells sustaining genetic damage by inducing a cell cycle arrest or apoptosis. Activation of p53 is accompanied by stabilization of the protein, resulting in accumulation to high levels within the cell. p53 is normally degraded through the proteasome following ubiquitination, although the mechanisms which regulate this proteolysis in normal cells and how the p53 protein becomes stabilized following DNA damage are not well understood. We show here that p53 can also be a substrate for cleavage by the calcium-activated neutral protease, calpain, and that a preferential site for calpain cleavage exists within the N terminus of the p53 protein. Treatment of cells expressing wild-type p53 with an inhibitor of calpain resulted in the stabilization of the p53 protein. By contrast, in vitro or in vivo degradation mediated by human papillomavirus E6 protein was unaffected by the calpain inhibitor, indicating that the stabilization did not result from inhibition of the proteasome. These results suggest that calpain cleavage plays a role in regulating p53 stability.

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