Inhibition Of Telomerase Is a Novel and Effective Therapy In MLL-Rearranged Acute Myeloid Leukemia (AML)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2887-2887
Author(s):  
Claudia Bruedigam ◽  
Frederik Otzen Bagger ◽  
Catherine Paine Kuhn ◽  
Therese Vu ◽  
Rebecca Austin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Prognostic Impact ◽  
Hematopoietic Stem ◽  
Telomerase Inhibition ◽  
Replicative Stress ◽  
Cycle Arrest

Abstract Telomerase is activated to maintain the long-term replicative potential in many human cancers including AML and novel inhibitors of telomerase have recently entered clinical trials for a variety of malignancies. We investigated the therapeutic potential of telomerase inhibition on MLL-rearranged AML using genetic and pharmacological approaches in murine and humanized models. Telomerase-deficient AML was generated by retroviral transduction of G3 Terc-/- LKS+ (Lin-Kit+Sca1+, enriched for hematopoietic stem cells) with pMIG-MLL-AF9 and compared to wild type (WT) controls. Transformed Terc-/- LKS+ colony-forming units (CFU) were mildly reduced at early passage (week 1 Terc-/- 13.1 ± 1 vs. WT 32.7 ± 4 per 1000 cells input, p < 0.01) but became progressively extinguished with serial replating (week 6 Terc-/- 3.8 ± 0.4 vs. WT 27.0 ± 2.7 per 1000 input, p < 0.01). Loss of CFU correlated with enforced differentiation (reduced Kit, increased Gr1), cell cycle arrest and preferential apoptosis of Kit+ cells. In vivo, AML developed with delayed latency, but was fully penetrant in recipients of G3 Terc-/- and WT cells (Terc-/- 64 days vs. WT 45 days, p < 0.01). Leukemic burden and leukemia stem cell (LSC, GFP+Lin-Sca1-Kit+FcgR+CD34-) frequency were similar between Terc-/- and WT AML. To determine the consequences of telomerase loss on AML LSCs, we performed gene expression profiling of purified LSCs. MLL-AF9-Terc-/- LSCs revealed enrichment of pathways controlling DNA damage/repair, cell cycle and apoptosis. Upstream analysis predicted activation of p53, Rbl1 and Cdkn2a, and inhibition of E2f1 in Terc-/- LSCs. Functionally, shRNA-mediated knockdown of p53 in Terc-/- LSCs partially rescued in vitro CFU, differentiation, cell cycle arrest and apoptosis. The phenotypic changes in Terc-/- AML were amplified by serial passage, suggesting that replicative stress may exacerbate the deleterious effects of telomerase loss on LSC function. To enforce replicative stress in vivo, we performed serial transplantation of Terc-/- AML vs. WT AML. Terc-/- LSCs were unable to generate secondary AML (survival Terc-/- not reached vs. WT 28 days, p < 0.01) and this was confirmed by limiting dilution analysis (Terc-/- LSC frequency 1:224,000 vs. WT 1:184 p < 0.001). Initial engraftment was similar between Terc-/- and WT LSCs. In vivo leukemogenesis was prevented by cell cycle arrest, DNA damage and massive apoptosis (Terc-/- 76.8% ± 3.6% vs. WT 22.49% ± 2.3%, p < 0.0001). Together, these findings demonstrate that in this murine model, telomerase loss eradicates LSC in vivo. To validate these findings in human AML we performed lentiviral shRNA knockdown of hTERT in the MLL-AF9-containing AML cell line Monomac6, followed by transplantation into NSGS (NOD/SCID/IL2Rgamma-/- transgenic for hSCF/hIL3/hGMCSF) xenograft recipients. Two independent shTERT constructs revealed significantly increased survival compared to non-transduced and non-targeting controls (sh-hTERT 149.5 days and 146 days vs. non-transduced 47 days, non-targeting 53.5 days, p< 0.01 for both sh-hTERT). hTERT knockdown correlated with reduced hCD45 engraftment, induction of DNA damage, cell cycle arrest and apoptosis compared to non-transduced or non-targeting shRNA controls. Pharmacological inhibition of telomerase (Telomerase Inhibitor IX, T-IX, Sigma) reduced growth of multiple human AML cell lines in vitro. Treatment with T-IX followed by transplantation of equal numbers of Monomac6 cells prolonged NSGS xenograft survival (T-IX 96.5 days vs. DMSO 59 days, p < 0.05). Finally, we examined the prognostic impact of telomerase-regulated genes in a large cohort of patients with AML. The top 140 differentially expressed genes (p < 0.001) between murine Terc-/- and WT LSCs predicted survival in 2 independent AML clinical trial cohorts. Computational modelling using a random forest approach was able to identify 10 key telomerase-regulated human homologues that could cluster AML patients into prognostically relevant groups and was reproducible in multiple independent datasets. These findings provide new mechanistic understanding into the effects of telomerase inhibition on MLL-rearranged AML and identify the telomerase complex as a novel therapeutic target for AML. Disclosures: No relevant conflicts of interest to declare.

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

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 All-Purpose Nanostrategy Based on Dose Deposition Enhancement, Cell Cycle Arrest, DNA Damage and ROS Production as Prostate Cancer Radiosensitizer for Potential Clinical Translation

2021 ◽  
Author(s):  
Xiao-xiao Guo ◽  
Zhen-hu Guo ◽  
Meng Wu ◽  
Jing-song Lu ◽  
Wen-sheng Xie ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Clinical Translation ◽  
Ros Production ◽  
Cycle Arrest ◽  
Dose Deposition

Abstract Background Radiotherapy (RT) is one of the main treatments for men with prostate cancer (PCa). Yet, to date, with numerous sophisticated nano-formulations as radiosensitizers have been synthesized with inspiring therapeutic effect both in vitro and in vivo, there still lacks the successful clinical translation of such nanosystems. Meanwhile, almost all the attention has been paid on the enhanced dose deposition effect by secondary electrons of nanomaterials with high atomic numbers (Z), despite that cell-cycle arrest, DNA damage and also reactive oxygen species (ROS) production are critical working mechanisms accounting for radiosensitization. Methods Herein, an ‘all-purpose’ nanostrategy based on dose deposition enhancement, cell cycle arrest and ROS production as prostate cancer radiosensitizer for potential clinical translation was proposed. The rather simple structure of docetaxel loaded Au nanoparticles (NPs) with prostate specific membrane antigen (PSMA) ligand conjugation have been successfully synthesized by a rather facile protocol. Results Enhanced cellular uptake achieved via selective internalization of the NPs by PCa cells with positive PSMA expression could guarantee the enhanced dose deposition. Moreover, the as-synthesized nanosystem could arrest cell cycle at G2/M phases, which would reduce the ability of DNA damage repair for more irradiation sensitive of the PCa cells. Meanwhile, G2/M phases arrest would further promote cascade retention and enrichment of the NPs within the cells. Furthermore, ROS generation and double strand breaks greatly promoted by the NPs under irradiation (IR) could also provide an underlying basis for effective radiosensitizers. Conclusions Investigations from in vitro and in vivo confirmed the as-synthesized NPs as an effective nano-radiosensitizer with ideal safety. More importantly, all the moieties within the present nanosystem have been approved by FDA for the purpose of PCa treatment, thus making the it highly attractive for clinical translation.

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 Menadione reduces CDC25B expression and promotes tumor shrinkage in gastric cancer

2020 ◽  
Vol 13 ◽  
pp. 175628481989543
Author(s):  
Amanda Braga Bona ◽  
Danielle Queiroz Calcagno ◽  
Helem Ferreira Ribeiro ◽  
José Augusto Pereira Carneiro Muniz ◽  
Giovanny Rebouças Pinto ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Specific Inhibitor ◽  
Gastric Carcinogenesis ◽  
In Vivo Experiments ◽  
Cycle Arrest

Background: Gastric cancer is one of the most incident types of cancer worldwide and presents high mortality rates and poor prognosis. MYC oncogene overexpression is a key event in gastric carcinogenesis and it is known that its protein positively regulates CDC25B expression which, in turn, plays an essential role in the cell division cycle progression. Menadione is a synthetic form of vitamin K that acts as a specific inhibitor of the CDC25 family of phosphatases. Methods: To better understand the menadione mechanism of action in gastric cancer, we evaluated its molecular and cellular effects in cell lines and in Sapajus apella, nonhuman primates from the new world which had gastric carcinogenesis induced by N-Methyl-N-nitrosourea. We tested CDC25B expression by western blot and RT-qPCR. In-vitro assays include proliferation, migration, invasion and flow cytometry to analyze cell cycle arrest. In in-vivo experiments, in addition to the expression analyses, we followed the preneoplastic lesions and the tumor progression by ultrasonography, endoscopy, biopsies, histopathology and immunohistochemistry. Results: Our tests demonstrated menadione reducing CDC25B expression in vivo and in vitro. It was able to reduce migration, invasion and proliferation rates, and induce cell cycle arrest in gastric cancer cell lines. Moreover, our in-vivo experiments demonstrated menadione inhibiting tumor development and progression. Conclusions: We suggest this compound may be an important ally of chemotherapeutics in the treatment of gastric cancer. In addition, CDC25B has proven to be an effective target for investigation and development of new therapeutic strategies for this malignancy.

Bound polyphenol extracted from jujube pulp triggers mitochondria-mediated apoptosis and cell cycle arrest of HepG2 cell in vitro and in vivo

2019 ◽  
Vol 53 ◽  
pp. 187-196 ◽  
Author(s):  
Shuhua Shan ◽  
Yue Xie ◽  
Huiling Zhao ◽  
Jinping Niu ◽  
Sheng Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Hepg2 Cell ◽  
Cycle Arrest

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.

Lobetyol activate MAPK pathways associated with G 1 /S cell cycle arrest and apoptosis in MKN45 cells in vitro and in vivo

2016 ◽  
Vol 81 ◽  
pp. 120-127 ◽  
Author(s):  
Jie Shen ◽  
XinGang Lu ◽  
WangChun Du ◽  
Jun Zhou ◽  
HongFu Qiu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Mapk Pathways ◽  
Cycle Arrest
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