Platelet Factor 4 (PF4) Causes Cell Cycle Arrest in Megakaryocytes (Megs) by Inactivating CDC2 (CDK1) and CDK2

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1238-1238
Author(s):  
Liqing Xiao ◽  
Mortimer Poncz ◽  
Michele Lambert
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Platelet Count ◽  
Steady State ◽  
Cell Cycle Arrest ◽  
Cell Line ◽  
Cell Surface Receptor ◽  
Brdu Incorporation ◽  
Cell Cycle Regulators ◽  
Cycle Arrest

Abstract Abstract 1238 PF4 (CXCL4), a platelet specific chemokine released in large amounts from activated platelet α -granules, is a negative regulator of megakaryopoiesis. In mouse studies, we have shown that PF4 levels regulate steady-state platelet count and impact chemotherapy and radiation-induced thrombocytopenia. In a clinical study in leukemia patients, we found that PF4 levels were inversely related to steady-state platelet count and to recovery after chemotherapy. The molecular basis for the effect of PF4 in megakaryopoiesis is largely unknown. Our studies in cell models suggested that PF4 might act through the cell surface receptor low-density lipoprotein related protein-1 (LRP1). Using an early megakaryoblastic cell line, which expresses LRP1, Meg-like cell line (Meg01), we show that PF4 exerts an anti-proliferative effect on the cells through inactivation of cell cycle regulators CDC2 (CDK1) and CDK2. PF4 treatment (200 μg/ml for 48 hrs) of Meg01 cells induced a decrease in cells in G1 (from 68% of cells to 51%, p=0.001) with a concurrent increase in the percentage of cells in S (12% of cells to 21%, p = 0.02 for no PF4 vs. PF4 treatment) and G2 (from 20% to 28% of cells) phase, without significant bromodeoxyuridine (BrdU) incorporation by the cells in the S phase, suggesting that PF4 causes a cell cycle arrest resulting in decreased cell proliferation. The cell cycle arrest and lack of BrDU incorporation was confirmed in primary murine Megs. No apoptosis was detected in PF4 treated Meg01 or primary cells. To determine the molecular mechanisms by which PF4 causes cell cycle arrest, we used Western blots interrogating cell cycle proteins. We detected a transient increase in the inhibitory phosphorylation (at Tyr15) of CDC2 after PF4 treatment, as well as a decrease in phosphorylation of the activating site (Thr160) on CDK2. In addition, we found PF4 treatment resulted in the degradation of Cdc25c, the upstream phosphatase of Tyr15 of CDC2. In primary murine Megs, we detected a significant decrease of total CDC2, biologically equivalent to the CDC2 inactivation seen in Meg01 cells. The CDK inhibitor Roscovitine inhibited Meg01 cell proliferation and had minimum additive effect with PF4. Overexpression of the constitutively active CDC2 mutant CDC2AF with the inhibitory phosphorylation sites Thr14 and Tyr15 replaced by Ala and Phe, respectively, desensitized the cells to PF4 treatment. These results suggested that PF4 inhibits megakaryopoiesis by decreasing the proliferation of megakaryocytes in their early developmental stage by inactivating cell cycle regulators CDC2 and CDK2. Unraveling the mechanisms by which PF4 inhibits megakaryopoiesis may lead to the development of novel therapeutics to regulate platelet counts. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Knockout of caspase-7 gene improves the expression of recombinant protein in CHO cell line through the cell cycle arrest in G2/M phase

2022 ◽  
Vol 55 (1) ◽  
Author(s):  
Fatemeh Safari ◽  
Bahman Akbari
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Enzymatic Activity ◽  
Cell Cycle Arrest ◽  
Cell Line ◽  
Caspase 3 ◽  
Cho Cell ◽  
Cycle Arrest ◽  
M Phase ◽  
Caspase 7

Abstract Background Chinese hamster ovary cell line has been used routinely as a bioproduction factory of numerous biopharmaceuticals. So far, various engineering strategies have been recruited to improve the production efficiency of this cell line such as apoptosis engineering. Previously, it is reported that the caspase-7 deficiency in CHO cells reduces the cell proliferation rate. But the effect of this reduction on the CHO cell productivity remained unclear. Hence, in the study at hand the effect of caspase-7 deficiency was assessed on the cell growth, viability and protein expression. In addition, the enzymatic activity of caspase-3 was investigated in the absence of caspase-7. Results Findings showed that in the absence of caspase-7, both cell growth and cell viability were decreased. Cell cycle analysis illustrated that the CHO knockout (CHO-KO) cells experienced a cell cycle arrest in G2/M phase. This cell cycle arrest resulted in a 1.7-fold increase in the expression of luciferase in CHO-KO cells compared to parenteral cells. Furthermore, in the apoptotic situation the enzymatic activity of caspase-3 in CHO-KO cells was approximately 3 times more than CHO-K1 cells. Conclusions These findings represented that; however, caspase-7 deficiency reduces the cell proliferation rate but the resulted cell cycle arrest leads to the enhancement of recombinant protein expression. Moreover, increasing in the caspase-3 enzymatic activity compensates the absence of caspase-7 in the caspase cascade of apoptosis.

scholarly journals Dimethylfumarate Inhibits Colorectal Carcinoma Cell Proliferation: Evidence for Cell Cycle Arrest, Apoptosis and Autophagy

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1329 ◽  
Author(s):  
Kaluzki ◽  
Hailemariam-Jahn ◽  
Doll ◽  
Kaufmann ◽  
Balermpas ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Colon Cancer ◽  
Cell Proliferation ◽  
Colorectal Carcinoma ◽  
Cell Cycle Arrest ◽  
Cell Line ◽  
Carcinoma Cell ◽  
Human Colon ◽  
G1 Phase ◽  
Cycle Arrest

Recent studies have proven that Dimethylfumarate (DMF) has a marked anti-proliferative impact on diverse cancer entities e.g., on malignant melanoma. To explore its anti-tumorigenic potential, we examined the effects of DMF on human colon carcinoma cell lines and the underlying mechanisms of action. Human colon cancer cell line HT-29 and human colorectal carcinoma cell line T84 were treated with or without DMF. Effects of DMF on proliferation, cell cycle progression, and apoptosis were analyzed mainly by Bromodeoxyuridine (BrdU)- and Lactatdehydrogenase (LDH)assays, caspase activation, flowcytometry, immunofluorescence, and immunoblotting. In addition, combinational treatments with radiation and chemotherapy were performed. DMF inhibits cell proliferation in both cell lines. It was shown that DMF induces a cell cycle arrest in G0/G1 phase, which is accompanied by upregulation of p21 and downregulation of cyclin D1 and Cyclin dependent kinase (CDK)4. Furthermore, upregulation of autophagy associated proteins suggests that autophagy is involved. In addition, the activation of apoptotic markers provides evidence that apoptosis is involved. Our results show that DMF supports the action of oxaliplatin in a synergetic manner and failed synergy with radiation. We demonstrated that DMF has distinct antitumorigenic, cell dependent effects on colon cancer cells by arresting cell cycle in G0/G1 phase as well as activating both the autophagic and apoptotic pathways and synergizes with chemotherapy.

scholarly journals AdipoR2 inhibits human glioblastoma cell growth through the AMPK/mTOR pathway

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Chen Jie ◽  
Wang Xuan ◽  
Han-Dong Feng ◽  
Ding-Mao Hua ◽  
Wang Bo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Cell Cycle Arrest ◽  
Cell Line ◽  
Expression Profiles ◽  
Mtor Pathway ◽  
Negative Regulator ◽  
Cycle Arrest ◽  
U251 Cells

Abstract Background AdipoR2, which belongs to the seven-transmembrane-domain receptor family, has been shown to play an important role in the development of human tumours, but the underlying mechanisms are poorly understood. In this study, we found that AdipoR2 expression correlates with glioma grade. In addition, we also investigated the mechanisms behind the antiproliferative effects of AdipoR2 in U251 cells (a human glioma cell line) using colony formation and WST-8 growth assays. Methods The U251 cell line was cultured in vitro. Western blotting was used to detect the expression of relevant proteins. Quantitative RT-PCR was used to detect AdipoR1 and AdipoR2 expression. Flow cytometry was used to detect cell cycle assay results. The gene expression profiles of glioma samples from the CGGA database were analysed by MATLAB and GSEA software. Results The AMPK/mTOR pathway plays a central role in the regulation of cell proliferation, differentiation and migration and may promote tumorigenesis. Therefore, we can control cancer progression by modulating the AMPK/mTOR pathway. However, there is no information on the relationship between AdipoR and AMPK/mTOR in central nervous system tumours such as GBM. In this study. We found 648 upregulated genes and 436 downregulated genes correlated with AdipoR2 expression in 158 glioma samples. GSEA suggested that AdipoR2 is a cell cycle-associated gene. The results of the flow cytometry analysis indicated that AdipoR2 induced G0/G1 cell cycle arrest in U251 cells. Furthermore, we identified the AMPK/mTOR signalling axis to be involved in AdipoR2-induced cell cycle arrest. Conclusions Our results suggest that AdipoR2 may represent a novel endogenous negative regulator of GBM cell proliferation. These findings also suggest that AdipoR2 may be a promising therapeutic target in GBM patients.

scholarly journals Trichostatin A inhibits cell proliferation and induces cell cycle arrest in human esophageal carcinoma cell line EC1

2009 ◽  
Vol 17 (34) ◽  
pp. 3534
Author(s):  
Jun-Fen Ma ◽  
Ya-Nan Jiang ◽  
Ji-Min Zhao ◽  
You-Tian Huang ◽  
Ming-Yao Zhao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Esophageal Carcinoma ◽  
Cell Cycle Arrest ◽  
Cell Line ◽  
Carcinoma Cell ◽  
Trichostatin A ◽  
Carcinoma Cell Line ◽  
Cycle Arrest

Overexpression of Lhx8 inhibits cell proliferation and induces cell cycle arrest in PC12 cell line

2014 ◽  
Vol 51 (4) ◽  
pp. 329-335 ◽  
Author(s):  
Haoming Li ◽  
Jianbing Qin ◽  
Guohua Jin ◽  
Linqing Zou ◽  
Jinhong Shi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Cycle Arrest ◽  
Cell Line ◽  
Pc12 Cell ◽  
Pc12 Cell Line ◽  
Cycle Arrest

Repression of ROCKII by GATA-1 Inhibits Cell Proliferation during Erythroid Maturation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3345-3345
Author(s):  
Peilin Ma ◽  
Reuben Kapur
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Estrogen Receptor ◽  
Steady State ◽  
Cell Cycle Arrest ◽  
Dominant Negative ◽  
Hormone Binding ◽  
Cycle Arrest ◽  
Hormone Binding Domain

Abstract Rho family GTPases are key regulators of many different biological processes including cell motility, growth, and differentiation. Cdc42, Rac and Rho are the most extensively studied members of this family. Although the role of GTPases is becoming increasingly clear in blood cells, virtually nothing is known about the role of downstream effectors of Rho GTPases in hematopoiesis. Rho activates the serine/threonine protein kinases ROCKI and ROCKII. To determine the role of ROCK kinases in hematopoiesis, we generated mice deficient in the expression of ROCKI. Under steady state conditions, deficiency of ROCKI did not demonstrate any significant changes in peripheral blood white cell counts nor defects in the production of red cells compared to controls were observed. Since deficiency of ROCKI alone did not result in perturbed steady state erythropoiesis, we examined the role of ROCKII in this process. For these studies, we used an erythroid progenitor cell line derived from GATA-1 (G1E) null mice. These cells proliferate continuously in culture as developmentally arrested erythroid precursors and upon restoration of GATA-1 activity, undergo cell cycle arrest and terminal maturation in a fashion that largely recapitulates normal erythropoiesis. Using these cells, we generated a conditionally regulated system to study the consequences of ROCKII inactivation on erythropoiesis. We utilized a retroviral vector encoding the activated and kinase dead version of ROCKII by swapping the COOH-terminal negative regulatory portion of ROCKII with the estrogen receptor hormone binding domain. Specifically, we utilized a fused active and kinase dead ROCKII mutant to EGFP and to the estrogen receptor hormone binding domain, which can be stimulated by the estrogen analogs tamoxifen or 4-hydroxytamoxifen (4-HT). In contrast to a lack of a positive role for ROCKI in erythropoiesis, inactivation of ROCKII in erythroid progenitors in the absence of GATA-1 activation resulted in complete repression of cytokine mediated growth (n=4, p<0.001). Consistently, a dose dependent repression in the growth of these cells was also observed in the presence of two distinct ROCK kinase inhibitors (n=3, p<0.001). Importantly, when these cells were induced to differentiate by conditionally activating GATA-1, cells expressing the empty vector or the dominant negative version of ROCKII under went cell cycle arrest and terminal maturation as expected. In contrast, cells expressing the activated version of ROCKII, also under went terminal differentiation, however these cells continued to proliferate throughout the course of differentiation (n=3, p<0.005). To determine the mechanism(s) of growth repression in the absence of GATA-1 activation, we conducted survival and cell cycle analysis. Surprisingly, expression of dominant negative (DN) ROCKII in these cells did not induce apoptosis, but significantly impaired cell cycle entry, with majority of cells falling in the G1/G0 phase of cell cycle (n=5, p<0.005). The extensive arrest in cell cycle progression due to ROCKII inactivation in these cells was associated with complete loss of cyclinD1 expression. Our results reveal that ROCKI and ROCKII play distinct roles in erythropoiesis. Furthermore, in addition to its well established function as an activator of erythroid genes, GATA-1 also participates in a genetic program that inhibits cell proliferation by repressing the activation of ROCKII kinase. In summary, we demonstrate a novel aspect of cross talk between GATA-1 and Rho kinase ROCKII in erythropoietic development.

Anticancer Effect of Amygdalin (Vitamin B-17) on Hepatocellular Carcinoma Cell Line (HepG2) in the Presence and Absence of Zinc

2020 ◽  
Vol 20 (4) ◽  
pp. 486-494
Author(s):  
Mohamed A. El-Desouky ◽  
Abdelgawad A. Fahmi ◽  
Ibrahim Y. Abdelkader ◽  
Karima M. Nasraldin
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Cytochrome C ◽  
Cell Cycle Arrest ◽  
Cell Line ◽  
Hepg2 Cell ◽  
Caspase 3 ◽  
Vitamin B ◽  
Cycle Arrest ◽  
Hepg2 Cell Lines

Background: Amygdalin (Vitamin B-17) is a naturally occurring vitamin found in the seeds of the fruits of Prunus Rosacea family including apricot, bitter almond, cherry, and peach. Objective: The purpose of this study was to examine the effect of amygdalin with and without zinc on hepatocellular carcinoma (HepG2) cell line. Methods: MTT assay was used to evaluate the cytotoxicity of amygdalin without zinc, amygdalin + 20μmol zinc, and amygdalin + 800μmol zinc on HepG2 cell lines. The cell cycle distribution assay was determined by flow cytometry. Apoptosis was confirmed by Annexin V-FITC/PI staining assay. Moreover, the pathway of apoptosis was determined by the percentage of change in the mean levels of P53, Bcl2, Bax, cytochrome c, and caspase-3. Results: Amygdalin without zinc showed strong anti-HepG2 activity. Furthermore, HepG2 cell lines treatment with amygdalin + 20μmol zinc and amygdalin + 800μmol zinc showed a highly significant apoptotic effect than the effect of amygdalin without zinc. Amygdalin treatment induced cell cycle arrest at G2/M and increased the levels of P53, Bax, cytochrome c, and caspase-3 significantly, while it decreased the level of anti-apoptotic Bcl2. Conclusion: Amygdalin is a natural anti-cancer agent, which can be used for the treatment of hepatocellular carcinoma. It promotes apoptosis via the intrinsic cell death pathway (the mitochondria-initiated pathway) and cell cycle arrest at G/M. The potency of amygdalin in HepG2 treatment increased significantly by the addition of zinc.

scholarly journals Induction of apoptosis and cell cycle arrest by chloroform fraction of Juniperus phoenicea and chemical constituents analysis

2021 ◽  
Vol 19 (1) ◽  
pp. 119-127
Author(s):  
Ibrahim O. Barnawi ◽  
Fahd A. Nasr ◽  
Omar M. Noman ◽  
Ali S. Alqahtani ◽  
Mohammed Al-zharani ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Cycle Arrest ◽  
Cancer Cells ◽  
Chloroform Fraction ◽  
Active Fraction ◽  
Cycle Arrest ◽  
Juniperus Phoenicea ◽  
Medicinal Properties ◽  
Mcf 7

Abstract Different phytochemicals from various plant species exhibit promising medicinal properties against cancer. Juniperus phoenicea is a plant species that has been found to present medicinal properties. Herein, crude extract and fractions of J. phoenicea were examined to determine its anticancer properties against several cancer cells. The active fraction was chosen to assess its activity on cell cycle progression and apoptosis induction by annexin and propidium iodide (PI) biomarkers. Further, phytochemical screening for possible contents of active fraction using gas chromatography–mass spectrometry (GC-MS) analysis was conducted. It was demonstrated that cell proliferation was suppressed, and the MCF-7 cell line was the most sensitive to J. phoenicea chloroform fraction (JPCF), with the IC50 values of 24.5 μg/mL. The anti-proliferation activity of JPCF in MCF-7 cells was linked to the aggregation of cells in the G1 phase, increases in early and late apoptosis as well as necrotic cell death. Contents analysis of JPCF using GC-MS analysis identified 3-methyl-5-(2′,6′,6′-trimethylcyclohex-1′-enyl)-1-penten-3-ol (16.5%), methyl 8-oxooctanoate (15.61%), cubenol (13.48%), and 7-oxabicyclo [2.2.1] heptane (12.14%) as major constituents. Our present study provides clear evidence that J. phoenicea can inhibit cell proliferation, trigger cell cycle arrest, and induce apoptosis in tested cancer cells.

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