scholarly journals Cell Cycle Synchrony in Giardia intestinalis Cultures Achieved by Using Nocodazole and Aphidicolin

2008 ◽  
Vol 7 (4) ◽  
pp. 569-574 ◽  
Author(s):  
Marianne K. Poxleitner ◽  
Scott C. Dawson ◽  
W. Zacheus Cande
Keyword(s):  
Cell Cycle ◽  
Model Organism ◽  
Protozoan Parasite ◽  
Giardia Intestinalis ◽  
Mitotic Spindles ◽  
Intestinal Protozoan ◽  
Cycle Arrest ◽  
Cycle Dependent ◽  
Dependent Processes

ABSTRACT Giardia intestinalis is a ubiquitous intestinal protozoan parasite and has been proposed to represent the earliest diverging lineage of extant eukaryotes. Despite the importance of Giardia as a model organism, research on Giardia has been hampered by an inability to achieve cell cycle synchrony for in vitro cultures. This report details successful methods for attaining cell cycle synchrony in Giardia cultures. The research presented here demonstrates reversible cell cycle arrest in G1/S and G2/M with aphidicolin and nocodazole, respectively. Following synchronization, cells were able to recover completely from drug treatment and remained viable and maintained synchronous growth for 6 h. These techniques were used to synchronize Giardia cultures to increase the percentages of mitotic spindles in the cultures. This method of synchronization will enhance our ability to study cell cycle-dependent processes in G. intestinalis.

scholarly journals Cyclin B2 undergoes cell cycle-dependent nuclear translocation and, when expressed as a non-destructible mutant, causes mitotic arrest in HeLa cells

1992 ◽  
Vol 117 (1) ◽  
pp. 213-224 ◽  
Author(s):  
P Gallant ◽  
EA Nigg
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Subcellular Distribution ◽  
Nuclear Translocation ◽  
Mitotic Spindles ◽  
Cycle Arrest ◽  
Regulatory Machinery ◽  
Essential Components ◽  
Cycle Dependent ◽  
Monospecific Antibodies

Cyclin proteins form complexes with members of the p34cdc2 kinase family and they are essential components of the cell cycle regulatory machinery. They are thought to determine the timing of activation, the subcellular distribution, and/or the substrate specificity of cdc2-related kinases, but their precise mode of action remains to be elucidated. Here we report the cloning and sequencing of avian cyclin B2. Based on the use of monospecific antibodies raised against bacterially expressed protein, we also describe the subcellular distribution of cyclin B2 in chick embryo fibroblasts and in DU249 hepatoma cells. By indirect immunofluorescence microscopy we show that cyclin B2 is cytoplasmic during interphase of the cell cycle, but undergoes an abrupt translocation to the cell nucleus at the onset of mitotic prophase. Finally, we have examined the phenotypic consequences of expressing wild-type and mutated versions of avian cyclin B2 in HeLa cells. We found that expression of cyclin B2 carrying a mutation at arginine 32 (to serine) caused HeLa cells to arrest in a pseudomitotic state. Many of the arrested cells displayed multiple mitotic spindles, suggesting that the centrosome cycle had continued in spite of the cell cycle arrest.

In vitro studies of a FLT3 inhibitor combined with chemotherapy: sequence of administration is important to achieve synergistic cytotoxic effects

Blood ◽  
2004 ◽  
Vol 104 (4) ◽  
pp. 1145-1150 ◽  
Author(s):  
Mark Levis ◽  
Rosalyn Pham ◽  
B. Douglas Smith ◽  
Donald Small
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Chemotherapeutic Agents ◽  
In Vitro Cytotoxicity ◽  
High Risk Group ◽  
Cycle Arrest ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors ◽  
Cycle Dependent

AbstractPatients with acute myeloid leukemia (AML) harboring internal tandem duplication mutations of the FLT3 receptor (FLT3/ITD mutations) have a poor prognosis compared to patients lacking such mutations. Incorporation of FLT3 inhibitors into existing chemotherapeutic regimens has the potential to improve clinical outcomes in this high-risk group of patients. CEP-701, an indolocarbazole-derived selective FLT3 inhibitor, potently induces apoptosis in FLT3/ITD-expressing cell lines and primary leukemic blasts. We conducted a series of in vitro cytotoxicity experiments combining CEP-701 with chemotherapy using the FLT3/ITD-expressing cell lines MV4-11 and BaF3/ITD as well as a primary blast sample from a patient with AML harboring a FLT3/ITD mutation. CEP-701 induced cytotoxicity in a synergistic fashion with cytarabine, daunorubicin, mitoxantrone, or etoposide if used simultaneously or immediately following exposure to the chemotherapeutic agent. In contrast, the combination of pretreatment with CEP-701 followed by chemotherapy was generally antagonistic, particularly with the more cell cycle-dependent agents such as cytarabine. This effect appears to be due to CEP-701 causing cell cycle arrest. We conclude that in FLT3/ITD-expressing leukemia cells, CEP-701 is synergistic with standard AML chemotherapeutic agents, but only if used simultaneously with or immediately following the chemotherapy. These results should be considered when designing trials combining chemotherapy with each of the FLT3 inhibitors currently in clinical development. (Blood. 2004; 104:1145-1150)

scholarly journals Identification of cell cycle dependent methotrexate resistance in placenta site trophoblastic tumors

2020 ◽  
Author(s):  
Jing Xu ◽  
Ling Zhang ◽  
Qiyu Liu ◽  
Luyao Ren ◽  
Ke Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Fold Increase ◽  
Cycle Arrest ◽  
Pdx Model ◽  
Ic50 Value ◽  
Cycle Dependent

Abstract Background The purpose is to study the mechanism of chemotherapy resistance in Placental site trophoblastic tumor(PSTT).Methods We established PSTT cell lines by primary culture of a surgically resected PSTT tissues and identified the expression of immune-phenotype markers(HLA-G, β-catenin, CD146, Muc4, hPL, hCG) by immunofluorescence. We measured the IC50 value of methotrexate(MTX), etoposide(VP-16), actinomycin-D(Act-D), cisplatin(DDP), fluorouracil(5-FU) and paclitaxel(TAX) in PSTTs and used a special Mini patient-derived xenograft (Mini PDX) model to evaluate effectiveness of these drugs in vivo. Given that MTX is a cell cycle-dependent chemotherapeutic, we analyzed cell cycle characteristics of PSTT and choriocarcinoma cell lines by flow cytometry and then analyzed RNA profiles and WGS data of the PSTT cell lines to identify the potential mechanism.Results We identified the expression of HLA-G, β-catenin, CD146, hPL and hCG in PSTT cell lines. The IC50 value of MTX was 4.922 mg/ml in PSTT-1, 4.525 mg/ml in PSTT-2, 5.117 mg/ml in PSTT-3, 0.0166 µg/ml in JEG-3 cells (p༜0.001), and 0.01 µg/ml in JAR cells (p༜0.001), with nearly 50,000-fold increase in PSTTs than in choriocarcinoma, indicating that PSTTs are resistant to MTX in vitro. The Mini PDX model revealed that PSTTs are also resistant to MTX in vivo. Cell cycle analysis showed dysregulation of G1/S transition and cell cycle arrest in PSTT cell lines. RNA sequencing profile also identified cell cycle-associated genes which were differentially expressed in PSTT cells than in choriocarcinoma cell.Conclusions We found PSTTs are resistant to MTX in vitro and in vivo compared to choriocarcinoma. Mechanisms could be focused on dysregulation of the G1/S transition and cell cycle arrest.

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 HIV-1 Vpr activates host CRL4-DCAF1 E3 ligase to degrade histone deacetylase SIRT7

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Xiaohong Zhou ◽  
Christina Monnie ◽  
Maria DeLucia ◽  
Jinwoo Ahn
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Histone Deacetylase ◽  
E3 Ligase ◽  
Cycle Arrest ◽  
Wide Range ◽  
In Vitro Reconstitution ◽  
Hiv 1

Abstract Background Vpr is a virion-associated protein that is encoded by lentiviruses and serves to counteract intrinsic immunity factors that restrict infection. HIV-1 Vpr mediates proteasome-dependent degradation of several DNA repair/modification proteins. Mechanistically, Vpr directly recruits cellular targets onto DCAF1, a substrate receptor of Cullin 4 RING E3 ubiquitin ligase (CRL4) for poly-ubiquitination. Further, Vpr can mediate poly-ubiquitination of DCAF1-interacting proteins by the CRL4. Because Vpr-mediated degradation of its known targets can not explain the primary cell-cycle arrest phenotype that Vpr expression induces, we surveyed the literature for DNA-repair-associated proteins that interact with the CRL4-DCAF1. One such protein is SIRT7, a deacetylase of histone 3 that belongs to the Sirtuin family and regulates a wide range of cellular processes. We wondered whether Vpr can mediate degradation of SIRT7 via the CRL4-DCAF1. Methods HEK293T cells were transfected with cocktails of plasmids expressing DCAF1, DDB1, SIRT7 and Vpr. Ectopic and endogeneous levels of SIRT7 were monitered by immunoblotting and protein–protein interactions were assessed by immunoprecipitation. For in vitro reconstitution assays, recombinant CRL4-DCAF1-Vpr complexes and SIRT7 were prepared and poly-ubiqutination of SIRT7 was monitored with immunoblotting. Results We demonstrate SIRT7 polyubiquitination and degradation upon Vpr expression. Specifically, SIRT7 is shown to interact with the CRL4-DCAF1 complex, and expression of Vpr in HEK293T cells results in SIRT7 degradation, which is partially rescued by CRL inhibitor MNL4924 and proteasome inhibitor MG132. Further, in vitro reconstitution assays show that Vpr induces poly-ubiquitination of SIRT7 by the CRL4-DCAF1. Importantly, we find that Vpr from several different HIV-1 strains, but not HIV-2 strains, mediates SIRT7 poly-ubiquitination in the reconstitution assay and degradation in cells. Finally, we show that SIRT7 degradation by Vpr is independent of the known, distinctive phenotype of Vpr-induced cell cycle arrest at the G2 phase, Conclusions Targeting histone deacetylase SIRT7 for degradation is a conserved feature of HIV-1 Vpr. Altogether, our findings reveal that HIV-1 Vpr mediates down-regulation of SIRT7 by a mechanism that does not involve novel target recruitment to the CRL4-DCAF1 but instead involves regulation of the E3 ligase activity.

Simvastatin Induces Death of Multiple Myeloma Cell Lines

2004 ◽  
Vol 52 (5) ◽  
pp. 335-344 ◽  
Author(s):  
Naomi Gronich ◽  
Liat Drucker ◽  
Hava Shapiro ◽  
Judith Radnay ◽  
Shai Yarkoni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Cytoplasmic Calcium ◽  
Multiple Myeloma Cell ◽  
Cycle Arrest ◽  
Rpmi 8226

BackgroundAccumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy.MethodsU266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 μM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity.ResultsExposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 μM for ARH77, 8 μM for RPMI 8226, and 13 μM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected.ConclusionsSimvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.

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