Correlating cell cycle with apoptosis in a cell line expressing a tandem green fluorescent protein substrate specific for group II caspases

Cytometry ◽  
2001 ◽  
Vol 45 (3) ◽  
pp. 225-234 ◽  
Author(s):  
Christopher J. Donahue ◽  
Maxine Santoro ◽  
Donald Hupe ◽  
Jay M. Jones ◽  
Brian Pollok ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Cell Line ◽  
Fluorescent Protein ◽  
Protein Substrate ◽  
A Cell ◽  
Group Ii ◽  
Green Fluorescent

scholarly journals Establishment of a Stable Cell Line Coexpressing Dengue Virus-2 and Green Fluorescent Protein for Screening of Antiviral Compounds

2011 ◽  
Vol 17 (3) ◽  
pp. 283-292 ◽  
Author(s):  
V. Leardkamolkarn ◽  
W. Sirigulpanit
Keyword(s):  
Green Fluorescent Protein ◽  
Dengue Virus ◽  
Cell Line ◽  
Fluorescent Protein ◽  
Antiviral Drug ◽  
Stable Cell Line ◽  
Entry Site ◽  
Mouth Disease Virus ◽  
A Cell ◽  
Green Fluorescent

This study aimed to generate a stable cell line harboring subgenomic dengue virus replicon and a green fluorescent gene (DENV/GFP) for a cell-based model to screen anti-DENV compounds. The gene-encoding envelope protein of DENV-2 was deleted and then replaced with fragments of the GFP gene and a foot-and-mouth-disease virus 2A–derived cleavage site. The human cytomegalovirus immediate early and antisense hepatitis delta virus ribozyme sequences were added at the 5′- and 3′-ends. An internal ribosome entry site and neomycin resistance genes were placed upstream and next to the NS1 gene. The recombinant plasmids were propagated in a mammalian cell line. A stable cell line with the brightest green fluorescent protein and the highest viral protein and RNA expression was selected from six clones. The clone was then examined for effectiveness in an antiviral drug screening assay with compounds isolated from the local plants using two known antiviral agents as controls. Two novel flavones, PMF and TMF, were discovered having DENV-inhibitory properties. The data were validated by a conventional plaque titration assay. The results indicate that this newly developed cell line is efficient for use as a cell-based model for primary screening of anti-DENV compounds.

scholarly journals Cell Cycle-Dependent Changes in Microtubule Dynamics in Living Cells Expressing Green Fluorescent Protein-α Tubulin

2001 ◽  
Vol 12 (4) ◽  
pp. 971-980 ◽  
Author(s):  
Nasser M. Rusan ◽  
Carey J. Fagerstrom ◽  
Anne-Marie C. Yvon ◽  
Patricia Wadsworth
Keyword(s):  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Dynamic Instability ◽  
Microtubule Dynamics ◽  
A Cell ◽  
Cycle Dependent ◽  
Green Fluorescent ◽  
Quantitative Immunoblotting

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-α tubulin construct and a cell line permanently expressing GFP-α tubulin was established (LLCPK-1α). The mitotic index and doubling time for LLCPK-1α were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1α cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1α and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1α cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.

scholarly journals Novel Real-Time Monitoring System for Human Cytomegalovirus- Infected Cells In Vitro That Uses a Green Fluorescent Protein-PML-Expressing Cell Line

2006 ◽  
Vol 50 (8) ◽  
pp. 2806-2813 ◽  
Author(s):  
T. Ueno ◽  
Y. Eizuru ◽  
H. Katano ◽  
T. Kurata ◽  
T. Sata ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Line ◽  
Real Time ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Fluorescent Protein ◽  
Clinical Isolates ◽  
Pml Bodies ◽  
Infected Cells ◽  
Green Fluorescent

ABSTRACT Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for “immediate-early 1”) protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

scholarly journals Derivation and Cloning of a Novel Rhesus Embryonic Stem Cell Line Stably Expressing Tau-Green Fluorescent Protein

Stem Cells ◽  
2008 ◽  
Vol 26 (6) ◽  
pp. 1444-1453 ◽  
Author(s):  
Florence Wianny ◽  
Agnieszka Bernat ◽  
Cyril Huissoud ◽  
Guillaume Marcy ◽  
Suzy Markossian ◽  
...  
Keyword(s):  
Stem Cell ◽  
Green Fluorescent Protein ◽  
Embryonic Stem Cell ◽  
Cell Line ◽  
Fluorescent Protein ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Stem Cell Line ◽  
Green Fluorescent

Autophagic and Apoptotic Effects of Tyrosine Kinase Inhibitors in Myeloid Leukemia: Comparison of Three Generation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4915-4915
Author(s):  
Cagla Kayabasi ◽  
Cigir Biray Avci ◽  
Sunde Yilmaz Susluer ◽  
Tugce Balci ◽  
Yusuf Baran ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Control Group ◽  
Green Fluorescent

Abstract Abstract 4915 The aim of the study was to evaluate the differences in cytotoxicity, apoptosis and autophagy levels in myeloid leukemia cell lines treated with tyrosine kinase inhibitors compared to cell line resistant to imatinib and control group. Chronic myeloid leukemia model was created by using cell lines as K-562 cell line for Ph+ chronic myeloid leukemia model, HL-60 cell line for acute promyelocytic Ph- leukemia model. NCI-BL2171 normal cell line was used as a control group while K562/ima3 cell line was used as an imatinib resistant model. Imatinib (STI571), Dasatinib (BMS-354825), Ponatinib (AP24534) were used as tyrosine kinase inhibitors in this study. Cytotoxicity analysis was conducted by WST-1 analysis. Apoptotis was evaluated by AnnexinV-enhanced green fluorescent protein (EGFP) and by Mitoprobe JC-1 for Mitochondrial Potential Detection. Autophagy was analyzed by The Premo Autophagy Tb/GFP TR-FRET LC3B assay which measures autophagy in cells expressing green fluorescent protein (GFP)-tagged LC3B using a Tb-based TR-FRET immunoassay approach. By using IC50 doses of tyrosine kinase inhibitors, autophagic effect of these drugs on cell lines were examined at 24th hours. Cells not treated with the active substance or chloroquine were considered as control groups. Chloroquine-treated cells were used as positive control for autophagy. LC3B-II increase is an indicator of autophagic suppression. Cells treated with chloroquine were compared with cells treated with active substances and concentrations of BacMam that displayed the highest LC3B-II increase were selected. Autophagic suppression ratio of the drugs was evaluated among the control group. Cytotoxicity, apoptosis and autophagy analysis results were provided in Table. Compared to control group, 30 μM chloroquine repressed autophagy 1. 93, 1. 48, 2. 74 and 1. 54 fold in K562, HL-60, K562/ima3 and NCI-BL 2171 cells, respectively. In HL-60 cells while Imatinib represented 0. 77 fold autophagy, it repressed autophagy 1. 77 and 3. 49 fold in K562 and K562/ima3 cells respectively. Dasatinib repressed autophagy 2. 11, 1. 95 and 4. 62 fold and Ponatinib repressed autophagy 2. 09, 1. 60 and 9. 15 fold in K562, HL-60, K562/ima3 cells respectively. Imatinib, Dasatinib and Ponatinib did not repressed autophagy in NCI-BL 2171 cells. In conclusion, apoptosis and autophagy paradox was illuminated in myeloid leukemia cells via tyrosine kinase inhibitors and autophagy may be a new strategy for targeted therapy in myeloid leukemia after clarifying responsible genes and proteins in signal transduction pathways. Cytotoxicity Apoptosis Autophagy WST-1 IC50 (nM) Annexin V JC-1 Premo Autophagy Ýmatinib Dasatinib Ponatinib Ýmatinib Dasatinib Ponatinib Ýmatinib Dasatinib Ponatinib Ýmatinib Dasatinib Ponatinib K562 24th hour 1.70 3.65 3.05 3.07 1.37 1.35 1.43 1.77 2.11 2.09 48th hour 650.00 0.24 2.67 2.51 2.32 2.03 2.35 2.06 72nd hour 4.53 4.81 3.00 2.97 3.07 2.50 HL-60 24th hour 1.33 1.26 1.32 1.29 1.22 1.34 0.77 1.95 1.60 48th hour 18000.00 1.39 1.23 1.41 1.61 1.92 1.96 72nd hour 896.00 607.00 2.21 1.80 2.82 1.58 1.73 2.23 K562/ima3 24th hour 1.33 0.76 1.69 1.51 1.36 1.59 3.49 4.62 9.15 48th hour 18350.00 1830.00 9.87 1.80 1.94 2.03 2.82 1.22 1.40 72nd hour 1.34 1.44 1.41 2.61 1.40 1.56 NCI-BL 2171 24th hour 48.00 2.48 2.79 2.62 3.99 4.04 4.25 1.01 0.88 0.90 48th hour 274.00 30.00 4.11 4.33 4.15 5.05 2.75 3.11 72nd hour 6.14 6.04 6.03 8.27 3.71 3.95 Disclosures: No relevant conflicts of interest to declare.

scholarly journals Conditional Transgenic System for Mouse Aurora A Kinase: Degradation by the Ubiquitin Proteasome Pathway Controls the Level of the Transgenic Protein

2005 ◽  
Vol 25 (12) ◽  
pp. 5270-5281 ◽  
Author(s):  
Tomokazu Fukuda ◽  
Yuji Mishina ◽  
Michael P. Walker ◽  
Richard P. DiAugustine
Keyword(s):  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Aurora A ◽  
Mitotic Kinase ◽  
Mitotic Stage ◽  
Conditional Expression ◽  
Ubiquitin Proteasome ◽  
Green Fluorescent

ABSTRACT Aurora A is a mitotic kinase that localizes to centrosomes. Expression of this protein is normally limited to the mitotic stage (G2-M) of the cell cycle, whereas human cancer cells frequently exhibit overexpression of Aurora A protein regardless of the cell cycle stage. In the present study, Aurora A transgenic mouse lines were generated with a new conditional expression system (cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter-Z-enhanced green fluorescent protein) in order to analyze the function of this protein. Although transcripts for Aurora A were elevated in multiple organs of the transgenic mice, the corresponding protein was not detected in extracts analyzed by immunoblotting. The treatment of transgenic-derived embryonic fibroblasts (MEF) with proteasome inhibitors markedly increased the protein level of transgenic Aurora A, indicating that the transgenic Aurora A protein is readily degraded in normal mouse tissues. Under the exponential growth conditions of MEF cells, transgenic Aurora A was detected within the mitotic stage of the cell cycle and localized to centrosomes. In contrast, the marker of the transgenic promoter (enhanced green fluorescent protein) was continuously expressed throughout the cell cycle, indicating the constitutive transcription of transgenic mRNA. These results indicate that transgenic Aurora A is protected from degradation within G2-M but is immediately degraded after translation in the G1-S stage of the cell cycle. The findings obtained with this transgenic model and derived cells support that the transition from protection to degradation by the ubiquitin proteasome system at the end of mitosis is an important step in controlling the level of Aurora A protein during the cell cycle.

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