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

Donahue, Christopher J.; Santoro, Maxine Fico; Hupe, Donald; Jones, Jay M.; Pollok, Brian A.; Heim, Roger; Giegel, David A.

doi:10.1002/1097-0320(20011101)45:3<225::aid-cyto1166>3.0.co;2-4

Cited by 8 publications

(5 citation statements)

References 30 publications

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“…While this agrees with some previous observations , , other studies have indicated S phase arrest , apoptosis during the G1−S transition , or surviving cells preferentially being in the S+G2/M phase following anti-Fas treatment of Jurkat cells. One study concluded that at low anti-Fas concentrations G0/G1 phase cells have a greater tendency to go into apoptosis, while at higher anti-Fas concentrations, no cell cycle preferences could be detected . However, these differences may be due to the use of different strains of Jurkat cells, different anti-Fas antibodies or other Fas ligands, and various concentrations of cells and antibodies and/or ligands.…”

Section: Discussionmentioning

confidence: 99%

Histone H1 Dephosphorylation Is Not a General Feature in Early Apoptosis

et al. 2008

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Histone H1 is a family of nucleosomal proteins that exist in a number of subtypes. These subtypes can be modified after translation in various ways, above all by phosphorylation. Increasing levels of H1 phosphorylation has been correlated with cell cycle progression, while both phosphorylation and dephosphorylation of histone H1 have been linked to the apoptotic process. Such conflicting results may depend on which various apoptosis-inducing agents cause apoptosis via different apoptotic pathways and often interfere with cell proliferation. Therefore, we investigated the relation between apoptosis and H1 phosphorylation in Jurkat cells after apoptosis induction via both the extrinsic and intrinsic pathways and by taking cell cycle effects into account. After apoptosis induction by anti-Fas, no significant dephosphorylation, as measured by capillary electrophoresis, or cell cycle-specific effects were detected. In contrast, H1 subtypes were rapidly dephosphorylated when apoptosis was induced by camptothecin. We conclude that histone H1 dephosphorylation is not connected to apoptosis in general but may be coupled to apoptosis by the intrinsic pathway or to concomitant growth inhibitory signaling.

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Section: Discussionmentioning

confidence: 99%

Histone H1 Dephosphorylation Is Not a General Feature in Early Apoptosis

et al. 2008

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“…Moreover, when exemplary using transfected cell lines expressing fluorescent reporter genes such as eGFP or mCherry, the choice of the right fluorophore coupled to AV is much more limited [47]. Additionally, a more specific and broader analysis of apoptotic cells regarding their cell cycle or division rate might be carried out simultaneously to the AV-assay.…”

Section: Plos Onementioning

confidence: 99%

Using the SNAP-Tag technology to easily measure and demonstrate apoptotic changes in cancer and blood cells with different dyes

et al. 2020

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In vitro and ex vivo development of novel therapeutic agents requires reliable and accurate analyses of the cell conditions they were preclinical tested for, such as apoptosis. The detection of apoptotic cells by annexin V (AV) coupled to fluorophores has often shown limitations in the choice of the dye due to interference with other fluorescent-labeled cell markers. The SNAP-tag technology is an easy, rapid and versatile method for functionalization of proteins and was therefore used for labeling AV with various fluorophores. We generated the fusion protein AV-SNAP and analyzed its capacity for the specific display of apoptotic cells in various assays with therapeutic agents. AV-SNAP showed an efficient coupling reaction with five different fluorescent dyes. Two selected fluorophores were tested with suspension, adherent and peripheral blood cells, treated by heat-shock or apoptosis-inducing therapeutic agents. Flow cytometry analysis of apoptotic cells revealed a strong visualization using AV-SNAP coupled to these two fluorophores exemplary, which was comparable to a commercial AV-Assay-kit. The combination of the apoptosis-specific binding protein AV with the SNAP-tag provides a novel solid method to facilitate protein labeling using several, easy to change, fluorescent dyes at once. It avoids high costs and allows an ordinary exchange of dyes and easier use of other fluorescent-labeled cell markers, which is of high interest for the preclinical testing of therapeutic agents in e.g. cancer research.

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“…84 In another example, He and colleagues applied a CFP-LEVD-YFP 49 probe to monitor caspase activity in living cells using flow cytometry, 93 while Onuki et al developed a YFP-Bid-CFP 50 probe designed for cleavage by for caspase-8 and studied the relationship between caspase-8 activation and β-amyloid toxicity (Bid is a pro-apoptotic member of the Bcl-2 protein family and an endogenous substrate of caspase-8). 94 CFP-YFP based probes were also used in studies correlating cell cycle with apoptosis in Jurkat cells 95 and for drug screening. 96 Mahajan and colleagues employed probes with a CFP-YFP donor-acceptor pair with caspase-1 and caspase-3 cleavage sequences to determine activity in COS-7 cells, while for in vitro studies probes with blue fluorescent protein (BFP) and green fluorescent protein (GFP) pair were utilized.…”

Section: Caspase Substratesmentioning

confidence: 99%

Small Molecule Active Site Directed Tools for Studying Human Caspases

et al. 2015

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Caspases are proteases of clan CD and were described for the first time more than two decades ago. They play critical roles in the control of regulated cell death pathways including apoptosis and inflammation. Due to their involvement in the development of various diseases like cancer, neurodegenerative diseases or autoimmune disorders, caspases have been intensively investigated as potential drug targets, both in academic and industrial laboratories. This review presents a thorough, deep, and systematic assessment of all technologies developed over the years for the investigation of caspase activity and specificity using substrates and inhibitors, as well as activity based probes, which in recent years have attracted considerable interest due to their usefulness in the investigation of biological functions of this family of enzymes.

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

Cited by 8 publications

References 30 publications

Histone H1 Dephosphorylation Is Not a General Feature in Early Apoptosis

Histone H1 Dephosphorylation Is Not a General Feature in Early Apoptosis

Using the SNAP-Tag technology to easily measure and demonstrate apoptotic changes in cancer and blood cells with different dyes

Small Molecule Active Site Directed Tools for Studying Human Caspases

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