scholarly journals Dual excitation multi-fluorescence flow cytometry for detailed analyses of viability and apoptotic cell transition

10.4081/838 ◽  
2009 ◽  
Vol 47 (4) ◽  
pp. 289 ◽  
Author(s):  
G Mazzini ◽  
C Ferrari ◽  
E Erba
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Membrane ◽  
Apoptotic Cell ◽  
Intact Cell ◽  
Specific Cell ◽  
Apoptotic Cells ◽  
Apoptotic Pathway ◽  
Culture Models ◽  
Cell Cycle Phases

The discrimination of live/dead cells as well as the detection of apoptosis is a frequent need in many areas of experimental biology. Cell proliferation is linked to apoptosis and controlled by several genes. During the cell life, specific events can stimulate proliferation while others may trigger the apoptotic pathway. Very few methods (i.e. TUNEL) are now available for studies aimed at correlation between apoptosis and proliferation. Therefore, there is interest in developing new methodological approaches that are able to correlate apoptosis to the cell cycle phases. Recently new approaches have been proposed to detect and enumerate apoptotic cells by flow cytometry. Among these, the most established and applied are those based on the cell membrane modifications induced in the early phases of the apoptotic process. The dye pair Hoechst 33342 (HO) and Propidium Iodide (PI), thanks to their peculiar characteristics to be respectively permeable and impermeable to the intact cell membrane, seems to be very useful. Unfortunately the spectral interaction of these dyes generates a consistent “energy transfer” from HO to PI. The co-presence of the dyes in a nucleus results in a modification in the intensity of both the emitted fluorescences. In order to designate the damaged cells (red fluorescence) to the specific cell cycle phases (blue fluorescence), we have tested different staining protocols aimed to minimize the interference of these dyes as much as possible. In cell culture models, we are able to detect serum-starved apoptotic cells as well as to designate their exact location in the cell cycle phases using a very low PI concentration. Using a Partec PAS flow cytometer equipped with HBO lamp and argon ion laser, a double UV/blue excitation has been performed. This analytical approach is able to discriminate live blue cells from the damaged (blue-red) ones even at 0.05 ?g/mL PI. The same instrumental setting allows performing other multi-colour analyses including AnnexinV-FITC as well as the possibility to make a correlated analysis to phenotype markers.

scholarly journals Data Driven Cell Cycle Model to Quantify the Efficacy of Cancer Therapeutics Targeting Specific Cell-Cycle Phases From Flow Cytometry Results

2021 ◽  
Vol 1 ◽  
Author(s):  
David W. James ◽  
Andrew Filby ◽  
M. Rowan Brown ◽  
Huw D. Summers ◽  
Lewis W. Francis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Phase Transitions ◽  
Mechanism Of Action ◽  
Cell Cycle Phase ◽  
Data Driven ◽  
Cycle Phase ◽  
Cell Populations ◽  
Specific Cell ◽  
Cell Cycle Phases

Many chemotherapeutic drugs target cell processes in specific cell cycle phases. Determining the specific phases targeted is key to understanding drug mechanism of action and efficacy against specific cancer types. Flow cytometry experiments, combined with cell cycle phase and division round specific staining, can be used to quantify the current cell cycle phase and number of mitotic events of each cell within a population. However, quantification of cell interphase times and the efficacy of cytotoxic drugs targeting specific cell cycle phases cannot be determined directly. We present a data driven computational cell population model for interpreting experimental results, where in-silico populations are initialized to match observable results from experimental populations. A two-stage approach is used to determine the efficacy of cytotoxic drugs in blocking cell-cycle phase transitions. In the first stage, our model is fitted to experimental multi-parameter flow cytometry results from untreated cell populations to identify parameters defining probability density functions for phase transitions. In the second stage, we introduce a blocking routine to the model which blocks a percentage of attempted transitions between cell-cycle phases due to therapeutic treatment. The resulting model closely matches the percentage of cells from experiment in each cell-cycle phase and division round. From untreated cell populations, interphase and intermitotic times can be inferred. We then identify the specific cell-cycle phases that cytotoxic compounds target and quantify the percentages of cell transitions that are blocked compared with the untreated population, which will lead to improved understanding of drug efficacy and mechanism of action.

Flow cytometric detection of apoptotic bone marrow cells with fractional DNA content after application of WR-2721, cyclophosphamide, cisplatin, and exposure of mice to gamma rays

2002 ◽  
Vol 21 (6) ◽  
pp. 335-341 ◽  
Author(s):  
L Mazur ◽  
A Czyzewska ◽  
M Bochenek
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Gamma Rays ◽  
Bone Marrow Cells ◽  
Apoptotic Cell ◽  
Apoptotic Cells ◽  
Chemotherapeutic Drugs ◽  
Normal Cells ◽  
Dna Damaging Agents ◽  
Flow Cytometric

Little is known about the mechanisms of apoptosis triggered in normal cells of the haemopoietic system by the aminothiol WR-2721 (Amifostine), chemotherapeutic drugs, and ionizing radiation; thus, the present study was undertaken to evaluate the effects of WR-2721, cyclophosphamide (CP), cisplatin (CDDP), and 60Co gamma rays on induction of apoptotic DNA degradation in bone marrow cells. Adult male Swiss mice were treated with WR-2721 (400 mg/kg b.wt.), CP (200 mg/kg b.wt.), and CDDP (10 mg/ kg b.wt.), and exposed to 6 Gy 60Co gamma rays. Alterations in the number of apoptotic cells with fractional DNA content and also the cell cycle position of the non-apoptotic cells were determined in the bone marrow at 7 and 24 hours after treatment of mice with these agents, using flow cytometric assay of the controlled extraction of low-MW DNA from apoptotic cells. The chemotherapeutic drugs CP and CDDP and 60Co gamma rays triggered apoptosis and affected the cell cycle position of the non-apoptotic cells in the mouse bone marrow. The pretreatment of mice with WR-2721 resulted in the modulatory action of the aminothiol on induction of apoptotic cell death and changes in the cell cycle distribution of the non-apoptotic cells caused by the DNA-damaging agents. The patterns of changes in the frequency of apoptotic cells and the cell cycle position of the non-apoptotic cells, observed in the bone marrow, were dependent on the agent(s) applied and the time interval after application of the drug(s) and exposure of mice to gamma rays. Understanding of the mechanisms responsible for triggering of apoptotic cell death and disturbing of the cell cycle by the DNA-damaging agents, and modulation of the apoptotic and cell cycle pathways by the aminothiol WR-2721, can lead to more effective therapy and chemo-and radio-protection of normal cells.

scholarly journals Wee1-Regulated Apoptosis Mediated by the Crk Adaptor Protein in Xenopus Egg Extracts

2000 ◽  
Vol 151 (7) ◽  
pp. 1391-1400 ◽  
Author(s):  
Jesse J. Smith ◽  
Erica K. Evans ◽  
Monica Murakami ◽  
Mary B. Moyer ◽  
M. Arthur Moseley ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Apoptotic Cell ◽  
Specific Interaction ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Caspase Activation ◽  
Apoptotic Pathway ◽  
Apoptotic Signaling ◽  
Cell Cycle Regulator ◽  
Egg Extracts

Many of the biochemical reactions of apoptotic cell death, including mitochondrial cytochrome c release and caspase activation, can be reconstituted in cell-free extracts derived from Xenopus eggs. In addition, because caspase activation does not occur until the egg extract has been incubated for several hours on the bench, upstream signaling processes occurring before full apoptosis are rendered accessible to biochemical manipulation. We reported previously that the adaptor protein Crk is required for apoptotic signaling in egg extracts (Evans, E.K., W. Lu, S.L. Strum, B.J. Mayer, and S. Kornbluth. 1997. EMBO (Eur. Mol. Biol. Organ.) J. 16:230–241). Moreover, we demonstrated that removal of Crk Src homology (SH)2 or SH3 interactors from the extracts prevented apoptosis. We now report the finding that the relevant Crk SH2-interacting protein, important for apoptotic signaling in the extract, is the well-known cell cycle regulator, Wee1. We have demonstrated a specific interaction between tyrosine-phosphorylated Wee1 and the Crk SH2 domain and have shown that recombinant Wee1 can restore apoptosis to an extract depleted of SH2 interactors. Moreover, exogenous Wee1 accelerated apoptosis in egg extracts, and this acceleration was largely dependent on the presence of endogenous Crk protein. As other Cdk inhibitors, such as roscovitine and Myt1, did not act like Wee1 to accelerate apoptosis, we propose that Wee1–Crk complexes signal in a novel apoptotic pathway, which may be unrelated to Wee1's role as a cell cycle regulator.

scholarly journals Role of Cell Death in Cellular Processes During Odontogenesis

2021 ◽  
Vol 9 ◽  
Author(s):  
John Abramyan ◽  
Poongodi Geetha-Loganathan ◽  
Marie Šulcová ◽  
Marcela Buchtová
Keyword(s):  
Cell Death ◽  
Tooth Development ◽  
Apoptotic Cell ◽  
Tooth Germ ◽  
Oral Epithelium ◽  
Special Focus ◽  
Apoptotic Cells ◽  
Molecular Signaling ◽  
Apoptotic Pathway ◽  
Cellular Processes

The development of a tooth germ in a precise size, shape, and position in the jaw, involves meticulous regulation of cell proliferation and cell death. Apoptosis, as the most common type of programmed cell death during embryonic development, plays a number of key roles during odontogenesis, ranging from the budding of the oral epithelium during tooth initiation, to later tooth germ morphogenesis and removal of enamel knot signaling center. Here, we summarize recent knowledge about the distribution and function of apoptotic cells during odontogenesis in several vertebrate lineages, with a special focus on amniotes (mammals and reptiles). We discuss the regulatory roles that apoptosis plays on various cellular processes during odontogenesis. We also review apoptosis-associated molecular signaling during tooth development, including its relationship with the autophagic pathway. Lastly, we cover apoptotic pathway disruption, and alterations in apoptotic cell distribution in transgenic mouse models. These studies foster a deeper understanding how apoptotic cells affect cellular processes during normal odontogenesis, and how they contribute to dental disorders, which could lead to new avenues of treatment in the future.

The Role of Urokinase Receptor (uPAR) In Efferocytosis: uPAR Engulfs Apoptotic Cells via a Phosphatidylserine Pathway Mediated by Plasma High Molecular Weight Kininogen

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 929-929 ◽  
Author(s):  
Aizhen Yang ◽  
Jihong Dai ◽  
Raymond B. Birge ◽  
Yi Wu
Keyword(s):  
Molecular Weight ◽  
Flow Cytometry ◽  
Cell Surface ◽  
High Molecular Weight ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Apoptotic Cells ◽  
High Molecular Weight Kininogen ◽  
Viable Cells

Abstract Abstract 929 Phagocytosis of apoptotic cells by phagocytes, also known as efferocytosis, is essential for maintaining normal tissue homeostasis and regulating immune responses. Defects in rapid clearance of apoptotic cells lead to the release of immunogenic cellular contents, which may cause tissue damage and autoimmune disease. Phagocytic receptors differentiate apoptotic cells from viable cells by recognizing ‘don't eat- or eat-me’ signals on the cell surface. Recently, we and others have reported the role of uPAR in mediating efferocytosis. In this study, we examined the mechanism by which uPAR recognizes and internalizes apoptotic cells. By flow cytometry-based in vivo and in vitro phagocytosis assay, we found that in knockout mice the lack of uPAR expression on macrophages decreased their apoptotic cell engulfing activity by >35%. Conversely, soluble uPAR and polyclonal anti-uPAR antibodies (Ab) suppressed the internalization of apoptotic cells by macrophages. However, there was no defect in uPAR-/- macrophage uptake of viable cells, suggesting that uPAR plays a specific role in phagocytosis of apoptotic cells. We established a HEK 293 cell line expressing human full-length uPAR (293-uPAR). In these cells, uPAR-mediated phagocytosis of apoptotic cells was completely blocked by annexin V in the presence of calcium. The effect of annexin V was not observed in the absence of calcium, indicating that uPAR internalizes apoptotic cells through a phosphatidylserine pathway. We also found that uPAR-mediated uptake of apoptotic cells was completely prevented under serum-free conditions. To identify plasma proteins that may opsonize the uPAR function, we used immunodepletion method to test three known uPAR-binding proteins, vitronectin, uPA and high molecular weight kininogen (HK). Depletion of HK from serum by a polyclonal anti-HK Ab significantly reduced the engulfment of apoptotic cells by either macrophages or 293-uPAR cells in a co-culture system. In contrast, depletion of vitronectin or uPA from serum had little effect. uPAR is a GPI-anchored protein. Upon sucrose gradient ultracentrifugation, the majority of uPAR molecules were co-localized with membrane-bound HK in lipid rafts. The binding capacity of HK to apoptotic cell surface was further analyzed by flow cytometry. Phycoerythrin-labeled HK bound to apoptotic cells in a concentration-dependent manner, saturating at 300 nM. In contrast, HK did not bind to viable cells at concentrations up to 1200 nM. It is known that HK is a key component of the plasma contact system and that apoptotic cells potentiate factor Xa formation. Our new findings of the uPAR-HK-phosphatidylserine axis in efferocytosis suggest that this pathway may modulate the coagulation cascade on the surface of apoptotic cells. This pathway may also play a role in the pathogenesis of autoimmune and thrombotic disease. Disclosures: No relevant conflicts of interest to declare.

Phosphatidylcholine catabolism in the MCF-7 cell cycle

2006 ◽  
Vol 84 (5) ◽  
pp. 737-744 ◽  
Author(s):  
Weiyang Lin ◽  
Gilbert Arthur
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Specific Cell ◽  
Synchronized Cells ◽  
M Phase ◽  
G2 Phase ◽  
Cell Cycle Phases ◽  
Kinetics Of ◽  
Mcf 7

The catabolism of phosphatidylcholine (PtdCho) appears to play a key role in regulating the net accumulation of the lipid in the cell cycle. Current protocols for measuring the degradation of PtdCho at specific cell-cycle phases require prolonged periods of incubation with radiolabelled choline. To measure the degradation of PtdCho at the S and G2 phases in the MCF-7 cell cycle, protocols were developed with radiolabelled lysophosphatidylcholine (lysoPtdCho), which reduces the labelling period and minimizes the recycling of labelled components. Although most of the incubated lysoPtdCho was hydrolyzed to glycerophosphocholine (GroPCho) in the medium, the kinetics of the incorporation of label into PtdCho suggests that the labelled GroPCho did not contribute significantly to cellular PtdCho formation. A protocol which involved exposing the cells twice to hydroxyurea, was also developed to produce highly synchronized MCF-7 cells with a profile of G1:S:G2/M of 90:5:5. An analysis of PtdCho catabolism in the synchronized cells following labelling with lysoPtdCho revealed that there was increased degradation of PtdCho in early to mid-S phase, which was attenuated in the G2/M phase. The results suggest that the net accumulation of PtdCho in MCF-7 cells may occur in the G2 phase of the cell cycle.

scholarly journals Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points

2017 ◽  
Vol 216 (3) ◽  
pp. 607-621 ◽  
Author(s):  
Yael Nechemia-Arbely ◽  
Daniele Fachinetti ◽  
Karen H. Miga ◽  
Nikolina Sekulic ◽  
Gautam V. Soni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein A ◽  
Epigenetic Mark ◽  
Specific Cell ◽  
Entry And Exit ◽  
Satellite Repeat ◽  
Reference Models ◽  
Centromere Protein A ◽  
Cell Cycle Phases ◽  
Dna Unwrapping

Chromatin assembled with centromere protein A (CENP-A) is the epigenetic mark of centromere identity. Using new reference models, we now identify sites of CENP-A and histone H3.1 binding within the megabase, α-satellite repeat–containing centromeres of 23 human chromosomes. The overwhelming majority (97%) of α-satellite DNA is found to be assembled with histone H3.1–containing nucleosomes with wrapped DNA termini. In both G1 and G2 cell cycle phases, the 2–4% of α-satellite assembled with CENP-A protects DNA lengths centered on 133 bp, consistent with octameric nucleosomes with DNA unwrapping at entry and exit. CENP-A chromatin is shown to contain equimolar amounts of CENP-A and histones H2A, H2B, and H4, with no H3. Solid-state nanopore analyses show it to be nucleosomal in size. Thus, in contrast to models for hemisomes that briefly transition to octameric nucleosomes at specific cell cycle points or heterotypic nucleosomes containing both CENP-A and histone H3, human CENP-A chromatin complexes are octameric nucleosomes with two molecules of CENP-A at all cell cycle phases.

A Cyclin D2-derived peptide acts on specific cell cycle phases by activating ERK1/2 to cause the death of breast cancer cells

2017 ◽  
Vol 151 ◽  
pp. 24-32 ◽  
Author(s):  
Lilian C. Russo ◽  
Christiane B. Araujo ◽  
Leo K. Iwai ◽  
Emer S. Ferro ◽  
Fabio L. Forti
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Specific Cell ◽  
Cyclin D2 ◽  
Cell Cycle Phases
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