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Zbigniew Darzynkiewicz - One of the best experts on this subject based on the ideXlab platform.
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DNA Damage Response Resulting from Replication Stress Induced by Synchronization of Cells by Inhibitors of DNA Replication: Analysis by Flow Cytometry.
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: Dorota Halicka, Monika Podhorecka, Hong Zhao, Jorge Garcia, Zbigniew DarzynkiewiczAbstract:Cell Synchronization is often achieved by transient inhibition of DNA replication. When cultured in the presence of such inhibitors as hydroxyurea, aphidicolin or excess of thymidine the Cells that become arrested at the entrance to S-phase upon release from the block initiate progression through S then G2 and M. However, exposure to these inhibitors at concentrations commonly used to synchronize Cells leads to activation of ATR and ATM protein kinases as well as phosphorylation of Ser139 of histone H2AX. This observation of DNA damage signaling implies that Synchronization of Cells by these inhibitors is inducing replication stress. Thus, a caution should be exercised while interpreting data obtained with use of Cells synchronized this way since they do not represent unperturbed Cell populations in a natural metabolic state. This chapter critically outlines virtues and vices of most Cell Synchronization methods. It also presents the protocol describing an assessment of phosphorylation of Ser139 on H2AX and activation of ATM in Cells treated with aphidicolin, as a demonstrative of one of several DNA replication inhibitors that are being used for Cell Synchronization. Phosphorylation of Ser139H2AX and Ser1981ATM in individual Cells is detected immunocytochemically with phospho-specific Abs and intensity of immunofluorescence is measured by flow cytometry. Concurrent measurement of Cellular DNA content followed by multiparameter analysis allows one to correlate the extent of phosphorylation of these proteins in response to aphidicolin with the Cell cycle phase.
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Cell Synchronization by inhibitors of DNA replication induces replication stress and DNA damage response: analysis by flow cytometry.
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: Zbigniew Darzynkiewicz, H. Dorota Halicka, Hong Zhao, Monika PodhoreckaAbstract:Cell Synchronization is often achieved by inhibition of DNA replication. The Cells cultured in the presence of such inhibitors as hydroxyurea, aphidicolin, or thymidine become arrested at the entrance to S phase and upon release from the block they synchronously progress through S, G(2), and M. We recently reported that exposure of Cells to these inhibitors at concentrations commonly used to synchronize Cell populations led to phosphorylation of histone H2AX on Ser139 (induction of γH2AX) through activation of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR). These findings imply that the induction of DNA replication stress by these inhibitors activates the DNA damage response signaling pathways and caution about interpreting data obtained with use of Cells synchronized such way as representing unperturbed Cells. The protocol presented in this chapter describes the methodology of assessment of phosphorylation of histone H2AX-Ser139, ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains as well as ataxia telangiectasia mutated (ATM) protein kinase in Cells treated with inhibitors of DNA replication. Phosphorylation of these proteins is detected in individual Cell immunocytochemically with phospho-specific antibody (Ab) and measured by flow cytometry. Concurrent measurement of Cellular DNA content and phosphorylated proteins followed by multiparameter cytometric analysis allows one to correlate extent of their phosphorylation with Cell cycle phase.
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Cell cycle synchronizing properties of staurosporine
Methods in Cell Science, 1996Co-Authors: Silvia Bruno, Frank Traganos, Zbigniew DarzynkiewiczAbstract:Staurosporine (SSP) is a microbial alkaloid isolated from cultures of Streptomyces. For its ability to specifically inhibit protein kinase C and other serine/threonine and tyrosine protein kinases, SSP has been utilized recently to synchronize Cells in G1 and/or G2 phases of the Cell cycle. In the present paper we focus on the synchronizing properties of SSP with respect to three human normal Cell types (PHA-activated peripheral blood lymphocytes, HS-68 and WI-38) and eight human tumor Cell cultures (SV-40 transformed WI-38, SW48, SW480, A253, T-24, A549, MOLT-4 and HL-60). We describe procedures for maintenance and Synchronization of these Cells in culture. We provide the DNA/RNA flow cytometric methodology to verify Cell Synchronization and to evaluate the position of Cell accumulation in specific phase and the level of synchrony. Synchronization in G1 is being achieved in the three normal Cell lines after 24h SSP treatment with low concentrations (5–10 ng/ml) and in G2 after 24h treatment at higher SSP concentrations (50–100 ng/ml) in four of the eight tumor Cell lines (A253, SW48, SW480 and A549). The other four tumor Cell cultures (SV-40, WI-38, T-24, MOLT-4 and HL-60) show, at 50–100 ng/ml SSP concentration, an apparent G2 block, which is actually due to the presence of Cells entering higher DNA ploidy levels. All other Cell type/SSP dose combinations fail to induce Cell Synchronization. We also report a summary of the literature data about Cell Synchronization with SSP in other Cell lines. Our results, together with the results from the literature, suggest that, while SSP may be useful for synchronizing normal Cells in G1, its application for synchronizing tumor and/or transformed Cells is limited. Critical comments follow, as well as suggestions and words of caution addressed to the future users of SSP as a Cell synchronizing agent.
Monika Podhorecka - One of the best experts on this subject based on the ideXlab platform.
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DNA Damage Response Resulting from Replication Stress Induced by Synchronization of Cells by Inhibitors of DNA Replication: Analysis by Flow Cytometry.
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: Dorota Halicka, Monika Podhorecka, Hong Zhao, Jorge Garcia, Zbigniew DarzynkiewiczAbstract:Cell Synchronization is often achieved by transient inhibition of DNA replication. When cultured in the presence of such inhibitors as hydroxyurea, aphidicolin or excess of thymidine the Cells that become arrested at the entrance to S-phase upon release from the block initiate progression through S then G2 and M. However, exposure to these inhibitors at concentrations commonly used to synchronize Cells leads to activation of ATR and ATM protein kinases as well as phosphorylation of Ser139 of histone H2AX. This observation of DNA damage signaling implies that Synchronization of Cells by these inhibitors is inducing replication stress. Thus, a caution should be exercised while interpreting data obtained with use of Cells synchronized this way since they do not represent unperturbed Cell populations in a natural metabolic state. This chapter critically outlines virtues and vices of most Cell Synchronization methods. It also presents the protocol describing an assessment of phosphorylation of Ser139 on H2AX and activation of ATM in Cells treated with aphidicolin, as a demonstrative of one of several DNA replication inhibitors that are being used for Cell Synchronization. Phosphorylation of Ser139H2AX and Ser1981ATM in individual Cells is detected immunocytochemically with phospho-specific Abs and intensity of immunofluorescence is measured by flow cytometry. Concurrent measurement of Cellular DNA content followed by multiparameter analysis allows one to correlate the extent of phosphorylation of these proteins in response to aphidicolin with the Cell cycle phase.
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Cell Synchronization by inhibitors of DNA replication induces replication stress and DNA damage response: analysis by flow cytometry.
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: Zbigniew Darzynkiewicz, H. Dorota Halicka, Hong Zhao, Monika PodhoreckaAbstract:Cell Synchronization is often achieved by inhibition of DNA replication. The Cells cultured in the presence of such inhibitors as hydroxyurea, aphidicolin, or thymidine become arrested at the entrance to S phase and upon release from the block they synchronously progress through S, G(2), and M. We recently reported that exposure of Cells to these inhibitors at concentrations commonly used to synchronize Cell populations led to phosphorylation of histone H2AX on Ser139 (induction of γH2AX) through activation of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR). These findings imply that the induction of DNA replication stress by these inhibitors activates the DNA damage response signaling pathways and caution about interpreting data obtained with use of Cells synchronized such way as representing unperturbed Cells. The protocol presented in this chapter describes the methodology of assessment of phosphorylation of histone H2AX-Ser139, ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains as well as ataxia telangiectasia mutated (ATM) protein kinase in Cells treated with inhibitors of DNA replication. Phosphorylation of these proteins is detected in individual Cell immunocytochemically with phospho-specific antibody (Ab) and measured by flow cytometry. Concurrent measurement of Cellular DNA content and phosphorylated proteins followed by multiparameter cytometric analysis allows one to correlate extent of their phosphorylation with Cell cycle phase.
Jerôme Bertherat - One of the best experts on this subject based on the ideXlab platform.
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Link between steroidogenesis, the Cell cycle, and PKA in adrenocortical tumor Cells
Molecular and Cellular Endocrinology, 2020Co-Authors: Marthe Rizk-rabin, Sabrina Chaoui-ibadioune, Anna Vaczlavik, Christopher Ribes, Michel Polak, Bruno Ragazzon, Jerôme BertheratAbstract:Adrenocortical tumors (ACTs) frequently cause steroid excess and present Cell-cycle dysregulation. cAMP/PKA signaling is involved in steroid synthesis and play a role in Cell-cycle regulation. We investigated, by Cell Synchronization in the different phases of the Cell-cycle, the control of steroidogenesis and the contribution of PKA in adrenocortical Cells (H295R and culture of primary pigmented nodular adrenocortical disease Cells). Cells showed increased steroidogenesis and a maximal PKA activity at G2 phase, and a reduction at G1 phase. PRKACA overexpression, or cAMP stimulation, enhanced PKA activity and induced steroidogenesis in all synchronized groups but is not sufficient to drive Cell-cycle progression. PRKAR1A inactivation enhanced PKA activity and induced STAR gene expression, only in Cells in G1, and triggered Cell-cycle progression in all groups. These findings provide evidence for a tight association between steroidogenesis and Cell-cycle in ACTs. Moreover, PRKAR1A is essential for mediating the function of PKA activity on both steroidogenesis and Cell-cycle progression in adrenocortical Cells.
Hong Zhao - One of the best experts on this subject based on the ideXlab platform.
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DNA Damage Response Resulting from Replication Stress Induced by Synchronization of Cells by Inhibitors of DNA Replication: Analysis by Flow Cytometry.
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: Dorota Halicka, Monika Podhorecka, Hong Zhao, Jorge Garcia, Zbigniew DarzynkiewiczAbstract:Cell Synchronization is often achieved by transient inhibition of DNA replication. When cultured in the presence of such inhibitors as hydroxyurea, aphidicolin or excess of thymidine the Cells that become arrested at the entrance to S-phase upon release from the block initiate progression through S then G2 and M. However, exposure to these inhibitors at concentrations commonly used to synchronize Cells leads to activation of ATR and ATM protein kinases as well as phosphorylation of Ser139 of histone H2AX. This observation of DNA damage signaling implies that Synchronization of Cells by these inhibitors is inducing replication stress. Thus, a caution should be exercised while interpreting data obtained with use of Cells synchronized this way since they do not represent unperturbed Cell populations in a natural metabolic state. This chapter critically outlines virtues and vices of most Cell Synchronization methods. It also presents the protocol describing an assessment of phosphorylation of Ser139 on H2AX and activation of ATM in Cells treated with aphidicolin, as a demonstrative of one of several DNA replication inhibitors that are being used for Cell Synchronization. Phosphorylation of Ser139H2AX and Ser1981ATM in individual Cells is detected immunocytochemically with phospho-specific Abs and intensity of immunofluorescence is measured by flow cytometry. Concurrent measurement of Cellular DNA content followed by multiparameter analysis allows one to correlate the extent of phosphorylation of these proteins in response to aphidicolin with the Cell cycle phase.
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Cell Synchronization by inhibitors of DNA replication induces replication stress and DNA damage response: analysis by flow cytometry.
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: Zbigniew Darzynkiewicz, H. Dorota Halicka, Hong Zhao, Monika PodhoreckaAbstract:Cell Synchronization is often achieved by inhibition of DNA replication. The Cells cultured in the presence of such inhibitors as hydroxyurea, aphidicolin, or thymidine become arrested at the entrance to S phase and upon release from the block they synchronously progress through S, G(2), and M. We recently reported that exposure of Cells to these inhibitors at concentrations commonly used to synchronize Cell populations led to phosphorylation of histone H2AX on Ser139 (induction of γH2AX) through activation of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR). These findings imply that the induction of DNA replication stress by these inhibitors activates the DNA damage response signaling pathways and caution about interpreting data obtained with use of Cells synchronized such way as representing unperturbed Cells. The protocol presented in this chapter describes the methodology of assessment of phosphorylation of histone H2AX-Ser139, ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains as well as ataxia telangiectasia mutated (ATM) protein kinase in Cells treated with inhibitors of DNA replication. Phosphorylation of these proteins is detected in individual Cell immunocytochemically with phospho-specific antibody (Ab) and measured by flow cytometry. Concurrent measurement of Cellular DNA content and phosphorylated proteins followed by multiparameter cytometric analysis allows one to correlate extent of their phosphorylation with Cell cycle phase.
Z. Weng - One of the best experts on this subject based on the ideXlab platform.
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Early and Late Replicative Chromosomal Banding Patterns of Gallus domesticus
The Journal of heredity, 1992Co-Authors: F. A. Ponce De Leon, Z. WengAbstract:Early and late replicating chromosomal banding patterns of Gallus domesticus were investigated by Cell Synchronization and incorporation of 5'-bromodeoxyuridine during early and late DNA synthesis. The early replicating chromosomal banding patterns observed, as revealed by either acridine orange or Hoechst 33258/propidium iodide staining, were similar to the structural G-banding patterns obtained by trypsin digestion and Giemsa staining. Late replicating chromosomal banding showed extensive reverse band complementarity to the G-banding pattern. Cell Synchronization increased the number of prometaphase and metaphase plates available for analysis. G-banding obtained by Hoechst 33258/propidium iodide staining was investigated due to the fact that it is compatible with chromosomal in situ hybridization procedures that use nonisotopically-labeled DNA probes. Standard replicative G-banded and R-banded idiograms, as obtained after Cell Synchronization, are proposed.