Flow cytometry to sort mammalian cells in cytokinesis

Gasnereau, Isabelle; Ganier, Olivier; Bourgain, Florence; Gramont, Armand de; Gendron, Marie Claude; Sobczak-Thépot, Joëlle

doi:10.1002/cyto.a.20352

Cited by 19 publications

(17 citation statements)

References 51 publications

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“…In contrast, no Chk1-Ser 345 phosphorylation was observed in the Adv transduction control. To further verify this finding and test whether the activation of Chk1 induced by Vpr indeed starts in S phase, HeLa cells were synchronized in the M phase (Figure 1B) by treatment with 100 ng/mL of Nocodazole [50]. Cell cycle profiles and Chk1-Ser 345 phosphorylation were then detected.…”

Section: Resultsmentioning

confidence: 99%

“…HeLa cells were synchronized to the G1/S boundary of the cell cycle using a previously described double thymidine (DT) block method [49]. Synchronized mitotic cells were obtained following treatment with Nocodazole for 20 hrs (100ng/ml, Sigma) [50]. To induce DNA checkpoints, cells were treated either with HU (10mM) or UV (10 sec at the dose rate of 3 J/m 2 ) immediately after cell release from the DT block.…”

Section: Methodsmentioning

confidence: 99%

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Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

Park

Dong

et al. 2010

Retrovirology

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BackgroundCell cycle G2 arrest induced by HIV-1 Vpr is thought to benefit viral proliferation by providing an optimized cellular environment for viral replication and by skipping host immune responses. Even though Vpr-induced G2 arrest has been studied extensively, how Vpr triggers G2 arrest remains elusive.ResultsTo examine this initiation event, we measured the Vpr effect over a single cell cycle. We found that even though Vpr stops the cell cycle at the G2/M phase, but the initiation event actually occurs in the S phase of the cell cycle. Specifically, Vpr triggers activation of Chk1 through Ser345 phosphorylation in an S phase-dependent manner. The S phase-dependent requirement of Chk1-Ser345 phosphorylation by Vpr was confirmed by siRNA gene silencing and site-directed mutagenesis. Moreover, downregulation of DNA replication licensing factors Cdt1 by siRNA significantly reduced Vpr-induced Chk1-Ser345 phosphorylation and G2 arrest. Even though hydroxyurea (HU) and ultraviolet light (UV) also induce Chk1-Ser345 phosphorylation in S phase under the same conditions, neither HU nor UV-treated cells were able to pass through S phase, whereas vpr-expressing cells completed S phase and stopped at the G2/M boundary. Furthermore, unlike HU/UV, Vpr promotes Chk1- and proteasome-mediated protein degradations of Cdc25B/C for G2 induction; in contrast, Vpr had little or no effect on Cdc25A protein degradation normally mediated by HU/UV.ConclusionsThese data suggest that Vpr induces cell cycle G2 arrest through a unique molecular mechanism that regulates host cell cycle regulation in an S-phase dependent fashion.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

Park

Dong

et al. 2010

Retrovirology

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“…To synchronize HeLa cells in mitosis, exponentially growing cells were treated with 5 ng/ml Nocodazole (Sigma) for 16 h [35]. Mitotic cells were collected by shake-off in cold PBS, washed twice in PBS, and further cultured for 2–3 hours in complete DMEM without nocodazole, so as to proceed in G1 phase before drug treatment.…”

Section: Methodsmentioning

confidence: 99%

Cdt1 Is Differentially Targeted for Degradation by Anticancer Chemotherapeutic Drugs

et al. 2012

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BackgroundMaintenance of genome integrity is crucial for the propagation of the genetic information. Cdt1 is a major component of the pre-replicative complex, which controls once per cell cycle DNA replication. Upon DNA damage, Cdt1 is rapidly targeted for degradation. This targeting has been suggested to safeguard genomic integrity and prevent re-replication while DNA repair is in progress. Cdt1 is deregulated in tumor specimens, while its aberrant expression is linked with aneuploidy and promotes tumorigenesis in animal models. The induction of lesions in DNA is a common mechanism by which many cytotoxic anticancer agents operate, leading to cell cycle arrest and apoptosis.Methodology/Principal FindingIn the present study we examine the ability of several anticancer drugs to target Cdt1 for degradation. We show that treatment of HeLa and HepG2 cells with MMS, Cisplatin and Doxorubicin lead to rapid proteolysis of Cdt1, whereas treatment with 5-Fluorouracil and Tamoxifen leave Cdt1 expression unaffected. Etoposide affects Cdt1 stability in HepG2 cells and not in HeLa cells. RNAi experiments suggest that Cdt1 proteolysis in response to MMS depends on the presence of the sliding clamp PCNA.Conclusion/SignificanceOur data suggest that treatment of tumor cells with commonly used chemotherapeutic agents induces differential responses with respect to Cdt1 proteolysis. Information on specific cellular targets in response to distinct anticancer chemotherapeutic drugs in different cancer cell types may contribute to the optimization of the efficacy of chemotherapy.

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“…Cell cycle progression was determined by flow cytometry as described (23). The cell cycle profile was determined from the DNA fluorescence data using the Multicycle software (Phoenix Flow System Inc., San Diego, CA).…”

Section: Methodsmentioning

confidence: 99%

Priming and potentiation of DNA damage response by fibronectin in human colon cancer cells and tumor-derived myofibroblasts

et al. 2011

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We have previously shown that the genotoxin-induced apoptosis in mouse embryo fibroblasts was enhanced by the extracellular matrix protein fibronectin (FN). In the present study, we tested the hypothesis that FN regulates the DNA damage response (DDR) signaling pathways in HCT116 (p53-wt) and HT29 (p53-mut) human colon cancer cells and tumor-derived myofibroblasts. DNA damage recognition mechanisms were analyzed by immunofluorescence staining, cell cycle analysis and immunoblotting addressed at specific molecular sensors and executors involved in the DDR pathways. The results show that FN, but not collagen type IV or Matrigel, initiates and potentiates the DDR to the anticancer drug cisplatin in an α5 integrin and cell cycle-dependent manner (S and G2/M phases) in human colon cancer cells. Accordingly, we demonstrate that adhesion of HCT116 cells to FN upregulated the expression of α5 integrin FN receptors at the cell surface. These FN-induced DDR pathways include the concerted phosphorylation of histone H2AX on Ser139 detected as nuclear foci (γ-H2AX, 15 and 25 kDa forms), of ataxia telangiectasia mutated (ATM-Ser1981), checkpoint kinase 2 (CHK2-Thr68, 62 and 67 kDa) and p53-Ser15. These FN-induced γ-H2AX signals were interrupted or attenuated by selective inhibitors acting on the DDR pathway kinases, including wortmannin (targeting the phosphatidylinositol-3-kinase-related protein kinases, PIKK), KU55933 (ATM), NU7026 (DNA-dependent protein kinase catalytic subunit, DNA-PK-cs) and SP600125 (JNK2, stress activated protein kinase/c-Jun N-terminal kinase-2). Adhesion to FN also potentiated the γ-H2AX signals and the cytotoxic effects of cisplatin in human colon tumor-derived myofibroblasts. These data provide evidence that FN promotes DNA damage recognition and chemosensitization to cisplatin via the potentiation of the DNA damage signaling responses in human colon cancer cells and tumor-derived myofibroblasts.

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Flow cytometry to sort mammalian cells in cytokinesis

Cited by 19 publications

References 51 publications

Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

Cdt1 Is Differentially Targeted for Degradation by Anticancer Chemotherapeutic Drugs

Priming and potentiation of DNA damage response by fibronectin in human colon cancer cells and tumor-derived myofibroblasts

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